**Bone Marrow Infiltration in Neuroblastoma: Characteristics of Infiltrating Cells and Role of the Microenvironment**

Fabio Morandi, Paola Scaruffi, Sara Stigliani, Barbara Carlini and Maria Valeria Corrias

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55774

#### **1. Introduction**

Neuroblastoma (NB) is a pediatric tumor that arises from peripheral nervous system. The clinical presentation of NB is highly heterogeneous, ranging from asymptomatic tumor masses requiring little, if any, treatment to metastatic disease requiring intensive multimodal therapies (see [1] for review). Also the outcome of NB patients is highly variable. The 5-years overall survival ranges from 98-100% for stage 1 infants without *MYCN* amplification to less than 20% for children with stage 4 *MYCN* amplified tumors [2]. The main prognostic factors are indeed stage, age at diagnosis and *MYCN* oncogene status [3].

At diagnosis, about 50% of cases present metastatic spread that mainly involves vascularized tissues, such as bone marrow (BM) and bone. According to the International Neuroblastoma Staging System (INSS [4]), patients with metastatic disease are categorized as stage 4, whereas in the absence of metastatic spread patients are categorized as stage 1, 2 and 3, depending on the extent of the primary tumor (within or across the midline), the involvement of ipsilateral or controlateral lymph nodes, and the surgical possibility of resection. Recently, the INSS has been replaced by the International Neuroblastoma Risk Group-Stage System (INRG-SS) based on image-defined surgical risk [5]. According to the INRG-SS criteria, patients with metastatic spread have stage M disease, while patients with localized disease have stage L1 or L2, depending on the level of surgical risk.

© 2013 Morandi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **2. Role of bone marrow infiltration in staging of NB patients**

Since the spread of tumor cells to the BM is a grim prognostic indicator for patients with NB, the search for BM infiltration is of utmost importance for both staging and therapeutic purposes. According to the INSS, presence of metastases is assessed by appropriate imaging, including 123I-MIBG scintigraphy, and morphological examination of both BM smears and trephine biopsies [4]. In spite of the limited sensitivity of morphological analysis, alternative methods for NB cell detection, such as flow cytometry, immunocytology (IC) and reverse transcription-polymerase chain reaction (RT-PCR) for markers selectively expressed in NB cells, are not included into the staging system. The reason for this choice depends on the good survival rate of children with localized NB [2], suggesting that, even if present, few circulating tumor cells are not clinically relevant. Only few patients with localized NB, in fact, relapse or die of the disease. Therefore, the introduction of more sensitive methods for the detection of BM-infiltrating NB cells may cause inappropriate overtreatment of patients with localized NB, resulting in unnecessary toxicity and long term side effects. These sensitive methods, however, are currently evaluated in ongoing clinical protocols for patients with metastatic disease for their potential prognostic value.

amplified for 35 cycles in a thermal cycler with primers specific for NB related markers, such as *Tyrosine hydroxylase* (*TH*) [7], *GD2 synthase* [8], *PHOX2B* [9], and for a housekeeping gene (*GAPDH, β2-microglobulin*). An aliquot of each PCR reaction is then loaded onto a 2% agarose gel and electrophoresed. The amplification products are visualized by staining with ethidium bromide. A sample is considered positive for the tested gene if an amplification product of the expected size is present, a sample is considered negative if product for the tested gene is absent

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**Figure 1.** Cytofluorimetric analysis of BM cells from a patient with stage 4 NB, showing the presence of GD2-CD45+ normal hematopoietic cells (upper left panel 74% of total cells) and GD2+CD45- NB metastatic cells (right bottom

panel 26% of total cells).

and the product for the housekeeping gene is present, as shown in Figure 3.

#### **3. Sensitive methods for detection of neuroblastoma cells in BM samples**

#### **3.1. Flow cytometry**

Total cells from BM aspirates are incubated with a monoclonal antibody (mAb) directed against a neuroblastoma specific antigen, as the disialoganglioside GD2, and with a mAb specific for the hematopoietic cells, as the pan leukocyte CD45, each one labeled with a different fluorochrome. After removal of the unbound mAbs and erythrocyte lysis, samples are analyzed in a flow cytometer. The GD2-positive CD45-negative cells, shown in Figure 1, are considered BM-infiltrating NB cells and their number relative to the total hematopoietic cells can be determined.

#### **3.2. Immunocytochemistry (IC)**

Ficoll-purified mononuclear cells from BM aspirates are spotted onto slides that are fixed and then incubated with an anti-GD2 mAb. After stain development, the stained GD2-positive cells can be counted by light microscopy, relative to a given number of total mononuclear cells. Standardized conditions for IC analysis and reporting have been developed [6]. An example of a BM slide with GD2-positive cells is shown in Figure 2.

#### **4. Molecular analysis (qualitative and quantitative RT-PCR)**

BM aspirates from the iliac crests are stored in tubes containing RNA preservative. Total RNA is then extracted and reverse transcribed (RT). For qualitative PCR analysis, the cDNA is amplified for 35 cycles in a thermal cycler with primers specific for NB related markers, such as *Tyrosine hydroxylase* (*TH*) [7], *GD2 synthase* [8], *PHOX2B* [9], and for a housekeeping gene (*GAPDH, β2-microglobulin*). An aliquot of each PCR reaction is then loaded onto a 2% agarose gel and electrophoresed. The amplification products are visualized by staining with ethidium bromide. A sample is considered positive for the tested gene if an amplification product of the expected size is present, a sample is considered negative if product for the tested gene is absent and the product for the housekeeping gene is present, as shown in Figure 3.

**2. Role of bone marrow infiltration in staging of NB patients**

their potential prognostic value.

**3.1. Flow cytometry**

166 Neuroblastoma

can be determined.

**3.2. Immunocytochemistry (IC)**

of a BM slide with GD2-positive cells is shown in Figure 2.

**4. Molecular analysis (qualitative and quantitative RT-PCR)**

Since the spread of tumor cells to the BM is a grim prognostic indicator for patients with NB, the search for BM infiltration is of utmost importance for both staging and therapeutic purposes. According to the INSS, presence of metastases is assessed by appropriate imaging, including 123I-MIBG scintigraphy, and morphological examination of both BM smears and trephine biopsies [4]. In spite of the limited sensitivity of morphological analysis, alternative methods for NB cell detection, such as flow cytometry, immunocytology (IC) and reverse transcription-polymerase chain reaction (RT-PCR) for markers selectively expressed in NB cells, are not included into the staging system. The reason for this choice depends on the good survival rate of children with localized NB [2], suggesting that, even if present, few circulating tumor cells are not clinically relevant. Only few patients with localized NB, in fact, relapse or die of the disease. Therefore, the introduction of more sensitive methods for the detection of BM-infiltrating NB cells may cause inappropriate overtreatment of patients with localized NB, resulting in unnecessary toxicity and long term side effects. These sensitive methods, however, are currently evaluated in ongoing clinical protocols for patients with metastatic disease for

**3. Sensitive methods for detection of neuroblastoma cells in BM samples**

Total cells from BM aspirates are incubated with a monoclonal antibody (mAb) directed against a neuroblastoma specific antigen, as the disialoganglioside GD2, and with a mAb specific for the hematopoietic cells, as the pan leukocyte CD45, each one labeled with a different fluorochrome. After removal of the unbound mAbs and erythrocyte lysis, samples are analyzed in a flow cytometer. The GD2-positive CD45-negative cells, shown in Figure 1, are considered BM-infiltrating NB cells and their number relative to the total hematopoietic cells

Ficoll-purified mononuclear cells from BM aspirates are spotted onto slides that are fixed and then incubated with an anti-GD2 mAb. After stain development, the stained GD2-positive cells can be counted by light microscopy, relative to a given number of total mononuclear cells. Standardized conditions for IC analysis and reporting have been developed [6]. An example

BM aspirates from the iliac crests are stored in tubes containing RNA preservative. Total RNA is then extracted and reverse transcribed (RT). For qualitative PCR analysis, the cDNA is

**Figure 1.** Cytofluorimetric analysis of BM cells from a patient with stage 4 NB, showing the presence of GD2-CD45+ normal hematopoietic cells (upper left panel 74% of total cells) and GD2+CD45- NB metastatic cells (right bottom panel 26% of total cells).

gene, in the presence of specific probes labeled with different fluorochromes. If a cDNA specific for the NB marker is present, at each amplification cycle the probe-specific fluorescence is released and detected by the instrument. At the end of 40 cycles, the cycle at whom the released fluorescence overcomes the threshold is used to quantitate, through an algorithm [10], the relative amount of the NB-specific amplified product. An example of quantitative analysis is shown in Figure 4. Standardized conditions for RT-qPCR analysis and reporting have been

Bone Marrow Infiltration in Neuroblastoma: Characteristics of Infiltrating Cells and Role of the Microenvironment

**Figure 4.** Representative amplification plots showing fluorescence amounts in relation to the number of cycles. Curves of the same color correspond to the replicates of the same sample. On the right side of the figure the plate layout is

**4.1. Sensitivity, specificity, diagnostic accuracy and prognostic values of different detection**

and bone trephine biopsy, respectively [12]. Sensitivity of flow cytometry, immunocytochem‐ istry and molecular analysis is evaluated in spiking experiments by mixing logarithmic dilutions of a NB cell line with fixed amount of mononuclear cells from a healthy donor.


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169

cells [13], whereas that of both IC and RT-qPCR is

for smear

The sensitivity of morphological analysis is approximately 1 NB cell out of 103

developed [11].

displayed.

**methods**

1 out of 106

Sensitivity of flow cytometry is 1 out of 104

cells [11, 12].

**Figure 2.** Immunocytological analysis of a BM smear from a patient with stage 4 NB, showing the presence of rosettes of NB cells stained in red and normal unstained hematopoietic cells (Magnification 20X).

**Figure 3.** Representative agarose gel electrophoresis of PCR products. Samples 1, 2 and 7 are positive for TH expres‐ sion, whereas samples 3, 4, 5 and 6 are negative. M: molecular weight DNA markers.

For quantitative analysis (qPCR), the cDNA is amplified for 40 cycles in triplicates in a realtime thermal cycler, using primers specific for a NB related marker and for a housekeeping gene, in the presence of specific probes labeled with different fluorochromes. If a cDNA specific for the NB marker is present, at each amplification cycle the probe-specific fluorescence is released and detected by the instrument. At the end of 40 cycles, the cycle at whom the released fluorescence overcomes the threshold is used to quantitate, through an algorithm [10], the relative amount of the NB-specific amplified product. An example of quantitative analysis is shown in Figure 4. Standardized conditions for RT-qPCR analysis and reporting have been developed [11].

**Figure 2.** Immunocytological analysis of a BM smear from a patient with stage 4 NB, showing the presence of rosettes

**Figure 3.** Representative agarose gel electrophoresis of PCR products. Samples 1, 2 and 7 are positive for TH expres‐

For quantitative analysis (qPCR), the cDNA is amplified for 40 cycles in triplicates in a realtime thermal cycler, using primers specific for a NB related marker and for a housekeeping

sion, whereas samples 3, 4, 5 and 6 are negative. M: molecular weight DNA markers.

of NB cells stained in red and normal unstained hematopoietic cells (Magnification 20X).

168 Neuroblastoma

**Figure 4.** Representative amplification plots showing fluorescence amounts in relation to the number of cycles. Curves of the same color correspond to the replicates of the same sample. On the right side of the figure the plate layout is displayed.

#### **4.1. Sensitivity, specificity, diagnostic accuracy and prognostic values of different detection methods**

The sensitivity of morphological analysis is approximately 1 NB cell out of 103 -104 for smear and bone trephine biopsy, respectively [12]. Sensitivity of flow cytometry, immunocytochem‐ istry and molecular analysis is evaluated in spiking experiments by mixing logarithmic dilutions of a NB cell line with fixed amount of mononuclear cells from a healthy donor. Sensitivity of flow cytometry is 1 out of 104 cells [13], whereas that of both IC and RT-qPCR is 1 out of 106 cells [11, 12].

Specificity of flow cytometry and immunocytochemistry is assessed by negative results obtained in samples from healthy donors. For molecular analysis specificity is assessed by negative results in samples reverse transcribed without reverse transcriptase and in samples amplified without cDNAs.

#### **4.2. Comparison of different techniques and markers**

The sensitivity and specificity of different techniques and of different markers have been compared [12, 14-19]. Following the development of standardized conditions [6], GD2 is the marker of choice for IC analysis [20]. GD2-IC is currently evaluated for its prognostic signifi‐ cance in ongoing protocols for stage 4 NB patients, both in Europe and North America.

To overcome NB tumor heterogeneity and increase sensitivity and specificity of RT-PCR analysis, different panels of various NB-specific markers have been developed thanks to the microarray technology [9, 21, 22]. To date the panels have not been compared, but each panel is currently tested for its prognostic role in ongoing therapeutic protocols for stage 4 NB patients.

## **5. BM infiltration in patients with localized NB detected by sensitive methods**

Patients with localized NB without *MYCN* amplification have good overall survival (OS) rates, following either surgery alone (stage 1 and 2, 95% and 86% OS, respectively), or standard-dose chemotherapy followed by surgery (stage 3, 65% OS) [23]. Both the histological features of the tumors [24] and the presence of no random genetic abnormalities [25] in the primary tumor are highly prognostic in patients with localized NB. Thus, these two parameters are presently evaluated at diagnosis to stratify the patients with localized disease into different therapeutic regimens. Some of the patients with favorable histology and genetics, however, relapse, making the search for new prognostic markers still necessary [3, 20].

**Figure 5.** Overall survival of patients with localized disease without MYCN amplification stratified by GD2 immunocy‐

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Peripheral blood (PB) samples can be obtained without patient's sedation with evident advantages as compared to BM samples. Thus, we tested whether RT-qPCR analysis for NBrelated markers in BM and PB samples from patients with localized NB had a prognostic impact on their survival [30]. The expression of seven different genes, previously shown to be specific for NB cells [7, 9, 13, 14, 18, 31-35], was evaluated and compared to those of healthy subjects. A high percentage of samples resulted positive for the various NB-related markers (Figure 6). Since the patients' cohort had a fairly good survival rate, in accordance with literature data [3, 36, 37], the positive results were likely related to low transcription levels by the PB hematopoietic cells [21, 22, 32, 38], or were due to the existence of so called dormant

tochemistry status

cells, i.e., tumor cells unable to proliferate [39].

Since conventional morphological methods have limited sensitivity, it has been suggested that some of the patients with localized NB could have a low number of metastatic cells that could be responsible for relapse. If such hypothesis was true, the use of sensitive methods, such as IC and RT-qPCR, may be helpful in identifying patients at risk of relapse and death. Indeed, we observed that in patients with localized NB the presence of GD2 positive cells in BM samples at diagnosis negatively associated with survival [12]. Since this finding was based on a small sample size with a relatively short follow-up, a further study performed in a larger cohort of patients confirmed the negative impact of BM GD2-positive cell infiltration on survival of patients with localized NB [26]. Moreover, the negative impact was demonstrated to be independent of *MYCN* amplification (Figure 5), the most important negative prognostic factor for these patients [3]. It is worth noting that *MYCN* amplification is a relatively rare event, occurring in about 10% of patients with localized NB [24, 27-29], making it inadequate to identify all the patients who will eventually relapse.

Specificity of flow cytometry and immunocytochemistry is assessed by negative results obtained in samples from healthy donors. For molecular analysis specificity is assessed by negative results in samples reverse transcribed without reverse transcriptase and in samples

The sensitivity and specificity of different techniques and of different markers have been compared [12, 14-19]. Following the development of standardized conditions [6], GD2 is the marker of choice for IC analysis [20]. GD2-IC is currently evaluated for its prognostic signifi‐ cance in ongoing protocols for stage 4 NB patients, both in Europe and North America.

To overcome NB tumor heterogeneity and increase sensitivity and specificity of RT-PCR analysis, different panels of various NB-specific markers have been developed thanks to the microarray technology [9, 21, 22]. To date the panels have not been compared, but each panel is currently tested for its prognostic role in ongoing therapeutic protocols for stage 4 NB

**5. BM infiltration in patients with localized NB detected by sensitive**

making the search for new prognostic markers still necessary [3, 20].

to identify all the patients who will eventually relapse.

Patients with localized NB without *MYCN* amplification have good overall survival (OS) rates, following either surgery alone (stage 1 and 2, 95% and 86% OS, respectively), or standard-dose chemotherapy followed by surgery (stage 3, 65% OS) [23]. Both the histological features of the tumors [24] and the presence of no random genetic abnormalities [25] in the primary tumor are highly prognostic in patients with localized NB. Thus, these two parameters are presently evaluated at diagnosis to stratify the patients with localized disease into different therapeutic regimens. Some of the patients with favorable histology and genetics, however, relapse,

Since conventional morphological methods have limited sensitivity, it has been suggested that some of the patients with localized NB could have a low number of metastatic cells that could be responsible for relapse. If such hypothesis was true, the use of sensitive methods, such as IC and RT-qPCR, may be helpful in identifying patients at risk of relapse and death. Indeed, we observed that in patients with localized NB the presence of GD2 positive cells in BM samples at diagnosis negatively associated with survival [12]. Since this finding was based on a small sample size with a relatively short follow-up, a further study performed in a larger cohort of patients confirmed the negative impact of BM GD2-positive cell infiltration on survival of patients with localized NB [26]. Moreover, the negative impact was demonstrated to be independent of *MYCN* amplification (Figure 5), the most important negative prognostic factor for these patients [3]. It is worth noting that *MYCN* amplification is a relatively rare event, occurring in about 10% of patients with localized NB [24, 27-29], making it inadequate

amplified without cDNAs.

patients.

170 Neuroblastoma

**methods**

**4.2. Comparison of different techniques and markers**

**Figure 5.** Overall survival of patients with localized disease without MYCN amplification stratified by GD2 immunocy‐ tochemistry status

Peripheral blood (PB) samples can be obtained without patient's sedation with evident advantages as compared to BM samples. Thus, we tested whether RT-qPCR analysis for NBrelated markers in BM and PB samples from patients with localized NB had a prognostic impact on their survival [30]. The expression of seven different genes, previously shown to be specific for NB cells [7, 9, 13, 14, 18, 31-35], was evaluated and compared to those of healthy subjects. A high percentage of samples resulted positive for the various NB-related markers (Figure 6). Since the patients' cohort had a fairly good survival rate, in accordance with literature data [3, 36, 37], the positive results were likely related to low transcription levels by the PB hematopoietic cells [21, 22, 32, 38], or were due to the existence of so called dormant cells, i.e., tumor cells unable to proliferate [39].

In the attempt to discriminate between illegitimate transcription by hematopoietic cells and transcription by low numbers of NB cells, ROC analysis was applied to find cut-off levels able to discriminate patients with localized NB from healthy subjects. Although the use of cut-off levels for each NB-related marker in PB and BM samples increased their specificity, the percentage of positive results that did not correlate with clinical events remained high. Also a modified ROC analysis [40] failed to improve the prognostic value of RT-qPCR analysis for any of the tested marker. However, *TH* expression in PB samples significantly correlated with

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**Figure 7.** Event-free survival of patients with localized disease stratified by TH status in peripheral blood samples

worse EFS of patients with localized NB (Figure 7).

**Figure 6.** Expression of various NB-related markers in BM and PB samples from patients with localized disease and healthy children

In the attempt to discriminate between illegitimate transcription by hematopoietic cells and transcription by low numbers of NB cells, ROC analysis was applied to find cut-off levels able to discriminate patients with localized NB from healthy subjects. Although the use of cut-off levels for each NB-related marker in PB and BM samples increased their specificity, the percentage of positive results that did not correlate with clinical events remained high. Also a modified ROC analysis [40] failed to improve the prognostic value of RT-qPCR analysis for any of the tested marker. However, *TH* expression in PB samples significantly correlated with worse EFS of patients with localized NB (Figure 7).

**Figure 7.** Event-free survival of patients with localized disease stratified by TH status in peripheral blood samples

**Figure 6.** Expression of various NB-related markers in BM and PB samples from patients with localized disease and

healthy children

172 Neuroblastoma

Indeed, *TH* expression was significantly higher in relapsing patients than in patients that remained in complete remission. Similar results were reported by Yanez *et al*. [41] that tested BM and PB samples from patients with localized NB for *TH* and *DCX* mRNA expression. Although the use of multiple markers has been recommended [20], the results obtained in these studies indicated that, in the subset of patients with localized NB, molecular analysis should be limited to *TH* expression in PB samples, because multiple target analysis did not add useful information to that obtained by *TH* analysis alone.

In conclusion, in patients with localized NB, detection of metastatic cells in BM by means of GD2-IC analysis and detection of *TH* mRNA expression in PB samples significantly associated with a higher risk of relapse. Therefore, both analyses may help individuating patients at risk of relapse that may require a closer follow-up.

#### **6. BM infiltration in patients with metastatic disease**

Presence of metastasis at diagnosis in children over 18 months of age is a powerful prognostic factor for a poor outcome [2, 3]. In order to understand the mechanisms responsible for such aggressive behavior, an extensive characterization of primary tumor cells from stage 4 NB patients has been performed. DNA abnormalities [42, 43], gene expression profiles [44-46], and non-coding RNAs expression [47, 48] have been pro‐ posed as sensitive indicators of NB tumor aggressiveness. Unfortunately, all the gene classifiers, although validated on independent patient cohorts [46, 49], did not appear to be helpful in stratifying stage 4 patients into different risk groups. Moreover, whole genome sequencing of primary NB tumors [50] demonstrated that no specific mutations or chromosomal alterations were present in NB tumors, suggesting that the mechanisms responsible for invasiveness and metastasis should be searched elsewhere.

**Figure 8.** Hierarchically clustered heat maps of differentially expressed genes in BM cells from stage 4 patients (infil‐ trated BMs, I), normal BMs (N) from healthy children, and BM cells from localized NB patients (non-infiltrated BMs, NI). Each color patch represents the expression level of genes (row) in that sample (column), with a continuum of expres‐

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Precisely,BMsamplesfromNBpatientssignificantlyoverexpressedgenesinvolvedintheinnate immune responses. In particular, all NB patients expressed an interferon (IFN) signature [57]. IFNs are pleiotropic cytokines involved in different biological processes [58], and IFN signa‐ tures have been associated with tumor progression of melanoma and colorectal cancer [59]. Moreover IFN signatures associated with the worse prognosis of African-American patients with prostate [60] and breast [61] cancer as compared to Caucasian patients. The IFN signature includes genes involved in the defense against bacterial and viral pathogens [62, 63]. Since all NB patients showed the IFN signature it was proposed that the NB primary tumor may release, or induce the release of, soluble factors as occurring during an infection. The BM microenvir‐ onment of NB patients up-regulated also the IFN related DNA damage resistance signature (IRDS), that was shown to be associated with resistance to radiation-induced DNA damage

sion levels from bright green (lowest) to bright red (highest).

[64], as summarized in Table 1.

#### **6.1. Role of the BM microenvironment for NB metastatic invasion**

The processes of invasion, survival and proliferation at distant sites may be mediated in part by the microenvironment. In the BM, several cell types, such as adipocytes, stromal and endothelial cells are present together with cells of all hematopoietic lineages. Each cell type may secrete factors that affect several signaling pathways leading to modifications in BM structural organization and cell function. Among these factors, the CXCL12 chemokine has been proposed to play an important role in NB invasiveness [51-53], but conflicting results have been obtained [54, 55]. Thus, we decided to compare the gene expression profiles of resident BM cells from healthy children to those of BM cells from patients with localized and metastatic NB. The results indicated that the resident BM cells from patients with either localized or metastatic NB have a different genetic signature from healthy children. However, the deregulation of transcription was more evident in the BM microenvironment of patients with metastatic stage 4 disease (Figure 8) [56],

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Indeed, *TH* expression was significantly higher in relapsing patients than in patients that remained in complete remission. Similar results were reported by Yanez *et al*. [41] that tested BM and PB samples from patients with localized NB for *TH* and *DCX* mRNA expression. Although the use of multiple markers has been recommended [20], the results obtained in these studies indicated that, in the subset of patients with localized NB, molecular analysis should be limited to *TH* expression in PB samples, because multiple target analysis did not

In conclusion, in patients with localized NB, detection of metastatic cells in BM by means of GD2-IC analysis and detection of *TH* mRNA expression in PB samples significantly associated with a higher risk of relapse. Therefore, both analyses may help individuating patients at risk

Presence of metastasis at diagnosis in children over 18 months of age is a powerful prognostic factor for a poor outcome [2, 3]. In order to understand the mechanisms responsible for such aggressive behavior, an extensive characterization of primary tumor cells from stage 4 NB patients has been performed. DNA abnormalities [42, 43], gene expression profiles [44-46], and non-coding RNAs expression [47, 48] have been pro‐ posed as sensitive indicators of NB tumor aggressiveness. Unfortunately, all the gene classifiers, although validated on independent patient cohorts [46, 49], did not appear to be helpful in stratifying stage 4 patients into different risk groups. Moreover, whole genome sequencing of primary NB tumors [50] demonstrated that no specific mutations or chromosomal alterations were present in NB tumors, suggesting that the mechanisms

The processes of invasion, survival and proliferation at distant sites may be mediated in part by the microenvironment. In the BM, several cell types, such as adipocytes, stromal and endothelial cells are present together with cells of all hematopoietic lineages. Each cell type may secrete factors that affect several signaling pathways leading to modifications in BM structural organization and cell function. Among these factors, the CXCL12 chemokine has been proposed to play an important role in NB invasiveness [51-53], but conflicting results have been obtained [54, 55]. Thus, we decided to compare the gene expression profiles of resident BM cells from healthy children to those of BM cells from patients with localized and metastatic NB. The results indicated that the resident BM cells from patients with either localized or metastatic NB have a different genetic signature from healthy children. However, the deregulation of transcription was more evident in the BM microenvironment of patients

add useful information to that obtained by *TH* analysis alone.

**6. BM infiltration in patients with metastatic disease**

responsible for invasiveness and metastasis should be searched elsewhere.

**6.1. Role of the BM microenvironment for NB metastatic invasion**

with metastatic stage 4 disease (Figure 8) [56],

of relapse that may require a closer follow-up.

174 Neuroblastoma

**Figure 8.** Hierarchically clustered heat maps of differentially expressed genes in BM cells from stage 4 patients (infil‐ trated BMs, I), normal BMs (N) from healthy children, and BM cells from localized NB patients (non-infiltrated BMs, NI). Each color patch represents the expression level of genes (row) in that sample (column), with a continuum of expres‐ sion levels from bright green (lowest) to bright red (highest).

Precisely,BMsamplesfromNBpatientssignificantlyoverexpressedgenesinvolvedintheinnate immune responses. In particular, all NB patients expressed an interferon (IFN) signature [57].

IFNs are pleiotropic cytokines involved in different biological processes [58], and IFN signa‐ tures have been associated with tumor progression of melanoma and colorectal cancer [59]. Moreover IFN signatures associated with the worse prognosis of African-American patients with prostate [60] and breast [61] cancer as compared to Caucasian patients. The IFN signature includes genes involved in the defense against bacterial and viral pathogens [62, 63]. Since all NB patients showed the IFN signature it was proposed that the NB primary tumor may release, or induce the release of, soluble factors as occurring during an infection. The BM microenvir‐ onment of NB patients up-regulated also the IFN related DNA damage resistance signature (IRDS), that was shown to be associated with resistance to radiation-induced DNA damage [64], as summarized in Table 1.


In conclusion, children with NB have evidence of chronic inflammation, more intense in the presence of infiltrating NB cells [56], that may reduce anti-tumor immune responses and

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Furthermore, resident BM cells from patients with NB down-regulated genes involved in cell adhesion, in erythrocyte, myeloid and platelet differentiation, and most importantly, CXCL12 expression. The CXCL12 down-regulation reached near complete silencing in patients with metastatic disease (Figure 9), likely explaining the anemia and platelet dysfunctions observed

**Figure 9.** Expression of CXCL12 in BM samples from healthy children (H\_BM), and children with localized (NI\_BM) and

The *CXCL12* mRNA down-regulation was independent of direct contact between neuroblasts and resident BM cells, as expected from the down-regulation observed in patients with localized NB. Since it is known that CXCL12 expression is regulated by the circadian secretion of noradrenaline [66], we speculated that CXCL12 down-regulation may be dependent on noradrenaline secretion by NB tumor cells [67]. Although, the CXCL12 chemokine has been proposed to play a pivotal role in promoting the homing of the CXCR4 positive NB cells in the

promote tumor progression [65].

in stage 4 patients.

metastatic (I\_BM) neuroblastoma.

**Table 1.** List of the most relevant genes of the IFN and IRDS signatures overexpressed by resident BM cells from patients with metastatic (I\_BM) and localized (NI\_BM) neuroblastoma.

In conclusion, children with NB have evidence of chronic inflammation, more intense in the presence of infiltrating NB cells [56], that may reduce anti-tumor immune responses and promote tumor progression [65].

**Gene Symbol IFN-α and IFN-β**

176 Neuroblastoma

**(IFN type I)**

**IFN-γ (IFN type II)**

*IFIT1* x x x I\_BM x

*MX1* x x x I\_BM x

*OAS3* x x x I\_BM x

*CD69* x x I\_BM, NI\_BM x *HLA-G* x x I\_BM, NI\_BM x

*IFIT3* x x x I\_BM, NI\_BM x

*IL1R2* x NI\_BM x

**Table 1.** List of the most relevant genes of the IFN and IRDS signatures overexpressed by resident BM cells from

patients with metastatic (I\_BM) and localized (NI\_BM) neuroblastoma.

*IFI44* x x I\_BM, NI\_BM

*PARP10* x I\_BM, NI\_BM *STAT3* x x I\_BM, NI\_BM *STRN* x I\_BM, NI\_BM *STXBP3* x I\_BM, NI\_BM *TAGAP* x I\_BM, NI\_BM *TRA2A* x I\_BM, NI\_BM *TRIM14* x x x I\_BM, NI\_BM *UPP1* x x I\_BM, NI\_BM *IFNG* x x NI\_BM

*EGR1* x I\_BM *GALNT1* x I\_BM *IFI44L* x x I\_BM *IFI6* x x x I\_BM

*ISG15* x x x I\_BM

*OAS2* x x x I\_BM

*OASL* x x x I\_BM *PLEC1* x x I\_BM *PML* x x I\_BM *PPIB* x x I\_BM *TAP1* x x I\_BM *TNF* x I\_BM *ADAMTSL4* x I\_BM, NI\_BM *B2M* x x x I\_BM, NI\_BM *CASP1* x x I\_BM, NI\_BM *CASP8* x x I\_BM, NI\_BM *CCL3* x I\_BM, NI\_BM *CCR3* x I\_BM, NI\_BM

**IFN-λ**

**(IFN type III) BM subgroup IRDS signature**

Furthermore, resident BM cells from patients with NB down-regulated genes involved in cell adhesion, in erythrocyte, myeloid and platelet differentiation, and most importantly, CXCL12 expression. The CXCL12 down-regulation reached near complete silencing in patients with metastatic disease (Figure 9), likely explaining the anemia and platelet dysfunctions observed in stage 4 patients.

**Figure 9.** Expression of CXCL12 in BM samples from healthy children (H\_BM), and children with localized (NI\_BM) and metastatic (I\_BM) neuroblastoma.

The *CXCL12* mRNA down-regulation was independent of direct contact between neuroblasts and resident BM cells, as expected from the down-regulation observed in patients with localized NB. Since it is known that CXCL12 expression is regulated by the circadian secretion of noradrenaline [66], we speculated that CXCL12 down-regulation may be dependent on noradrenaline secretion by NB tumor cells [67]. Although, the CXCL12 chemokine has been proposed to play a pivotal role in promoting the homing of the CXCR4 positive NB cells in the BM [51, 68], the absence of CXCL12 in the BM of NB patients makes highly unlikely that the axis CXCL12/CXCR4 play a role in the BM infiltration by NB cells. It is worth noting that we had previously shown that CXCR4 in freshly isolated human metastatic NB cells was not functional [54], and that Carlisle *et al.* [55] showed that CXCR4 expression was regulated independently of CXCL12.

In conclusion, NB tumor growth at the primary site can alter the BM microenvironment, and the presence of BM-infiltrating NB cells makes these alterations only more pronounced. Therefore, the BM microenvironment is unlikely to be responsible for the presence of NB cells in the BM.

#### **6.2. Characteristics of the BM-infiltrating NB cells**

After *in vitro* culture of BM samples from patients with metastatic disease, Hansford *et al.* [69] isolated cells endowed with high tumorigenic potential, suggesting that metastatic cells may be enriched in tumor initiating cells (TICs). A gene expression profiling of TICs has been reported [70]. However, it has been demonstrated that the isolated TICs were not NB cells [71], and Coulon *et al.* [72] had recently demonstrated that *in vitro* expanded stem-like NB cells were a dynamic and heterogeneous cell population, quite difficult to characterize because of the influence of external stimuli.

To avoid any modification or selection following *in vitro* culture, we thus decided to charac‐ terize freshly isolated BM-infiltrating NB cells. The metastatic cells expressed several costimulatory molecules [73] and were susceptible to NK cell-mediated lysis [74]. Moreover, as mentioned above, the CXCR4 expressed by these cells was not functional [54].

Since the proteins selectively over-expressed by the BM-infiltrating NB cells may represent novel prognostic markers and potential targets for biologically driven therapy for metastatic NB patients, we performed gene expression profiling of these cells, as compared to the cells in the primary tumors [75]. The results of the study showed that the BM-infiltrating GD2 positive cells were enriched in CD56-positive and NB84-positive mononuclear NB cells, had the same genetic aberration as the primary tumor cells, and expressed NB-specific genes as primary tumor cells. The BM-infiltrating GD2-positive cells up-regulated several genes normally expressed by different lineages of resident BM cells. Therefore, to ascribe the expression of the proteins encoded by these genes to the metastatic NB cells, we took advantage of multiple color cytofluorimetric analysis of unprocessed BM samples from stage 4 patients. While in unprocessed BM samples from healthy individuals the GD2 expression was absent [76], in BM samples from stage 4 NB patients the GD2-positive cells represented about 20-30% of mononuclear cells. These latter cells never express the pan-leukocyte CD45 antigen and always co-express the NB specific markers B7H3 and CD56 [77, 78] (Figure 10), thus confirming that the GD2-positive BM-infiltrating cells were indeed metastatic NB cells. All freshly isolated GD2-positive BM-infiltrating NB cells never expressed CD133, sometime co-expressed c-KIT, CD37 and CD177, but most importantly, they always expressed both HLA-G and calprotectin, as shown in Figure 10.

**Figure 10.** A representative two color cytofluorimetric analysis of a fresh BM sample from a patient with metastatic stage 4 NB, tested with anti-GD2 mAb and: anti-B7H3 (5B14), anti CD45, anti-CD56, anti-CD117 (c-kit), anti-CD37, an‐ ti-CD177, anti-HLA-G, and anti-calprotectin. Each plot shows specific mAb fluorescence intensity (Y axes) versus side-

Bone Marrow Infiltration in Neuroblastoma: Characteristics of Infiltrating Cells and Role of the Microenvironment

http://dx.doi.org/10.5772/55774

179

scatter (X axes), after gating on GD2 positive cells.

BM [51, 68], the absence of CXCL12 in the BM of NB patients makes highly unlikely that the axis CXCL12/CXCR4 play a role in the BM infiltration by NB cells. It is worth noting that we had previously shown that CXCR4 in freshly isolated human metastatic NB cells was not functional [54], and that Carlisle *et al.* [55] showed that CXCR4 expression was regulated

In conclusion, NB tumor growth at the primary site can alter the BM microenvironment, and the presence of BM-infiltrating NB cells makes these alterations only more pronounced. Therefore, the BM microenvironment is unlikely to be responsible for the presence of NB cells

After *in vitro* culture of BM samples from patients with metastatic disease, Hansford *et al.* [69] isolated cells endowed with high tumorigenic potential, suggesting that metastatic cells may be enriched in tumor initiating cells (TICs). A gene expression profiling of TICs has been reported [70]. However, it has been demonstrated that the isolated TICs were not NB cells [71], and Coulon *et al.* [72] had recently demonstrated that *in vitro* expanded stem-like NB cells were a dynamic and heterogeneous cell population, quite difficult to characterize because of the

To avoid any modification or selection following *in vitro* culture, we thus decided to charac‐ terize freshly isolated BM-infiltrating NB cells. The metastatic cells expressed several costimulatory molecules [73] and were susceptible to NK cell-mediated lysis [74]. Moreover, as

Since the proteins selectively over-expressed by the BM-infiltrating NB cells may represent novel prognostic markers and potential targets for biologically driven therapy for metastatic NB patients, we performed gene expression profiling of these cells, as compared to the cells in the primary tumors [75]. The results of the study showed that the BM-infiltrating GD2 positive cells were enriched in CD56-positive and NB84-positive mononuclear NB cells, had the same genetic aberration as the primary tumor cells, and expressed NB-specific genes as primary tumor cells. The BM-infiltrating GD2-positive cells up-regulated several genes normally expressed by different lineages of resident BM cells. Therefore, to ascribe the expression of the proteins encoded by these genes to the metastatic NB cells, we took advantage of multiple color cytofluorimetric analysis of unprocessed BM samples from stage 4 patients. While in unprocessed BM samples from healthy individuals the GD2 expression was absent [76], in BM samples from stage 4 NB patients the GD2-positive cells represented about 20-30% of mononuclear cells. These latter cells never express the pan-leukocyte CD45 antigen and always co-express the NB specific markers B7H3 and CD56 [77, 78] (Figure 10), thus confirming that the GD2-positive BM-infiltrating cells were indeed metastatic NB cells. All freshly isolated GD2-positive BM-infiltrating NB cells never expressed CD133, sometime co-expressed c-KIT, CD37 and CD177, but most importantly, they always expressed both HLA-G and calprotectin,

mentioned above, the CXCR4 expressed by these cells was not functional [54].

independently of CXCL12.

influence of external stimuli.

as shown in Figure 10.

**6.2. Characteristics of the BM-infiltrating NB cells**

in the BM.

178 Neuroblastoma

**Figure 10.** A representative two color cytofluorimetric analysis of a fresh BM sample from a patient with metastatic stage 4 NB, tested with anti-GD2 mAb and: anti-B7H3 (5B14), anti CD45, anti-CD56, anti-CD117 (c-kit), anti-CD37, an‐ ti-CD177, anti-HLA-G, and anti-calprotectin. Each plot shows specific mAb fluorescence intensity (Y axes) versus sidescatter (X axes), after gating on GD2 positive cells.

The heterodimer protein calprotectin, encoded by the *S100A8* and *S100A9* genes, is a member of the S100 family, composed of small (10–12 kDa) acidic calcium and zinc binding proteins. Calprotectin is normally expressed by phagocytes and polymorph nuclear leukocyte and it is released into biological fluids during inflammation. Calprotectin, in fact, is widely used as a biomarker of inflammation [79]. Calprotectin is a potent ligand of the Toll-like receptor 4 (TLR4) [80], which is responsible for specific response to endogenous danger signals. Thus, the expression of calprotectin by the BM-infiltrating metastatic cells may be responsible in part for the state of chronic inflammation of the BM microenviron‐ ment (see previous paragraph for details). Moreover, the calprotectin-TLR4 axis may also guide metastatic cell invasion, and facilitate the survival and proliferation of cancer cells at the metastatic site [79].

**Acknowledgements**

and centralized the samples.

by Ministero della Salute.

, Paola Scaruffi2

(2010). , 362(23), 2202-2211.

42(13), 2081-2091.

**Author details**

Fabio Morandi1

**References**

The Authors wish to thank the parents and legal guardians that have allowed the use of their children's samples for research studies and all the physicians and pathologists that collected

Bone Marrow Infiltration in Neuroblastoma: Characteristics of Infiltrating Cells and Role of the Microenvironment

The studies were supported in part by Fondazione Italiana per la Lotta al Neuroblastoma and

2 Center of Physiopathology of Human Reproduction, Obstetrics and Gynecology Unit,

[1] Maris, J. M. Recent advances in neuroblastoma. New England Journal of Medicine

[2] Spix, C, Pastore, G, Sankila, R, Stiller, C. A, & Steliarova-foucher, E. Neuroblastoma incidence and survival in European children (1978-1997): report from the Automated Childhood Cancer Information System project. European Journal of Cancer (2006). ,

[3] Cohn, S. L, Pearson, A. D, London, W. B, Monclair, T, Ambros, P. F, Brodeur, G. M, et al. The International Neuroblastoma Risk Group (INRG) classification system: an

[4] Brodeur, G. M, Pritchard, J, Berthold, F, Carlsen, N. L, Castel, V, Castelberry, R. P, et al. Revisions of the international criteria for neuroblastoma diagnosis, staging, and

[5] Monclair, T, Brodeur, G. M, Ambros, P. F, Brisse, H. J, Cecchetto, G, Holmes, K, et al. The International Neuroblastoma Risk Group (INRG) staging system: an INRG Task

INRG Task Force report. Journal of Clinical Oncology (2009). , 27(2), 289-297.

response to treatment. Journal of Clinical Oncology (1993). , 11(8), 1466-1477.

Force report. Journal of Clinical Oncology (2009). , 27(2), 298-303.

, Barbara Carlini1

and Maria Valeria Corrias1

http://dx.doi.org/10.5772/55774

181

BC is recipient of Fondazione Italiana per la Lotta al Neuroblastoma fellowship.

, Sara Stigliani2

IRCCS San Martino Hospital-National Cancer Research Institute, Genoa, Italy

1 Laboratory of Oncology, IRCCS Giannina Gaslini Institute, Genoa, Italy

The GD2-positive BM-infiltrating cells also expressed HLA-G, a monomorphic HLA class Ib molecule, whose over-expression facilitate tumor escape from host immune response in a wide variety of human cancers [81]. Since primary NB tumors did not express HLA-G [82], it is conceivable that HLA-G may contribute to the high aggressiveness of BMinfiltrating NB cells throughout its immunosuppressive activity.

#### **7. Conclusion**

Presence of NB cells within the BM is the most powerful negative prognostic factor for patients with NB. Presently, sensitive methods of detection of metastatic cells have been standardized [20], and prospective studies are ongoing to demonstrate their relative or combined prognostic role. In the near future these methods will help stratification of stage 4 patients into different risk groups. Conversely, these sensitive methods are of limited use in patients with localized NB.

To date, information about the role of the BM microenvironment in driving infiltration by metastatic cells is few and still conflicting. However, the finding that the BM microenviron‐ ment of patients with localized disease is not so different from that of patients with metastatic disease [56], strongly support the hypothesis that the invasion of the BM mainly depends on the characteristics of the metastatic cells, rather than on the properties of the BM microenvironment. In this regard the findings that the BM-infiltrating NB cells expressed proteins not found in the primary tumor cells is intriguing [75]. In fact, both calprotectin and HLA-G favor tumor escape from anti-tumor immune responses, likely contributing to the survival and proliferation of the metastatic cells in the BM. These proteins may be also responsible for the state of intense chronic inflammation observed in patients with metastatic NB [56]. Future studies are needed to elucidate the mechanisms responsible for the acquisition of the different properties of metastatic cells as compared to primary tumor cells. However, the proteins specifically expressed by BM-infiltrating metastatic NB cells could be new prognostic markers and novel therapeutic targets for high risk NB patients.

## **Acknowledgements**

The heterodimer protein calprotectin, encoded by the *S100A8* and *S100A9* genes, is a member of the S100 family, composed of small (10–12 kDa) acidic calcium and zinc binding proteins. Calprotectin is normally expressed by phagocytes and polymorph nuclear leukocyte and it is released into biological fluids during inflammation. Calprotectin, in fact, is widely used as a biomarker of inflammation [79]. Calprotectin is a potent ligand of the Toll-like receptor 4 (TLR4) [80], which is responsible for specific response to endogenous danger signals. Thus, the expression of calprotectin by the BM-infiltrating metastatic cells may be responsible in part for the state of chronic inflammation of the BM microenviron‐ ment (see previous paragraph for details). Moreover, the calprotectin-TLR4 axis may also guide metastatic cell invasion, and facilitate the survival and proliferation of cancer cells

The GD2-positive BM-infiltrating cells also expressed HLA-G, a monomorphic HLA class Ib molecule, whose over-expression facilitate tumor escape from host immune response in a wide variety of human cancers [81]. Since primary NB tumors did not express HLA-G [82], it is conceivable that HLA-G may contribute to the high aggressiveness of BM-

Presence of NB cells within the BM is the most powerful negative prognostic factor for patients with NB. Presently, sensitive methods of detection of metastatic cells have been standardized [20], and prospective studies are ongoing to demonstrate their relative or combined prognostic role. In the near future these methods will help stratification of stage 4 patients into different risk groups. Conversely, these sensitive methods are of limited use

To date, information about the role of the BM microenvironment in driving infiltration by metastatic cells is few and still conflicting. However, the finding that the BM microenviron‐ ment of patients with localized disease is not so different from that of patients with metastatic disease [56], strongly support the hypothesis that the invasion of the BM mainly depends on the characteristics of the metastatic cells, rather than on the properties of the BM microenvironment. In this regard the findings that the BM-infiltrating NB cells expressed proteins not found in the primary tumor cells is intriguing [75]. In fact, both calprotectin and HLA-G favor tumor escape from anti-tumor immune responses, likely contributing to the survival and proliferation of the metastatic cells in the BM. These proteins may be also responsible for the state of intense chronic inflammation observed in patients with metastatic NB [56]. Future studies are needed to elucidate the mechanisms responsible for the acquisition of the different properties of metastatic cells as compared to primary tumor cells. However, the proteins specifically expressed by BM-infiltrating metastatic NB cells could be new prognostic markers and novel therapeutic targets for high

infiltrating NB cells throughout its immunosuppressive activity.

at the metastatic site [79].

180 Neuroblastoma

**7. Conclusion**

risk NB patients.

in patients with localized NB.

The Authors wish to thank the parents and legal guardians that have allowed the use of their children's samples for research studies and all the physicians and pathologists that collected and centralized the samples.

The studies were supported in part by Fondazione Italiana per la Lotta al Neuroblastoma and by Ministero della Salute.

BC is recipient of Fondazione Italiana per la Lotta al Neuroblastoma fellowship.

## **Author details**

Fabio Morandi1 , Paola Scaruffi2 , Sara Stigliani2 , Barbara Carlini1 and Maria Valeria Corrias1

1 Laboratory of Oncology, IRCCS Giannina Gaslini Institute, Genoa, Italy

2 Center of Physiopathology of Human Reproduction, Obstetrics and Gynecology Unit, IRCCS San Martino Hospital-National Cancer Research Institute, Genoa, Italy

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91-104.

184 Neuroblastoma


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Bone Marrow Infiltration in Neuroblastoma: Characteristics of Infiltrating Cells and Role of the Microenvironment

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186 Neuroblastoma


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**Chapter 9**

**Neuroblastoma Integrins**

Shanique A. Young, Ryon Graf and

Additional information is available at the end of the chapter

In the body, cells are surrounded and supported by an intricate network of glycoproteins and proteoglycans that make up a complex extracellular matrix, or ECM. Many constituents, such as collagen, laminin, and fibronectin, are locally produced within the tissues, where they act as physical scaffolds, growth factor depots, and points of anchorage [1]. The local rigidity and

The ECM surrounding cells can be considered in two broad classes. On one hand, there exists a 'physiologic' ECM, present in all tissues, that aids in structuring and maintaining homeo‐ stasis. Typical ECM components include several collagens and laminins, as well as proteogly‐ cans. On the other hand, there is a provisional ECM that is deposited during wounding, hemostasis and tissue remodeling. This ECM is typically deposited, digested and replaced in a very dynamic manner, and contains proteins such as fibronectin, fibrin, vitronectin and even residual fragments of collagen and laminin. This type of ECM promotes tissue remodeling as well as cellular survival, proliferation and invasion. In both types of ECM, however, the diversity in the type and quantity of each individual ECM component present determines the physical properties of these tissues. In so doing, this modulates the mechanical forces sensed by cells that bind to the ECM, and provides yet another layer of information relayed to cells. This 'mechanosensation' requires integrins, receptors that can transmit extracellular forces to

Although many classes of receptors can interact with components of the ECM, the integrins are regarded as the principle receptors mediating anchorage and attachment to the ECM [2]. The name integrin was derived from initial observations that these receptors permitted a realignment of the actin cytoskeleton to match that of an underlying ECM. Integrins are transmembrane glycoprotein receptors that are composed of a heterodimer of α and β subunits

> © 2013 Young et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

composition of the matrix also provide environmental cues that govern cell behavior.

Dwayne G. Stupack

**1. Introduction**

the actin cytoskeleton.

http://dx.doi.org/10.5772/55991


**Chapter 9**

## **Neuroblastoma Integrins**

Shanique A. Young, Ryon Graf and Dwayne G. Stupack

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55991

#### **1. Introduction**

roblastoma cells undergo apoptosis following interaction with CD40L. British

[74] Castriconi, R, Dondero, A, Corrias, M. V, Lanino, E, Pende, D, Moretta, L, et al. Natu‐ ral killer cell-mediated killing of freshly isolated neuroblastoma cells: critical role of DNAX accessory molecule-1-poliovirus receptor interaction. Cancer Research

[75] Morandi, F, Scaruffi, P, Gallo, F, Stigliani, S, Moretti, S, Bonassi, S, et al. Bone mar‐ row-infiltrating human neuroblastoma cells express high levels of calprotectin and

[76] Martinez, C, Hofmann, T. J, Marino, R, Dominici, M, & Horwitz, E. M. Human bone marrow mesenchymal stromal cells express the neural ganglioside GD2: a novel sur‐

[77] Bassili, M, Birman, E, Schor, N. F, & Saragovi, H. U. Differential roles of Trk and neu‐ rotrophin receptors in tumorigenesis and chemoresistance ex vivo and in vivo. Can‐

[78] Castriconi, R, Dondero, A, Augugliaro, R, Cantoni, C, Carnemolla, B, Sementa, A. R, et al. Identification of 4Ig-B7-H3 as a neuroblastoma-associated molecule that exerts a protective role from an NK cell-mediated lysis. Proceeding National Academy of Sci‐

[79] Ghavami, S, Rashedi, I, Dattilo, B. M, Eshraghi, M, Chazin, W. J, Hashemi, M, et al. S100A8/A9 at low concentration promotes tumor cell growth via RAGE ligation and MAP kinase-dependent pathway. Journal of Leukocyte Biology (2008). , 83(6),

[80] Vogl, T, Tenbrock, K, Ludwig, S, Leukert, N, Ehrhardt, C, Van Zoelen, M. A, et al. Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal,

[81] Rouas-freiss, N, Moreau, P, & Menier, C. LeMaoult J, Carosella ED. Expression of tol‐ erogenic HLA-G molecules in cancer prevents antitumor responses. Seminars in Can‐

[82] Morandi, F, Levreri, I, Bocca, P, Galleni, B, Raffaghello, L, Ferrone, S, et al. Human neuroblastoma cells trigger an immunosuppressive program in monocytes by stimu‐

lating soluble HLA-G release. Cancer Research (2007). , 67(13), 6433-6441.

endotoxin-induced shock. Nature Medicine (2007). , 13(9), 1042-1049.

face marker for the identification of MSCs. Blood (2007). , 109(10), 4245-4248.

Journal of Cancer (2003). , 88(10), 1527-1536.

HLA-G proteins. Plos One. (2012). e29922.

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ence USA (2004). , 101(34), 12640-12645.

cer Biology (2007). , 17(6), 413-421.

1484-1492.

(2004). , 64(24), 9180-9184.

188 Neuroblastoma

In the body, cells are surrounded and supported by an intricate network of glycoproteins and proteoglycans that make up a complex extracellular matrix, or ECM. Many constituents, such as collagen, laminin, and fibronectin, are locally produced within the tissues, where they act as physical scaffolds, growth factor depots, and points of anchorage [1]. The local rigidity and composition of the matrix also provide environmental cues that govern cell behavior.

The ECM surrounding cells can be considered in two broad classes. On one hand, there exists a 'physiologic' ECM, present in all tissues, that aids in structuring and maintaining homeo‐ stasis. Typical ECM components include several collagens and laminins, as well as proteogly‐ cans. On the other hand, there is a provisional ECM that is deposited during wounding, hemostasis and tissue remodeling. This ECM is typically deposited, digested and replaced in a very dynamic manner, and contains proteins such as fibronectin, fibrin, vitronectin and even residual fragments of collagen and laminin. This type of ECM promotes tissue remodeling as well as cellular survival, proliferation and invasion. In both types of ECM, however, the diversity in the type and quantity of each individual ECM component present determines the physical properties of these tissues. In so doing, this modulates the mechanical forces sensed by cells that bind to the ECM, and provides yet another layer of information relayed to cells. This 'mechanosensation' requires integrins, receptors that can transmit extracellular forces to the actin cytoskeleton.

Although many classes of receptors can interact with components of the ECM, the integrins are regarded as the principle receptors mediating anchorage and attachment to the ECM [2]. The name integrin was derived from initial observations that these receptors permitted a realignment of the actin cytoskeleton to match that of an underlying ECM. Integrins are transmembrane glycoprotein receptors that are composed of a heterodimer of α and β subunits

© 2013 Young et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

[3]. There are 18 different α subunits and 8 β subunits, but there are a limited number of possible combinations that can form from these subunits. To date, at least 24 unique integrin complexes have been identified, each with its own binding specificity for different subsets of ligands (Figure 1). Cells will generally express only a limited number of integrins, perhaps 10 of these combinations. The particular repertoire of integrins expressed by a given cell varies, but is typically closely tied to a cell's particular extracellular microenvironment. Differences in integrin binding to a ligand can be subtle. For example, approximately one third of human integrins bind to an arginine-glycine-aspartic acid (RGD) sequence of amino acid residues, but this can be profoundly conformation specific, and thus not all 'RGD-binding' integrins are capable of binding all RGD sequences with appreciable affinity.

also have a small (~30-50 amino acid) cytosolic domain, with the singular exception being integrin β4, which has a large cytosolic domain that interacts with intermediate filaments [4]. Integrins are cysteine–rich proteins, and have extensive crosslinking within domains that stabilize domain structure. Thus, integrins appear at different sizes when analyzed on reducing and non-reducing gels, and detection of integrins by some antibodies may require either

Neuroblastoma Integrins http://dx.doi.org/10.5772/55991 191

The integrin extracellular domains are required for and sufficient to bind to ECM or to 'receptor-ligands' present on the surface of adjacent cells. However, the binding of integrins to their ligands is controlled by their conformation, which is influenced by the stalk and cytosolic regions of the molecule. Inactive integrins adapt a 'folded back' conformation at a region halfway up the stalk (at the 'genou,' or knee, between the thigh and leg). Active integrins are extended molecules with stalks separated, and intermediates between these states tend to have intermediate affinities for ligands. Integrin-ligand binding requires the presence of divalent cations, with a typical preference for manganese, magnesium and calcium, although the relative preference for optimal affinity varies among the different heterodimers. These divalent cations, and Mn+2 in particular, directly influence integrin conformation, stabilizing

With the exception of circulating hematopoietic cells, which tend to maintain their integrins in an inactive conformation, most cells that have been examined express both active and inactive integrins. Active integrins tend to form higher order clusters on the cell surface, which promotes their localization to sites of ligation. There, the integrins are further stabilized by interaction with ligand. The accumulation of integrins in these sites creates a 'Velcro-like' effect, with groups of integrins (rather than individual molecules) collaborating to strengthen anchorage and to induce downstream signaling points of extracellular matrix contact. This clustering effect is called integrin 'avidity' regulation, which is distinct from affinity. This permits the stable interaction with the ECM required for sustained cellular anchorage and signaling via the assembly of a 'focal adhesion complex' that accumulates proximal to the

The focal adhesion complex that forms is multifunctional, and is capable of signaling directly, scaffolding additional or alternative signals, and engaging the actin/myosin system. Thus, despite the absence of intrinsic kinase or proteolytic activity, integrins transform mechanical and chemical cues from the extracellular environment into intracellular signals that profound‐

The focal adhesion complex contains a complicated array of non-receptor kinases and adaptor proteins that mediate downstream signaling events. As will be discussed in more detail below, integrin effectors in the focal adhesion include diverse signaling elements such as: focal adhesion kinase (FAK), src kinase, cytoskeletal elements including talin, paxillin and vinculin, phosphoinositide 3 kinase, and small GTPases of the Ras and Rho families and their effectors [5, 6]. Importantly, as part of the clustering process, integrins tend to undergo lateral associa‐ tions with other cell surface receptors such as the receptor tyrosine kinases, EGFR and VEGFR, which are important for other global cellular signaling events. This type of signaling, in which

condition, depending upon the linearity or conformation dependence of an epitope.

them in the extended and high affinity conformation (Figure 2).

membrane.

ly impact cell behavior and function.

Fn, Fibronectin; Fg, Fibrinogen; Vn, Vitronectin; vWF, von Willebrand factor; Ln, Laminin; Cn, Collagen; Opn, Osteopontin; Tn, Tenascin; Tsp, Thrombospondin

**Figure 1. Integrin heterodimers and their ligands** This diagram shows the 24 known heterodimers and their li‐ gands. Integrin heterodimers are represented by an α and a β subunit connected by a black line. For example, the β1 subunit dimerizes with 12 different α subunits. The ligands for each heterodimer are written in purple.

#### **1.1. Integrin structure**

Each integrin is composed of a large extracellular region of 600-1000 amino acids, as well as a single transmembrane domain. The extracellular regions can be broadly thought of in terms of a head and stalk (leg/thigh) region; the head is the critical site for ligand binding and divalent cation binding, as well as heterodimerization between the α and β subunits. Most integrins

1

also have a small (~30-50 amino acid) cytosolic domain, with the singular exception being integrin β4, which has a large cytosolic domain that interacts with intermediate filaments [4]. Integrins are cysteine–rich proteins, and have extensive crosslinking within domains that stabilize domain structure. Thus, integrins appear at different sizes when analyzed on reducing and non-reducing gels, and detection of integrins by some antibodies may require either condition, depending upon the linearity or conformation dependence of an epitope.

[3]. There are 18 different α subunits and 8 β subunits, but there are a limited number of possible combinations that can form from these subunits. To date, at least 24 unique integrin complexes have been identified, each with its own binding specificity for different subsets of ligands (Figure 1). Cells will generally express only a limited number of integrins, perhaps 10 of these combinations. The particular repertoire of integrins expressed by a given cell varies, but is typically closely tied to a cell's particular extracellular microenvironment. Differences in integrin binding to a ligand can be subtle. For example, approximately one third of human integrins bind to an arginine-glycine-aspartic acid (RGD) sequence of amino acid residues, but this can be profoundly conformation specific, and thus not all 'RGD-binding' integrins are

α4

β2

β7

αE

Leukocyte Integrins

MadCAM-1, VCAM-1, Fn

> ICAM-1, Fg, iC3b, factor X, herapin

ICAM-2, Fg, Fn iC3b, factor X, herapin

ICAMs 1-5

αL

αM

αX

αD

VCAM-1, Vn, Plasminogen

E-cadherin

capable of binding all RGD sequences with appreciable affinity.

Ln Cn I & IV, Ln, Tn

α2

Ln, Tsp

α3

Ln, Tsp, CYR61

α5

Fn, VCAM-1, MadCAM-1, Opn

Fn, Tsp, Fibrillin-1

β4

Cn I & VI,

α1

β1

α7 α6

Ln, Fn, Opn, Vn

αv

Opn, Osteopontin; Tn, Tenascin; Tsp, Thrombospondin

Ln

Fn

α9

α8

Cn

α11

α10

Opn, Fg, Vn, Fn, Tsp

β5

Vn

**1.1. Integrin structure**

β8

VCAM-1, Tn

190 Neuroblastoma

Cn

Vn, Fn, Opn, Fg, vWF, Tsp Fibrillin-1

β3

Fg, Fn, vWF

subunit dimerizes with 12 different α subunits. The ligands for each heterodimer are written in purple.

β6

Fn, Fibronectin; Fg, Fibrinogen; Vn, Vitronectin; vWF, von Willebrand factor; Ln, Laminin; Cn, Collagen;

αΙΙb

**Figure 1. Integrin heterodimers and their ligands** This diagram shows the 24 known heterodimers and their li‐ gands. Integrin heterodimers are represented by an α and a β subunit connected by a black line. For example, the β1

1

Each integrin is composed of a large extracellular region of 600-1000 amino acids, as well as a single transmembrane domain. The extracellular regions can be broadly thought of in terms of a head and stalk (leg/thigh) region; the head is the critical site for ligand binding and divalent cation binding, as well as heterodimerization between the α and β subunits. Most integrins

Fn, Fg, Vn,Tn, Fibrillin-1

Ln

The integrin extracellular domains are required for and sufficient to bind to ECM or to 'receptor-ligands' present on the surface of adjacent cells. However, the binding of integrins to their ligands is controlled by their conformation, which is influenced by the stalk and cytosolic regions of the molecule. Inactive integrins adapt a 'folded back' conformation at a region halfway up the stalk (at the 'genou,' or knee, between the thigh and leg). Active integrins are extended molecules with stalks separated, and intermediates between these states tend to have intermediate affinities for ligands. Integrin-ligand binding requires the presence of divalent cations, with a typical preference for manganese, magnesium and calcium, although the relative preference for optimal affinity varies among the different heterodimers. These divalent cations, and Mn+2 in particular, directly influence integrin conformation, stabilizing them in the extended and high affinity conformation (Figure 2).

With the exception of circulating hematopoietic cells, which tend to maintain their integrins in an inactive conformation, most cells that have been examined express both active and inactive integrins. Active integrins tend to form higher order clusters on the cell surface, which promotes their localization to sites of ligation. There, the integrins are further stabilized by interaction with ligand. The accumulation of integrins in these sites creates a 'Velcro-like' effect, with groups of integrins (rather than individual molecules) collaborating to strengthen anchorage and to induce downstream signaling points of extracellular matrix contact. This clustering effect is called integrin 'avidity' regulation, which is distinct from affinity. This permits the stable interaction with the ECM required for sustained cellular anchorage and signaling via the assembly of a 'focal adhesion complex' that accumulates proximal to the membrane.

The focal adhesion complex that forms is multifunctional, and is capable of signaling directly, scaffolding additional or alternative signals, and engaging the actin/myosin system. Thus, despite the absence of intrinsic kinase or proteolytic activity, integrins transform mechanical and chemical cues from the extracellular environment into intracellular signals that profound‐ ly impact cell behavior and function.

The focal adhesion complex contains a complicated array of non-receptor kinases and adaptor proteins that mediate downstream signaling events. As will be discussed in more detail below, integrin effectors in the focal adhesion include diverse signaling elements such as: focal adhesion kinase (FAK), src kinase, cytoskeletal elements including talin, paxillin and vinculin, phosphoinositide 3 kinase, and small GTPases of the Ras and Rho families and their effectors [5, 6]. Importantly, as part of the clustering process, integrins tend to undergo lateral associa‐ tions with other cell surface receptors such as the receptor tyrosine kinases, EGFR and VEGFR, which are important for other global cellular signaling events. This type of signaling, in which the integrin ectodomain is ligated and transforms information from the extracellular environ‐ ment into cues for cytosolic signaling events has been termed "outside-in signaling."

**2. Integrins and development**

**2.1. Integrins in early development**

malities [7]. (Table 1)

subunits do not appear to be essential during development.

polarity [29], neurite outgrowth [30, 31] and myelination [32].

**2.2. Integrins in nervous system development**

The ability of cells to interact with their extracellular environment is crucial for most devel‐ opmental processes. Consequently, it is perhaps not surprising that integrins, as mediators of the interplay between cells, the ECM and the microenvironment, have critical roles in early development. The early physiological relevance is evident in defects observed in murine genetic models lacking proper integrin function or expression. Overall, the loss of the β1, α5, and α4 subunits leads to an embryonic lethal phenotype. The loss of the αv or α3 subunits permits initial and subsequent development, but results in perinatal lethality. Other integrin

Neuroblastoma Integrins http://dx.doi.org/10.5772/55991 193

Nonetheless, loss, misregulation, or improper function of integrins can lead to other abnor‐

The development of the nervous system is dependent on integrin function, in part, because it involves extensive migration of neuronal precursors which is mediated by integrins. During the process of neurulation, the neural crest forms in the region of the neural plate border. Upon formation of the neural tube, neural crest cells undergo an 'epithelial-to-mesenchymal-like transition' which permits them to move along migratory tracks. These tracks lead cells to a variety of destinations where they differentiate and help to form several different tissue types. During development, collagens, laminins, fibronectin and vitronectin are expressed along these migratory pathways [28]. Disruption of integrin-ligand binding inhibits neural crest cell migration and results in impaired function in the peripheral nervous system. Following the initial gross exodus of neurons from the neural crest, integrins also play other key roles in the development of the peripheral nervous system, including the establishment of Schwann cell

In addition to the requirement for integrins to support migration, integrins are also important for arresting migration at the proper time and place. In the central nervous system, for example, the presence of the α6 and β1 subunits appears to serves as stop signals for neuronal cells when they reach a laminin rich region. This is critical for cortical plate formation. In the absence of these integrins, neuronal precursors migrating outward to the outermost layer of the cortical

Neuroblastoma is a tumor that is considered to arise from ganglion or pre-ganglion cells. To begin to understand the pathological roles of integrins in this disease, it is helpful to be familiar with the normal expression patterns of these receptors in neural crest cells and how that expression changes over time. Neural crest cells express subsets of integrins that allow them to adhere to the fibrillar proteins that line their migratory pathways. Truncal neural crest cells, which give rise to dorsal root ganglia, sympathetic ganglia, and the adrenal medulla express

plate overshoot their destination and disrupt the cortical plate structure [33, 34].

**2.3. Integrin expression in the dorsal root ganglion and in neuroblasts**

However, in some cases, signals from inside the cell result in changes in integrin conformation. These are typically associated with cytosolic proteins binding to the cytosolic domains of the integrins. This type of regulation of integrin conformation is called "inside-out signaling." Both types of signaling are important for understanding the role of integrins in normal tissues and in disease pathology.

**Figure 2. Integrin structure and activation** Integrins are composed of a large extracellular domain and short intra‐ cellular tails (with the exception of the β4 tail). The extracellular domain comprises a head region and a stalk region, which includes the "thigh" and "leg" areas of the integrin. Ligand binding occurs at the head region and requires the presence of divalent cations such as manganese, magnesium, and calcium. Integrins on the cell surface can exist in a range of conformations that affect their affinity for ligand. In the low affinity conformation, the extracellular domain is folded back at the knee (between the thigh and leg areas) and the intracellular tails are clasped together. In the high affinity conformation the extracellular stalk is straight, the subunits are slightly separated and the tails shift apart as well. Conformations between these low and high affinity states confer intermediate affinity for ligand. Changes in conformation can be regulated by intracellular signaling events such as the binding of cytosolic proteins to integrin tails leading to integrin clustering, focal adhesion formation and further interaction of cytoskeletal proteins.

2

## **2. Integrins and development**

the integrin ectodomain is ligated and transforms information from the extracellular environ‐

However, in some cases, signals from inside the cell result in changes in integrin conformation. These are typically associated with cytosolic proteins binding to the cytosolic domains of the integrins. This type of regulation of integrin conformation is called "inside-out signaling." Both types of signaling are important for understanding the role of integrins in normal tissues and

Ligand

Migration

2

tails leading to integrin clustering, focal adhesion formation and further interaction of cytoskeletal proteins.

**Figure 2. Integrin structure and activation** Integrins are composed of a large extracellular domain and short intra‐ cellular tails (with the exception of the β4 tail). The extracellular domain comprises a head region and a stalk region, which includes the "thigh" and "leg" areas of the integrin. Ligand binding occurs at the head region and requires the presence of divalent cations such as manganese, magnesium, and calcium. Integrins on the cell surface can exist in a range of conformations that affect their affinity for ligand. In the low affinity conformation, the extracellular domain is folded back at the knee (between the thigh and leg areas) and the intracellular tails are clasped together. In the high affinity conformation the extracellular stalk is straight, the subunits are slightly separated and the tails shift apart as well. Conformations between these low and high affinity states confer intermediate affinity for ligand. Changes in conformation can be regulated by intracellular signaling events such as the binding of cytosolic proteins to integrin

Proliferation

Differentiation

Survival

Focal Adhesion Complex

Integrin Clustering

α β

α β

High Affinity

ment into cues for cytosolic signaling events has been termed "outside-in signaling."

α

Intracellular Signals

β

Low Affinity

in disease pathology.

192 Neuroblastoma

Divalent cations

Intermediate Affinity

α β

Head

Thigh

Leg

#### **2.1. Integrins in early development**

The ability of cells to interact with their extracellular environment is crucial for most devel‐ opmental processes. Consequently, it is perhaps not surprising that integrins, as mediators of the interplay between cells, the ECM and the microenvironment, have critical roles in early development. The early physiological relevance is evident in defects observed in murine genetic models lacking proper integrin function or expression. Overall, the loss of the β1, α5, and α4 subunits leads to an embryonic lethal phenotype. The loss of the αv or α3 subunits permits initial and subsequent development, but results in perinatal lethality. Other integrin subunits do not appear to be essential during development.

Nonetheless, loss, misregulation, or improper function of integrins can lead to other abnor‐ malities [7]. (Table 1)

#### **2.2. Integrins in nervous system development**

The development of the nervous system is dependent on integrin function, in part, because it involves extensive migration of neuronal precursors which is mediated by integrins. During the process of neurulation, the neural crest forms in the region of the neural plate border. Upon formation of the neural tube, neural crest cells undergo an 'epithelial-to-mesenchymal-like transition' which permits them to move along migratory tracks. These tracks lead cells to a variety of destinations where they differentiate and help to form several different tissue types. During development, collagens, laminins, fibronectin and vitronectin are expressed along these migratory pathways [28]. Disruption of integrin-ligand binding inhibits neural crest cell migration and results in impaired function in the peripheral nervous system. Following the initial gross exodus of neurons from the neural crest, integrins also play other key roles in the development of the peripheral nervous system, including the establishment of Schwann cell polarity [29], neurite outgrowth [30, 31] and myelination [32].

In addition to the requirement for integrins to support migration, integrins are also important for arresting migration at the proper time and place. In the central nervous system, for example, the presence of the α6 and β1 subunits appears to serves as stop signals for neuronal cells when they reach a laminin rich region. This is critical for cortical plate formation. In the absence of these integrins, neuronal precursors migrating outward to the outermost layer of the cortical plate overshoot their destination and disrupt the cortical plate structure [33, 34].

#### **2.3. Integrin expression in the dorsal root ganglion and in neuroblasts**

Neuroblastoma is a tumor that is considered to arise from ganglion or pre-ganglion cells. To begin to understand the pathological roles of integrins in this disease, it is helpful to be familiar with the normal expression patterns of these receptors in neural crest cells and how that expression changes over time. Neural crest cells express subsets of integrins that allow them to adhere to the fibrillar proteins that line their migratory pathways. Truncal neural crest cells, which give rise to dorsal root ganglia, sympathetic ganglia, and the adrenal medulla express


its ligands via blocking antibodies or ligand-mimicking peptides, leads to a marked reduction

Neuroblastoma Integrins http://dx.doi.org/10.5772/55991 195

As neural crest cells reach their target tissues and differentiate, their integrin expression changes. For example, neural crest cells do not express detectable levels of α6β1 until they differentiate into a peripheral nervous system cell type such as a Schwann cell precursor [39]. Conversely, neural crest cells express α1β1 but Schwann cell precursors do not [40, 41]. This induction of expression of one class of integrins while another is eliminated is not well understood, however, and further study will be required to elucidate additional neuroblast-

Similarly, the formation of the vascular system relies heavily on integrin function. During vasculogenesis, or *de novo* formation of blood vessels, and angiogenesis, the growth of new vessels from pre-existing vasculature, integrins play essential roles in endothelial cell migra‐ tion, adhesion to basement membranes and cell survival. Endothelial cells are known to express a large number of β1 integrin heterodimers including the α1 through α6 subunits as well as integrins α6β4, αvβ5, and αvβ3. The expression of different subsets of these integrins is dependent on the activation state of the endothelial cells. For example, integrins αvβ3 and α4β1 are primarily expressed on activated or angiogenic endothelial cells [42]. Knockout of integrin αv leads to perinatal lethality due to vessel malformation [15] and studies on the αvβ3 heterodimer show that it is essential for the survival of angiogenic endothelial cells [43]. In addition, knock-out of integrin α4 in mice is embryonic lethal by day 14.5 due to placental and cardiac defects [11], likely due to a lack of binding to the α4 ligand, vascular cell adhesion

molecule-1 (VCAM-1) which is present on endothelial and smooth muscle cells.

**3. Integrin expression during tumorigenesis and tumor progression**

As cells are transformed from a normal to malignant state, their integrin expression is modulated to support pathologic behaviors. In primary tumors, integrin signaling can impact cell growth, differentiation, and vascular infiltration and continues to be important as the cancer progresses through the stages of metastasis (Figure 3). The initial steps of the metastatic process involve the degradation and remodeling of extracellular matrix adjacent to primary

The formation of the vasculature, and angiogenesis in particular, is of interest to scientists who study neuroblastoma, which is typically a highly angiogenic disease. Although a focus has been placed on the roles of integrins in development of the neuronal and vascular systems, the ability of integrins to regulate such a large array of cellular functions renders them essential for most, if not all, developmental processes. Their roles may be directly associated with their adhesion and motility-related functions, or with the ability of integrins to indirectly enhance

in neural crest cell migration [37].

specific integrin expression and function.

**2.4. Integrins in vascular system development**

the efficiency of other signaling pathways [44].

**3.1. Tumors exploit integrins for local invasion**

\*Subunit found on neural crest cells but not yet reported on NB tumor cells

#### **Table 1.** Effects of Integrin Deletion in Murine Models

receptors for vitronectin (αvβ1, αvβ3, and αvβ5: [35]), laminin (α1β1, α3β1,: [36] [37]), and fibronectin and associated molecules (α4β1, α5β1, α8β1, αvβ1 and β8 integrin: [37], [38]. Antibody blockade of any one type of these integrins is unable to completely abolish cell migration, consistent with a multi-receptor and complex ligand system. However, in studies on avian truncal neural crest cells, the α3β1, α4β1, and αv integrins appear to be the most crucial to maintain migration [38]. In particular, inhibition of the interaction between α4β1 and its ligands via blocking antibodies or ligand-mimicking peptides, leads to a marked reduction in neural crest cell migration [37].

As neural crest cells reach their target tissues and differentiate, their integrin expression changes. For example, neural crest cells do not express detectable levels of α6β1 until they differentiate into a peripheral nervous system cell type such as a Schwann cell precursor [39]. Conversely, neural crest cells express α1β1 but Schwann cell precursors do not [40, 41]. This induction of expression of one class of integrins while another is eliminated is not well understood, however, and further study will be required to elucidate additional neuroblastspecific integrin expression and function.

#### **2.4. Integrins in vascular system development**

Similarly, the formation of the vascular system relies heavily on integrin function. During vasculogenesis, or *de novo* formation of blood vessels, and angiogenesis, the growth of new vessels from pre-existing vasculature, integrins play essential roles in endothelial cell migra‐ tion, adhesion to basement membranes and cell survival. Endothelial cells are known to express a large number of β1 integrin heterodimers including the α1 through α6 subunits as well as integrins α6β4, αvβ5, and αvβ3. The expression of different subsets of these integrins is dependent on the activation state of the endothelial cells. For example, integrins αvβ3 and α4β1 are primarily expressed on activated or angiogenic endothelial cells [42]. Knockout of integrin αv leads to perinatal lethality due to vessel malformation [15] and studies on the αvβ3 heterodimer show that it is essential for the survival of angiogenic endothelial cells [43]. In addition, knock-out of integrin α4 in mice is embryonic lethal by day 14.5 due to placental and cardiac defects [11], likely due to a lack of binding to the α4 ligand, vascular cell adhesion molecule-1 (VCAM-1) which is present on endothelial and smooth muscle cells.

The formation of the vasculature, and angiogenesis in particular, is of interest to scientists who study neuroblastoma, which is typically a highly angiogenic disease. Although a focus has been placed on the roles of integrins in development of the neuronal and vascular systems, the ability of integrins to regulate such a large array of cellular functions renders them essential for most, if not all, developmental processes. Their roles may be directly associated with their adhesion and motility-related functions, or with the ability of integrins to indirectly enhance the efficiency of other signaling pathways [44].

#### **3. Integrin expression during tumorigenesis and tumor progression**

#### **3.1. Tumors exploit integrins for local invasion**

receptors for vitronectin (αvβ1, αvβ3, and αvβ5: [35]), laminin (α1β1, α3β1,: [36] [37]), and fibronectin and associated molecules (α4β1, α5β1, α8β1, αvβ1 and β8 integrin: [37], [38]. Antibody blockade of any one type of these integrins is unable to completely abolish cell migration, consistent with a multi-receptor and complex ligand system. However, in studies on avian truncal neural crest cells, the α3β1, α4β1, and αv integrins appear to be the most crucial to maintain migration [38]. In particular, inhibition of the interaction between α4β1 and

**Integrin subunit Genetic Defect (KO) Expressed on NB**

194 Neuroblastoma

α1 Viable Yes Normal; [8]

**tumors**

α2 Viable Yes Abnormal mammary branching

α4 Lethal, by E14.5 Yes Abnormal placenta and heart formation;

α5 Lethal, E11 Yes Abnormal mesoderm morphogenesis; [12]

α8 Perinatal lethality No\* Abnormal kidneys and lungs; [15, 16]

αv Perinatal lethality Yes Brain and bladder, hemorrhages; [19] αL Viable No Impaired leukocyte recruitment; [18] αM Viable No Impaired phagocytosis; obesity; [18] αE Viable No Inflammatory skin lesions; [18] αIIb Viable No Impaired platelet aggregation; [18] β1 Lethal, E5.5 Yes Abnormal mesoderm morphogenesis; [20] β2 Viable No Impaired leukocyte recruitment; [21] β3 Viable Yes Glanzmann's thrombasthenia;

α3 Perinatal lethality Yes Abnormal kidneys; [10]

α6 Perinatal lethality Yes Skin blistering; [13] α7 Viable Yes Muscular dystrophy; [14]

α11 Viable No Dwarfism; [18]

β4 Perinatal lethality No Skin blistering; [23]

\*Subunit found on neural crest cells but not yet reported on NB tumor cells

**Table 1.** Effects of Integrin Deletion in Murine Models

β5 Viable Yes No apparent phenotype; [24]

β8 Lethal, E12 - birth No\* Abnormal placenta; defects in

β6 Viable No Macrophage infiltration in skin and lungs;

β7 Viable No No gut-associated lymphoid tissue; [26]

α9 Perinatal lethality No Bilateral chylothorax; [17] α10 Viable No Improper function of growth plate

**Notes**

[11]

morphogenesis; [9]

chondrocytes; [18]

osteosclerotic; [22]

neurovascular homeostasis; [27]

[25]

As cells are transformed from a normal to malignant state, their integrin expression is modulated to support pathologic behaviors. In primary tumors, integrin signaling can impact cell growth, differentiation, and vascular infiltration and continues to be important as the cancer progresses through the stages of metastasis (Figure 3). The initial steps of the metastatic process involve the degradation and remodeling of extracellular matrix adjacent to primary tumor cells, facilitating cancer cell migration into recruited blood vessels. This process is termed local invasion. Usually, for local invasion to begin, cells from the primary tumor shift from an epithelial or non-motile to a more mesenchymal phenotype. In addition, cells frequently create a pathway for themselves by inducing degradation of the matrix via enzymes such as matrix metalloproteases [45]. Integrins can regulate MMP expression and/or activity. For example, integrin α2β1 is a positive regulator of MMP-1 expression [46, 47].

by Stephen Paget in 1889 followed his observation of tissue-specific patterns of tumor meta‐ stasis in 735 breast cancer patients. Paget noted that the pattern of organs bearing metastases was not random, and suggested that certain tumor types preferentially metastasized to compatible environments [48]. He proposed that 'seeds' of tumors required compatible 'soil' to take root and grow. An alternative theory, by Ewing, suggests that tissue tropism is simply due to mechanical forces and circulatory patterns [49], and that tissue tropism results from this. These are not absolutely exclusive theories, and it is reasonable that blood flow patterns are important for the initial distribution of circulating tumor cells, while the propensity to invade, grow and survive may be dependent on the presence of the appropriate integrin

Neuroblastoma Integrins http://dx.doi.org/10.5772/55991 197

Though there have been no studies specifically linking integrins to site-specific metastasis in neuroblastoma, integrins have been shown to play a role in tissue tropism. The primary sites of neuroblastoma metastasis are bone marrow, bone, lymph node and liver. In general, certain integrins have been linked to metastasis to these sites. For instance, integrin α4β1 can promote homing to the bone [50] and has been shown to enhance bone metastasis in melanoma [51]. This effect may be due to expression of VCAM-1 on bone marrow stromal cells. Integrin α4β1 may also promote lymphatic metastasis by enhancing binding to VCAM-1 present on lym‐ phatic endothelial cells [52]. Integrin α2β1 is associated with enhanced liver metastasis. This

is potentially due to its binding to collagen type IV expressed in liver sinusoids [53].

Since the metastatic cascade involves several steps, including local tumor invasion, intrava‐ sation, survival in the lymphatics/blood stream, extravasation, invasion into the new tissue parenchyma and growth and establishment of metastatic nodules, there are many opportuni‐ ties for integrins to facilitate this process. The role of integrins in local invasion is clear. Once cells gain entry into the vasculature, integrins are important for cell-cell and cell-platelet adhesion leading to increased formation of cell emboli [54] and subsequent lodging in capillary beds. Integrins are also important for the endothelial transmigration that follows. At the site of distant metastasis, the microenvironment and composition of the extracellular matrix may be different from that of the native tissue of the invading tumor cells. Here, the balance of ligated and unligated integrins impacts cell behavior and survival, as discussed in Section 4.

The shedding of gangliosides also impacts neuroblastoma metastasis. Gangliosides are glycosphingolipids with one or more sialic acids linked to them. In circulation, gangliosides are associated with lipoproteins. There are several different types of gangliosides that are classified based on the number of associated sialic acids. Some of these gangliosides, such as GM3, are normally present in circulation. Conversely, elevated levels of circulating GD2, a disialoganglioside, have been found in neuroblastoma patients and its concentration is inversely related to progression-free survival. Shedding of gangliosides enhances integrin α2β1-dependent platelet activation, leading to platelet aggregation, and increased adhesion to vascular basement membranes [55]. These events can enhance tumor cell embolization impacting the occurrence of cells lodging in capillary beds and invading into surrounding

ligands as well as other pro-survival factors.

**3.3. Indirect roles for integrins in metastasis**

tissue.

**Figure 3. Roles Played by integrins in cancer progression** Integrins play key roles in each phase of cancer progres‐ sion. 1. Ligation of integrins promotes cell survival 2. Co-signaling with growth factor receptors impacts cell prolifera‐ tion 3. Endothelial cell integrins are important for tumor angiogenesis 4. Integrins modulate the expression of proteolytic enzymes such as matrix metalloproteinases, which play a role in matrix degradation during tumor cell inva‐ sion 5. Integrins are required for migration during invasion and binding to endothelial cells during intravasation (entry into the vasculature) 6. In circulation, tumor cells interact with platelets and leukocytes via integrins and form cell em‐ boli that can lodge in capillary beds of distant tissues 7. Binding of tumor cell integrins such as α4β1 to endothelial VCAM-1 can then promote extravasation of tumor cells into surrounding tissues.

#### **3.2. Integrins, tumor metastasis, and tissue tropism**

3 For many types of cancer, metastasizing cells spread to a specific subset of secondary locations for establishment of metastatic nodules. This phenomenon, termed tissue tropism, has historically been explained by two major theories. The "seed and soil" hypothesis proposed by Stephen Paget in 1889 followed his observation of tissue-specific patterns of tumor meta‐ stasis in 735 breast cancer patients. Paget noted that the pattern of organs bearing metastases was not random, and suggested that certain tumor types preferentially metastasized to compatible environments [48]. He proposed that 'seeds' of tumors required compatible 'soil' to take root and grow. An alternative theory, by Ewing, suggests that tissue tropism is simply due to mechanical forces and circulatory patterns [49], and that tissue tropism results from this. These are not absolutely exclusive theories, and it is reasonable that blood flow patterns are important for the initial distribution of circulating tumor cells, while the propensity to invade, grow and survive may be dependent on the presence of the appropriate integrin ligands as well as other pro-survival factors.

Though there have been no studies specifically linking integrins to site-specific metastasis in neuroblastoma, integrins have been shown to play a role in tissue tropism. The primary sites of neuroblastoma metastasis are bone marrow, bone, lymph node and liver. In general, certain integrins have been linked to metastasis to these sites. For instance, integrin α4β1 can promote homing to the bone [50] and has been shown to enhance bone metastasis in melanoma [51]. This effect may be due to expression of VCAM-1 on bone marrow stromal cells. Integrin α4β1 may also promote lymphatic metastasis by enhancing binding to VCAM-1 present on lym‐ phatic endothelial cells [52]. Integrin α2β1 is associated with enhanced liver metastasis. This is potentially due to its binding to collagen type IV expressed in liver sinusoids [53].

#### **3.3. Indirect roles for integrins in metastasis**

tumor cells, facilitating cancer cell migration into recruited blood vessels. This process is termed local invasion. Usually, for local invasion to begin, cells from the primary tumor shift from an epithelial or non-motile to a more mesenchymal phenotype. In addition, cells frequently create a pathway for themselves by inducing degradation of the matrix via enzymes such as matrix metalloproteases [45]. Integrins can regulate MMP expression and/or activity.

1. Cell Survival

3

For many types of cancer, metastasizing cells spread to a specific subset of secondary locations for establishment of metastatic nodules. This phenomenon, termed tissue tropism, has historically been explained by two major theories. The "seed and soil" hypothesis proposed

**Figure 3. Roles Played by integrins in cancer progression** Integrins play key roles in each phase of cancer progres‐ sion. 1. Ligation of integrins promotes cell survival 2. Co-signaling with growth factor receptors impacts cell prolifera‐ tion 3. Endothelial cell integrins are important for tumor angiogenesis 4. Integrins modulate the expression of proteolytic enzymes such as matrix metalloproteinases, which play a role in matrix degradation during tumor cell inva‐ sion 5. Integrins are required for migration during invasion and binding to endothelial cells during intravasation (entry into the vasculature) 6. In circulation, tumor cells interact with platelets and leukocytes via integrins and form cell em‐ boli that can lodge in capillary beds of distant tissues 7. Binding of tumor cell integrins such as α4β1 to endothelial

2. Proliferation & Growth Factor Signaling

> 3. Tumor Angiogenesis

4. Matrix Degradation

For example, integrin α2β1 is a positive regulator of MMP-1 expression [46, 47].

5. Cell Invasion & Intravasation

VCAM-1 can then promote extravasation of tumor cells into surrounding tissues.

**3.2. Integrins, tumor metastasis, and tissue tropism**

6. Embolization

196 Neuroblastoma

7. Extravasation

Since the metastatic cascade involves several steps, including local tumor invasion, intrava‐ sation, survival in the lymphatics/blood stream, extravasation, invasion into the new tissue parenchyma and growth and establishment of metastatic nodules, there are many opportuni‐ ties for integrins to facilitate this process. The role of integrins in local invasion is clear. Once cells gain entry into the vasculature, integrins are important for cell-cell and cell-platelet adhesion leading to increased formation of cell emboli [54] and subsequent lodging in capillary beds. Integrins are also important for the endothelial transmigration that follows. At the site of distant metastasis, the microenvironment and composition of the extracellular matrix may be different from that of the native tissue of the invading tumor cells. Here, the balance of ligated and unligated integrins impacts cell behavior and survival, as discussed in Section 4.

The shedding of gangliosides also impacts neuroblastoma metastasis. Gangliosides are glycosphingolipids with one or more sialic acids linked to them. In circulation, gangliosides are associated with lipoproteins. There are several different types of gangliosides that are classified based on the number of associated sialic acids. Some of these gangliosides, such as GM3, are normally present in circulation. Conversely, elevated levels of circulating GD2, a disialoganglioside, have been found in neuroblastoma patients and its concentration is inversely related to progression-free survival. Shedding of gangliosides enhances integrin α2β1-dependent platelet activation, leading to platelet aggregation, and increased adhesion to vascular basement membranes [55]. These events can enhance tumor cell embolization impacting the occurrence of cells lodging in capillary beds and invading into surrounding tissue.

Finally, it is worth noting that at any phase of tumor progression, cancer cells must evade the immune system. Some T-cell lysis mechanisms are dependent on integrin expression. For instance, binding of T-cell integrin LFA-1 (αLβ2) to its ligands ICAM-1 on tumor cells is important in CD3-mediated T-cell lysis [56, 57]. Of note, ICAM expression on neuroblastoma cells is associated with increased susceptibility to lymphokine-activated killer (LAK) cell lysis following interferon gamma treatment [58].

rarely able to form xenograft tumors [65]. S-type cells express fibronectin; it is therefore not surprising that they represent the group of neuroblastoma that express α5β1integrin, the

Neuroblastoma Integrins http://dx.doi.org/10.5772/55991 199

The third type of cells is the 'intermediate cells.' Noted as potential 'cancer stem cells' as early as 1989 by Ross and colleagues, these cells look like an intermediate between the N and S types via diverse measures including phase contrast microscopy, intermediate filament expression, tyrosine hydroxylase activity, and norepinephrine uptake [64]. Consistent with being a tumor stem-like cell (or tumor initiating cell), I-type cells are by far the most tumorigenic in mice and in *in vitro* surrogate assays of tumor formation. Treatment with 13-cis retinoic acid or 5 bromo-2'-deoxyuridine can differentiate I-type cells into N-type or S-type, respectively. Retinoic acid has significant effects on integrins, consistent with changes seen during neuronal differentiation, and can differentiate some neuroblastomas into a benign growth-arrested state [67]. Clinically, retinoic acid has also been demonstrated to improve event free and overall

survival in a long-term follow-up on a large cohort of neuroblastoma patients [68].

In addition to key roles in cell anchorage and migration, integrin-mediated ligation of the extracellular matrix results in the initiation of signaling events exerting both local and cellular effects. Thus, the extracellular matrix encodes information via the local milieu of cell surface or diffusible factors presented to the cell (Figure 4). Most of these signals have been studied in rigorously defined systems *in vitro* with cell lines, rather than primary *in vivo* investigation.

**4.1. Integrin ligation promotes the activation of the nonreceptor tyrosine kinases FAK and**

Signaling that follows the ligation of integrins by extracellular matrix components can be studied by introducing suspended neuroblastoma cells to a surface coated with an extracellular matrix component, such as fibronectin. This results in cell attachment and spreading. Con‐ current with these events, phosphorylation is observed on cytosolic nonreceptor tyrosine kinases like FAK (tyrosine residue 397) and Src (tyrosine residue 418), which indicate activation of the tyrosine kinases. At least some of this activity is physically present in the integrin associated focal adhesion complex, and these kinases can be co-purified with integrins from

FAK and Src can associate with each other and with an array of cytosolic adaptor proteins and other effectors. For example, FAK can associate with the cytoskeletal adaptor protein talin, which also binds to integrins. The adhesion of NB7 neuroblastoma cells to fibronectin or collagen has been shown to promote co-association of these molecules together in a complex with the protease calpain. Calpain in turn cleaves talin in a cell-adhesion dependent manner, which faciliates more rapid turn over of the focal adhesion, and promotes neuroblastoma cell migration. The same cleavage is observed in other neuroblastoma cells, including NB5 and

NB16, suggesting it may be a conserved pathway [69].

fibronectin receptor [66].

**4. Signaling by integrins**

**Src**

this complex.

#### **3.4. Trends in integrin expression with neuroblastoma stage and grade**

Since integrins impact cell differentiation and invasion, there has been an interest in linking the expression of subsets of integrins with a particular tumor stage, or more appropriately, with tumor 'risk.' Key risk predictors to date have been established by the Children's Oncology Group, and include status of the MYCN gene, the pathology of the tumor according to guidelines established by Shimada [59], and in some cases the relative ploidy of the tumor. Since integrins are associated with neuronal cell developmental stages and activities, it is reasonable that integrin expression could offer insights into tumor activities.

In pioneering studies, using 45 clinical samples, Favrot et al. showed that the α2 and α6 subunits were associated with low grade, well-differentiated neuroblastoma samples. The finding is consistent with observations of normal 'neural crest cell to neuronal' differentiation. The β1 subunit was expressed on all samples while the α5 subunit was not expressed on any samples examined. Samples expressing the α4, αv, β3, and β4 subunits revealed no N-Myc amplification, and were associated with a good prognosis. In addition, expression of α4 and β4 subunits was found selectively on Schwannian stromal cells [60].

Conversely, more recent studies have found that many neuroblastoma cell lines express integrin α4 and that α4 expression is associated with increased tumor stage (stages 3 and 4) in clinical samples [61]. At least on cell lines, integrin α5β1 also appears to be expressed [45] and integrin αvβ3 has been described to be present on some malignant neuroblastomas [62]. In addition, by flow cytometry, our lab consistently observes low levels of integrin αvβ5 on established neuroblastoma cell lines, although whether this is a tissue culture adaptation or reflects actual expression *in situ* remains unclear. Indeed, neuroblasts exhibit significant plasticity, and although integrins may be associated with specific stages of neuroblastoma, or specific developmental states where transformation of the neuroblast initially occurred, an alternative hypothesis is that neuroblastoma may retain the capacity to alter their relative integrin expression, and that this type of plasticity may itself be a malignancy factor.

Neuroblastomas fall into three common morphological/adhesive categories when grown *in vitro*: S (Substrate adherent), N (Neuroblastic), and I (Intermediate) types [63, 64]. These different types are sometimes ascribed to a particular cell line, though in many cases a cell line may contain cells of all three types. Studies using tissue culture cell lines have shown that, relative to S-type, N-type neuroblastomas exhibit decreased expression of β1 integrin and greater expression of αvβ3, and are more migratory *in vitro*. However, the expression of αvβ3 on these cells is still relatively low, at least when one compares with tissues well known to express αvβ3, such as angiogenic endothelium or melanoma. N-type cells also form more colonies in soft agar and are more tumorigenic when implanted in mice than S-type, which are rarely able to form xenograft tumors [65]. S-type cells express fibronectin; it is therefore not surprising that they represent the group of neuroblastoma that express α5β1integrin, the fibronectin receptor [66].

The third type of cells is the 'intermediate cells.' Noted as potential 'cancer stem cells' as early as 1989 by Ross and colleagues, these cells look like an intermediate between the N and S types via diverse measures including phase contrast microscopy, intermediate filament expression, tyrosine hydroxylase activity, and norepinephrine uptake [64]. Consistent with being a tumor stem-like cell (or tumor initiating cell), I-type cells are by far the most tumorigenic in mice and in *in vitro* surrogate assays of tumor formation. Treatment with 13-cis retinoic acid or 5 bromo-2'-deoxyuridine can differentiate I-type cells into N-type or S-type, respectively. Retinoic acid has significant effects on integrins, consistent with changes seen during neuronal differentiation, and can differentiate some neuroblastomas into a benign growth-arrested state [67]. Clinically, retinoic acid has also been demonstrated to improve event free and overall survival in a long-term follow-up on a large cohort of neuroblastoma patients [68].

## **4. Signaling by integrins**

Finally, it is worth noting that at any phase of tumor progression, cancer cells must evade the immune system. Some T-cell lysis mechanisms are dependent on integrin expression. For instance, binding of T-cell integrin LFA-1 (αLβ2) to its ligands ICAM-1 on tumor cells is important in CD3-mediated T-cell lysis [56, 57]. Of note, ICAM expression on neuroblastoma cells is associated with increased susceptibility to lymphokine-activated killer (LAK) cell lysis

Since integrins impact cell differentiation and invasion, there has been an interest in linking the expression of subsets of integrins with a particular tumor stage, or more appropriately, with tumor 'risk.' Key risk predictors to date have been established by the Children's Oncology Group, and include status of the MYCN gene, the pathology of the tumor according to guidelines established by Shimada [59], and in some cases the relative ploidy of the tumor. Since integrins are associated with neuronal cell developmental stages and activities, it is

In pioneering studies, using 45 clinical samples, Favrot et al. showed that the α2 and α6 subunits were associated with low grade, well-differentiated neuroblastoma samples. The finding is consistent with observations of normal 'neural crest cell to neuronal' differentiation. The β1 subunit was expressed on all samples while the α5 subunit was not expressed on any samples examined. Samples expressing the α4, αv, β3, and β4 subunits revealed no N-Myc amplification, and were associated with a good prognosis. In addition, expression of α4 and

Conversely, more recent studies have found that many neuroblastoma cell lines express integrin α4 and that α4 expression is associated with increased tumor stage (stages 3 and 4) in clinical samples [61]. At least on cell lines, integrin α5β1 also appears to be expressed [45] and integrin αvβ3 has been described to be present on some malignant neuroblastomas [62]. In addition, by flow cytometry, our lab consistently observes low levels of integrin αvβ5 on established neuroblastoma cell lines, although whether this is a tissue culture adaptation or reflects actual expression *in situ* remains unclear. Indeed, neuroblasts exhibit significant plasticity, and although integrins may be associated with specific stages of neuroblastoma, or specific developmental states where transformation of the neuroblast initially occurred, an alternative hypothesis is that neuroblastoma may retain the capacity to alter their relative

integrin expression, and that this type of plasticity may itself be a malignancy factor.

Neuroblastomas fall into three common morphological/adhesive categories when grown *in vitro*: S (Substrate adherent), N (Neuroblastic), and I (Intermediate) types [63, 64]. These different types are sometimes ascribed to a particular cell line, though in many cases a cell line may contain cells of all three types. Studies using tissue culture cell lines have shown that, relative to S-type, N-type neuroblastomas exhibit decreased expression of β1 integrin and greater expression of αvβ3, and are more migratory *in vitro*. However, the expression of αvβ3 on these cells is still relatively low, at least when one compares with tissues well known to express αvβ3, such as angiogenic endothelium or melanoma. N-type cells also form more colonies in soft agar and are more tumorigenic when implanted in mice than S-type, which are

**3.4. Trends in integrin expression with neuroblastoma stage and grade**

reasonable that integrin expression could offer insights into tumor activities.

β4 subunits was found selectively on Schwannian stromal cells [60].

following interferon gamma treatment [58].

198 Neuroblastoma

In addition to key roles in cell anchorage and migration, integrin-mediated ligation of the extracellular matrix results in the initiation of signaling events exerting both local and cellular effects. Thus, the extracellular matrix encodes information via the local milieu of cell surface or diffusible factors presented to the cell (Figure 4). Most of these signals have been studied in rigorously defined systems *in vitro* with cell lines, rather than primary *in vivo* investigation.

#### **4.1. Integrin ligation promotes the activation of the nonreceptor tyrosine kinases FAK and Src**

Signaling that follows the ligation of integrins by extracellular matrix components can be studied by introducing suspended neuroblastoma cells to a surface coated with an extracellular matrix component, such as fibronectin. This results in cell attachment and spreading. Con‐ current with these events, phosphorylation is observed on cytosolic nonreceptor tyrosine kinases like FAK (tyrosine residue 397) and Src (tyrosine residue 418), which indicate activation of the tyrosine kinases. At least some of this activity is physically present in the integrin associated focal adhesion complex, and these kinases can be co-purified with integrins from this complex.

FAK and Src can associate with each other and with an array of cytosolic adaptor proteins and other effectors. For example, FAK can associate with the cytoskeletal adaptor protein talin, which also binds to integrins. The adhesion of NB7 neuroblastoma cells to fibronectin or collagen has been shown to promote co-association of these molecules together in a complex with the protease calpain. Calpain in turn cleaves talin in a cell-adhesion dependent manner, which faciliates more rapid turn over of the focal adhesion, and promotes neuroblastoma cell migration. The same cleavage is observed in other neuroblastoma cells, including NB5 and NB16, suggesting it may be a conserved pathway [69].

**4.2. Integrin activation of the phosphoinositide 3' kinase signaling axis**

is likely due to improved pharmacokinetics associated with the targeting peptide.

**4.3. Interplay between integrins and signature neuroblastoma signaling pathways**

N-Myc is a transcription factor normally expressed during early lymphocyte development and in embryonic brain and kidney tissues [82], and is critical for survival of neural crest-derived neurons [83]. Amplification of greater than ten copies of the *MYCN* gene has long been recognized as a strong negative prognostic indicator of outcome in neuroblastoma [84]. N-Myc interacts with integrins in an antagonistic manner; while N-Myc seems to increase expression of FAK, it has also been shown to down-regulate the expression of integrins such as α3β1 and α1β1 [85-88]. Transcriptional analysis of the β3 and αv promoters have revealed negative transcriptional regulatory elements in their promoters by the closely related c-Myc [76], suggesting why αvβ3 is not highly expressed in neuroblastoma relative to other tumors. In fact, the loss of integrin expression may be important for survival in specific circumstances, particularly among tumors that retain intrinsic apoptotic capacity, as discussed below.

ALK is a tyrosine kinase that is expressed largely during development within the nervous system. ALK belongs to the 'insulin-like tyrosine kinase' family of receptors that is frequently upregulated or subject to oncogenic mutation in neuroblastoma [89]. Signaling by tyrosine kinases generally requires integrin ligation [5], activating downstream targets (such as FAK, Src, PI3K etc.). This suggests that there is an intrinsic requirement for ECM adhesion to permit a tumor to 'leverage' amplified ALK. However, mutant forms of ALK also exist, particularly

Integrins stabilized and ligated to correct ECM promote signaling via class I phosphoinisotol-3 kinases (PI3K). PI3K's are a family of lipid bound kinases found at the cell membrane or intracellular endosomes, and can promote cell motility, intracellular trafficking and survival. Among the four class I PI3K's, neuroblastoma tend to express P110α and p110β, with the latter more likely to be associated with N-Myc expressing tumors. Nonetheless, P110γ and p110δ are also sometimes detected [73]. Activation of the PI3K signaling axis promotes malignancy in numerous cancer cell lines and models of human cancer [74]. PI3K signaling also enhances turnover of pro-mitochondrial apoptotic proteins like Bad and promotes downstream prosurvival pathways, such as AKT and mTOR [75]. PTEN, a suppressor of PI3K, is frequently lost in cancer, although studies in neuroblastoma have shown a lesser degrees of loss, in the range of ~5% for homozygous deletion [76]. Mutations of PI3K that enhance kinase activity have been reported in other cancers [77], yet they have been proven to be infrequent in neuroblastoma [78]. Thus, the activity of PI3K appears to frequently depend upon extrinsic regulatory factors, mediated by receptor tyrosine kinases (eg., IGFR-1, ALK) and integrins. Given the lack of effective therapies for malignant neuroblastoma, it is perhaps not surprising that the PI3K pathway is being pursued for pharmacological intervention [79]. In neuroblas‐ toma, inhibition of PI3K has been demonstrated to decrease migration and survival of tumor cells *in vitro*, and inhibit tumor growth *in vivo* [80, 81]. The efficacy of pharmacological PI3K inhibition may be enhanced by combining a pro-drug with an RGDS peptide to target the agent to tumor sites [81]. The relative affinity for this linear peptide for integrin, however, is quite low, and it is improbable that enhanced efficacy is due to direct action on integrins, rather, it

Neuroblastoma Integrins http://dx.doi.org/10.5772/55991 201

**Figure 4. Signaling Pathways Downstream of Integrins** The continued survival of a single cell and its progeny in the wrong environment can disrupt the homeostasis of the tissue that contains them. Thus, the impetus of individual cells to live or die is critical for the continued homeostasis of an organism. Recognition of compatible ECM promotes stable ligation and clustering of integrins, as well as assembly of the heterogeneous and dynamic focal adhesion complex. Signaling from integrins and focal adhesion-associated receptor tyrosine kinases (RTK) leads to downstream pro-sur‐ vival signaling pathways such as the PI3K /AKT and Erk axes. By contrast, the presence of an incompatible ECM or of unligated or antagonized integrins promotes cell death via anoikis pathways, including integrin-mediated death. N-Myc exerts pleiotropic effects via transcription (or inhibition thereof) of many downstream genes, enhancing prolifera‐ tion and survival, and attenuating the expression of integrins, and therefore decreasing anoikis signaling.

FAK also associates with Grb2 and SoS [70], key regulators of Ras-GTP mediated activation of the Raf/MEK/ERK pathway of MAP kinase signaling. This pathway helps to drive proliferation of the tumor cells, and may account for adhesion-based induction of cyclin E in neuroblastoma (and other) cells [71]. FAK is perhaps best known for its capacity to support and promote integrin-mediated cell migration on an ECM, and performs this function in neuroblastoma cells as well, although this appears to be integrin specific [61]. For example, integrin α5β1 activates FAK and uses this kinase for migration, while integrin α4β1 migration is dependent upon the non-receptor kinase Src. Both integrins can bind to a fibronectin substrate, thus the particular integrin ligated can have an impact on the cells' response. Other effects of specific integrin ligation have been reported in non-neuroblastoma cell lines, such as the FAK and α5β1-induced expression of the pro-survival gene Bcl-2 [72]. Thus, signals from FAK can play a role in regulating cell survival in an ECM and integrin-dependent manner. 4

#### **4.2. Integrin activation of the phosphoinositide 3' kinase signaling axis**

Integrins stabilized and ligated to correct ECM promote signaling via class I phosphoinisotol-3 kinases (PI3K). PI3K's are a family of lipid bound kinases found at the cell membrane or intracellular endosomes, and can promote cell motility, intracellular trafficking and survival. Among the four class I PI3K's, neuroblastoma tend to express P110α and p110β, with the latter more likely to be associated with N-Myc expressing tumors. Nonetheless, P110γ and p110δ are also sometimes detected [73]. Activation of the PI3K signaling axis promotes malignancy in numerous cancer cell lines and models of human cancer [74]. PI3K signaling also enhances turnover of pro-mitochondrial apoptotic proteins like Bad and promotes downstream prosurvival pathways, such as AKT and mTOR [75]. PTEN, a suppressor of PI3K, is frequently lost in cancer, although studies in neuroblastoma have shown a lesser degrees of loss, in the range of ~5% for homozygous deletion [76]. Mutations of PI3K that enhance kinase activity have been reported in other cancers [77], yet they have been proven to be infrequent in neuroblastoma [78]. Thus, the activity of PI3K appears to frequently depend upon extrinsic regulatory factors, mediated by receptor tyrosine kinases (eg., IGFR-1, ALK) and integrins.

Given the lack of effective therapies for malignant neuroblastoma, it is perhaps not surprising that the PI3K pathway is being pursued for pharmacological intervention [79]. In neuroblas‐ toma, inhibition of PI3K has been demonstrated to decrease migration and survival of tumor cells *in vitro*, and inhibit tumor growth *in vivo* [80, 81]. The efficacy of pharmacological PI3K inhibition may be enhanced by combining a pro-drug with an RGDS peptide to target the agent to tumor sites [81]. The relative affinity for this linear peptide for integrin, however, is quite low, and it is improbable that enhanced efficacy is due to direct action on integrins, rather, it is likely due to improved pharmacokinetics associated with the targeting peptide.

#### **4.3. Interplay between integrins and signature neuroblastoma signaling pathways**

FAK also associates with Grb2 and SoS [70], key regulators of Ras-GTP mediated activation of the Raf/MEK/ERK pathway of MAP kinase signaling. This pathway helps to drive proliferation of the tumor cells, and may account for adhesion-based induction of cyclin E in neuroblastoma (and other) cells [71]. FAK is perhaps best known for its capacity to support and promote integrin-mediated cell migration on an ECM, and performs this function in neuroblastoma cells as well, although this appears to be integrin specific [61]. For example, integrin α5β1 activates FAK and uses this kinase for migration, while integrin α4β1 migration is dependent upon the non-receptor kinase Src. Both integrins can bind to a fibronectin substrate, thus the particular integrin ligated can have an impact on the cells' response. Other effects of specific integrin ligation have been reported in non-neuroblastoma cell lines, such as the FAK and α5β1-induced expression of the pro-survival gene Bcl-2 [72]. Thus, signals from FAK can play

tion and survival, and attenuating the expression of integrins, and therefore decreasing anoikis signaling.

4

**TUMOR PROGRESSION** 

**Figure 4. Signaling Pathways Downstream of Integrins** The continued survival of a single cell and its progeny in the wrong environment can disrupt the homeostasis of the tissue that contains them. Thus, the impetus of individual cells to live or die is critical for the continued homeostasis of an organism. Recognition of compatible ECM promotes stable ligation and clustering of integrins, as well as assembly of the heterogeneous and dynamic focal adhesion complex. Signaling from integrins and focal adhesion-associated receptor tyrosine kinases (RTK) leads to downstream pro-sur‐ vival signaling pathways such as the PI3K /AKT and Erk axes. By contrast, the presence of an incompatible ECM or of unligated or antagonized integrins promotes cell death via anoikis pathways, including integrin-mediated death. N-Myc exerts pleiotropic effects via transcription (or inhibition thereof) of many downstream genes, enhancing prolifera‐

Focal Adhesion Complex

F

C

ocal A

om

α β

RTK

FAK

**Survival Proliferation Migration**

Ligated

Integrins

Src Anoikis

Pathways

ikishways

edns

α β

**Cell Death**

Unligated

or Antagonized

Integrins

Incompatible ECM

α

200 Neuroblastoma

β

N-Myc

ERK PI3K

on

dhesion

mplex

Adhe

AKT

N-Myc

RTK

RT

α β

RTK

Compatible ECM

α

β

a role in regulating cell survival in an ECM and integrin-dependent manner.

N-Myc is a transcription factor normally expressed during early lymphocyte development and in embryonic brain and kidney tissues [82], and is critical for survival of neural crest-derived neurons [83]. Amplification of greater than ten copies of the *MYCN* gene has long been recognized as a strong negative prognostic indicator of outcome in neuroblastoma [84]. N-Myc interacts with integrins in an antagonistic manner; while N-Myc seems to increase expression of FAK, it has also been shown to down-regulate the expression of integrins such as α3β1 and α1β1 [85-88]. Transcriptional analysis of the β3 and αv promoters have revealed negative transcriptional regulatory elements in their promoters by the closely related c-Myc [76], suggesting why αvβ3 is not highly expressed in neuroblastoma relative to other tumors. In fact, the loss of integrin expression may be important for survival in specific circumstances, particularly among tumors that retain intrinsic apoptotic capacity, as discussed below.

ALK is a tyrosine kinase that is expressed largely during development within the nervous system. ALK belongs to the 'insulin-like tyrosine kinase' family of receptors that is frequently upregulated or subject to oncogenic mutation in neuroblastoma [89]. Signaling by tyrosine kinases generally requires integrin ligation [5], activating downstream targets (such as FAK, Src, PI3K etc.). This suggests that there is an intrinsic requirement for ECM adhesion to permit a tumor to 'leverage' amplified ALK. However, mutant forms of ALK also exist, particularly a F1174 mutation that drives neuroblastoma malignancy cooperatively with MYCN. In this case, it is unclear whether integrin-mediated adhesion is actually required for cell proliferation, although it is likely to enhance signaling in keeping with the rationale described above. MYCN also leads to increased expression of a close ALK relative, insulin-like growth factor I receptor (IGF-IR). In this case, crosstalk between IGF-IR and integrins is also observed [90].

fibrinogen, von Willebrand factor and others. Gladson et al. found that αv was present in all tumors they examined regardless of stage. While αvβ1 and αvβ5 heterodimers were found in normal adrenal tissues and ganglioneuroblastomas which exhibit lower levels of dissemina‐ tion, the αvβ3 integrin was found to be expressed in highly metastatic, undifferentiated neuroblastomas [62]. By contrast, we observe only very low levels of integrin αvβ3 on our neuroblastoma specimens relative to melanoma or cultured endothelial cells, which express robust levels of αvβ3. However, it remains possible that the techniques originally used by Gladson were simply very sensitive and detected this modest but important level of integrin expression. Indeed, αvβ3 is, in some systems, a stem cell marker, and this may reflect the

Neuroblastoma Integrins http://dx.doi.org/10.5772/55991 203

In addition, on a variety of tumor cells, αvβ3 expression has been demonstrated to promote tumor progression by its ability to bind to a wide array of different ligands, facilitating anchorage and invasion. Integrin αvβ3 also stimulates MMP activity, promotes the activation of receptor and non-receptor tyrosine kinases including src, and the release of growth factors such as TGF that promote tumor response. This vascularization provides the growing tumor with the nutrients it needs and brings tumor cells proximal to vessels, which may facilitate invasion and metastasis. As previously mentioned, αvβ3 is also expressed on angiogenic endothelial cells where it promotes cell survival and migration. One study showed that there is higher β3 expression on invasive and metastatic melanomas than on noninvasive melanomas [97], although the levels demonstrated in these cases appear to be logarithmically higher than

Integrin α4β1 is primarily known as a trafficking integrin, as it is present on most leukocytes. Binding to its ligand VCAM-1, present on activated endothelial cells, enhances the transen‐ dothelial migration of white blood cells into surrounding tissues. Cancer cells that express α4β1 acquire this same enhanced trafficking potential and show increased tumor cell arrest in circulation and increased extravasation and colony formation. α4β1 may also enhance invasion and metastasis through promotion of angiogenesis and lymphangiogenesis [99, 100]. In [97], α4β1 expression was found on 40% of invasive and metastatic melanomas, although not on

It is important to note that, though the expression of α4β1 can indeed promote extravasation, the overall role of integrin α4β1 in tumor progression and metastasis is highly controversial and is dependent on the level of expression and the phase of tumor progression. For example, high α4β1 expression in some primary tumors can enhance homotypic cell-cell adhesion [101], preventing cells from breaking away from the tumor and invading into surrounding tissues [102]. In addition, α4β1 expression can lead to a reduction in MMPs and impair the ability of the cells to degrade the matrix and create a pathway for invasion [103]. If cells do successfully metastasize to distant sites, α4β1 expression may promote or inhibit metastatic growth

advanced stage and poor prognosis of her positive cohort.

those seen on neuroblastoma cell lines [98].

**5.2. Integrin α4β1 and tumor spread**

non-malignant melanocytes.

depending on the microenvironment.

#### **4.4. Integrins and cell survival signaling**

Cells that lose anchorage for extended periods of time will typically undergo apoptosis. This phenomenon encompasses one aspect of anoikis (gr., homelessness), a phenomenon wherein a cell that finds itself in an inappropriate environment is signaled to undergo apoptosis. However, there is no 'central cell death pathway' associated with anoikis, and in fact many different pathways have been validated in the literature. This underscores the critical need for cell adhesion. One anoikis pathway is focused on the activation of caspase-9. Although many neuroblastomas lose expression of one copy of caspase-9 (as many are LOH1p21), this does not appear to impact the capacity of caspase-9 to activate [91]. Antagonism of b1 integrins on differentiated neuroblastoma, but not undifferentiated, promotes this apoptotic pathway [92].

Integrin-mediated death is an anoikis pathway in which the presence of unligated, or antago‐ nized,integrins onthe cell surfacepromote celldeathvia the activationof caspase-8.Neuroblas‐ toma avoid this death pathway via several mechanisms. First, the amplification of MYCN can lead to an overall decrease in integrin expression, which lowers the capacity of the pathway to trigger. Secondly, stage III and IV neuroblastoma tend to methylate, delete, or disrupt the caspase-8 gene [93, 94], preventing the triggering of the apoptotic pathway, and this results in a survival advantage *in vitro* and a metastasis advantage *in vivo*. Finally, neuroblastoma that are seeded as individual cells in an 'inappropriate' three dimensional matrix will tend to either die or, within only a couple days, find each other and form small cell clusters. These islands of cells promote their own survival and can persist, although they are sometimes surrounded by apoptotic bodies as errant progeny try to migrate away from the original cell mass.

Opposing the induction of death by unligated or antagonized integrins, it is worth noting that a cell that has a robust interaction with the ECM is more resistant to certain insults than others, and integrin ligation has been linked to chemo and radiation resistance. Mechanistically, this is likely to result from remodeling of the ECM, combined with transcriptional alterations of survival promoting genes such as Bcl-2 family members, IAPs and others. However, direct effects, such as maturation-inhibiting phosphorylation of procaspase-8, cannot be excluded from contributing to this effect [95, 96].

#### **5. Specific integrins in neuroblastoma progression**

#### **5.1. Integrin αvβ3**

Integrin αvβ3 is the most 'promiscuous' member of the integrin family, in that it binds a variety of different RGD conformations, and thus binds to ligands that include vitronectin, fibronectin, fibrinogen, von Willebrand factor and others. Gladson et al. found that αv was present in all tumors they examined regardless of stage. While αvβ1 and αvβ5 heterodimers were found in normal adrenal tissues and ganglioneuroblastomas which exhibit lower levels of dissemina‐ tion, the αvβ3 integrin was found to be expressed in highly metastatic, undifferentiated neuroblastomas [62]. By contrast, we observe only very low levels of integrin αvβ3 on our neuroblastoma specimens relative to melanoma or cultured endothelial cells, which express robust levels of αvβ3. However, it remains possible that the techniques originally used by Gladson were simply very sensitive and detected this modest but important level of integrin expression. Indeed, αvβ3 is, in some systems, a stem cell marker, and this may reflect the advanced stage and poor prognosis of her positive cohort.

In addition, on a variety of tumor cells, αvβ3 expression has been demonstrated to promote tumor progression by its ability to bind to a wide array of different ligands, facilitating anchorage and invasion. Integrin αvβ3 also stimulates MMP activity, promotes the activation of receptor and non-receptor tyrosine kinases including src, and the release of growth factors such as TGF that promote tumor response. This vascularization provides the growing tumor with the nutrients it needs and brings tumor cells proximal to vessels, which may facilitate invasion and metastasis. As previously mentioned, αvβ3 is also expressed on angiogenic endothelial cells where it promotes cell survival and migration. One study showed that there is higher β3 expression on invasive and metastatic melanomas than on noninvasive melanomas [97], although the levels demonstrated in these cases appear to be logarithmically higher than those seen on neuroblastoma cell lines [98].

#### **5.2. Integrin α4β1 and tumor spread**

a F1174 mutation that drives neuroblastoma malignancy cooperatively with MYCN. In this case, it is unclear whether integrin-mediated adhesion is actually required for cell proliferation, although it is likely to enhance signaling in keeping with the rationale described above. MYCN also leads to increased expression of a close ALK relative, insulin-like growth factor I receptor

Cells that lose anchorage for extended periods of time will typically undergo apoptosis. This phenomenon encompasses one aspect of anoikis (gr., homelessness), a phenomenon wherein a cell that finds itself in an inappropriate environment is signaled to undergo apoptosis. However, there is no 'central cell death pathway' associated with anoikis, and in fact many different pathways have been validated in the literature. This underscores the critical need for cell adhesion. One anoikis pathway is focused on the activation of caspase-9. Although many neuroblastomas lose expression of one copy of caspase-9 (as many are LOH1p21), this does not appear to impact the capacity of caspase-9 to activate [91]. Antagonism of b1 integrins on differentiated neuroblastoma, but not undifferentiated, promotes this apoptotic pathway [92].

Integrin-mediated death is an anoikis pathway in which the presence of unligated, or antago‐ nized,integrins onthe cell surfacepromote celldeathvia the activationof caspase-8.Neuroblas‐ toma avoid this death pathway via several mechanisms. First, the amplification of MYCN can lead to an overall decrease in integrin expression, which lowers the capacity of the pathway to trigger. Secondly, stage III and IV neuroblastoma tend to methylate, delete, or disrupt the caspase-8 gene [93, 94], preventing the triggering of the apoptotic pathway, and this results in a survival advantage *in vitro* and a metastasis advantage *in vivo*. Finally, neuroblastoma that are seeded as individual cells in an 'inappropriate' three dimensional matrix will tend to either die or, within only a couple days, find each other and form small cell clusters. These islands of cells promote their own survival and can persist, although they are sometimes surrounded by

apoptotic bodies as errant progeny try to migrate away from the original cell mass.

Opposing the induction of death by unligated or antagonized integrins, it is worth noting that a cell that has a robust interaction with the ECM is more resistant to certain insults than others, and integrin ligation has been linked to chemo and radiation resistance. Mechanistically, this is likely to result from remodeling of the ECM, combined with transcriptional alterations of survival promoting genes such as Bcl-2 family members, IAPs and others. However, direct effects, such as maturation-inhibiting phosphorylation of procaspase-8, cannot be excluded

Integrin αvβ3 is the most 'promiscuous' member of the integrin family, in that it binds a variety of different RGD conformations, and thus binds to ligands that include vitronectin, fibronectin,

(IGF-IR). In this case, crosstalk between IGF-IR and integrins is also observed [90].

**4.4. Integrins and cell survival signaling**

202 Neuroblastoma

from contributing to this effect [95, 96].

**5.1. Integrin αvβ3**

**5. Specific integrins in neuroblastoma progression**

Integrin α4β1 is primarily known as a trafficking integrin, as it is present on most leukocytes. Binding to its ligand VCAM-1, present on activated endothelial cells, enhances the transen‐ dothelial migration of white blood cells into surrounding tissues. Cancer cells that express α4β1 acquire this same enhanced trafficking potential and show increased tumor cell arrest in circulation and increased extravasation and colony formation. α4β1 may also enhance invasion and metastasis through promotion of angiogenesis and lymphangiogenesis [99, 100]. In [97], α4β1 expression was found on 40% of invasive and metastatic melanomas, although not on non-malignant melanocytes.

It is important to note that, though the expression of α4β1 can indeed promote extravasation, the overall role of integrin α4β1 in tumor progression and metastasis is highly controversial and is dependent on the level of expression and the phase of tumor progression. For example, high α4β1 expression in some primary tumors can enhance homotypic cell-cell adhesion [101], preventing cells from breaking away from the tumor and invading into surrounding tissues [102]. In addition, α4β1 expression can lead to a reduction in MMPs and impair the ability of the cells to degrade the matrix and create a pathway for invasion [103]. If cells do successfully metastasize to distant sites, α4β1 expression may promote or inhibit metastatic growth depending on the microenvironment.

#### **6. Drugs that target integrins**

The involvement of integrins in multiple stages of tumor progression makes them attractive therapeutic targets. Inhibition of integrin signaling can be achieved using several approaches including blocking ligand binding, preventing the formation of functional focal adhesion complexes and disrupting integrin association with the cytoskeleton. Because the structure of integrins has been extensively studied and because having an extracellular target eliminates the challenges of intracellular delivery, the most common approach has been to target the integrin ligand-binding site. This has been accomplished using blocking antibodies, cyclic and ligand-mimicking peptides, small molecule antagonists and disintegrins [104] (Table 2).

**6.1. Integrin αv**

indications.

**6.2. Integrin α4**

**6.3. Integrin αIIbβ3**

anemia and acute coronary syndromes [104].

**7. Summary and considerations**

The primary rationale for targeting integrin αvβ3 in cancer is to reduce primary tumor growth and metastasis via nutrient deprivation due to inhibition of tumor angiogenesis. Several αvβ3 antagonists have gone to clinical trials with the most notable being cilengitide. Cilengitide is a cyclic peptide containing the RGD integrin-binding motif. It inhibits both αvβ3 and αvβ5. Cilengitide produces both anti-angiogenic and anti-tumor effects through inhibition of VEGF stimulation and FAK-Src and Erk signaling, respectively [105]. *In vitro*, cilengitide reduces cell growth and survival and inhibits endothelial and tumor cell migration. In clinical trials, cilengitide has been evaluated as a single agent and in combination with radiation, DNAalkylating agents and gemcitibine. Importantly, cilengitide in combination with radiotherapy and temozolomide (a DNA-alkylating agent) has reached phase III trials in glioblastoma multiforme patients. Other small molecule antagonists are in development for noncancer

Neuroblastoma Integrins http://dx.doi.org/10.5772/55991 205

The integrin α4 subunit is predominantly expressed in lymphocytes and leukocytes and supports endothelial transmigration of these cells via binding to VCAM-1. Consequently, α4 is important for immune function and has been targeted in diseases such as multiple sclerosis (MS), Crohn's disease and asthma that are characterized by excessive inflammation or an improper immune response. Natalizumab, the only FDA approved α4 antagonist, is a humanized mouse monoclonal antibody that binds both α4 heterodimers. The use of natali‐ zumab was successful in clinical trials in MS [106, 107] and Crohn's disease [108] with the exception of rare cases of progressive multi-focal leukoencephalopathy (PML) caused by reactivation of latent JC virus associated with immunosuppression [109]. Unfortunately, this side effect was detrimental enough to lead to limitation of the use of natalizumab to patients who are unresponsive to other treatments. Other α4 antagonists under clinical evaluation include MLN-00002 (human α4β7 antibody), firategrast and IVL745 (small molecules: [104]). Though the rationale for the use of most α4 antagonists is to reduce excessive infiltration of immune cells, these therapies have the potential for use against cancer cells that exploit α4 for tumor cell extravasation. The success of targeting α4 in cancer will depend on the ability to minimize immunosuppression or to indirectly impair α4 function via downstream targets.

Integrin αIIbβ3 is also a frequently targeted integrin. This heterodimer is expressed selectively on platelets and megakaryocytes and is mostly known for its role in blood coagulation. Antagonists of this receptor are primarily employed in diseases such as stroke, sickle cell

Integrins are a unique group of receptors that provide anchorage, mediate cell migration and invasion, and signal via cell survival and proliferation pathways. Aptly named,


**Table 2.** Drugs that Target Integrins

#### **6.1. Integrin αv**

**6. Drugs that target integrins**

204 Neuroblastoma

c7E3 (Abciximab)

The involvement of integrins in multiple stages of tumor progression makes them attractive therapeutic targets. Inhibition of integrin signaling can be achieved using several approaches including blocking ligand binding, preventing the formation of functional focal adhesion complexes and disrupting integrin association with the cytoskeleton. Because the structure of integrins has been extensively studied and because having an extracellular target eliminates the challenges of intracellular delivery, the most common approach has been to target the integrin ligand-binding site. This has been accomplished using blocking antibodies, cyclic and ligand-mimicking peptides, small molecule antagonists and disintegrins [104] (Table 2).

**Target Antagonist Type Clinical Development**

antibody

CNTO 95 humanized antibody Phase II trials

L000845704 Small molecule Phase I trials

MLN-00002 human antibody Phase II trials Firategrast small molecule Phase II trials

α4β1 Natalizumab humanized antibody FDA approved (1994) for

antibody

antibody

α2β1 Rhodocetin disintegrin Pre-clinical

JSM6427 small molecule Phase I trials

αIIbβ3 c7E3 (Abciximab) Chimeric mouse- human

α5β1 Volociximab chimeric human-mouse

**Table 2.** Drugs that Target Integrins

Chimeric mouse- human

Cilengitide cyclic peptide Phase III trials for glioblastoma

SB273005 Small molecule Pre-clinical animal studies

Eptifibatide cyclic peptide FDA approved (1998) for use in

Tirofiban small molecule FDA approved in 1999

FDA approved (1994) for use in percutaneous coronary intervention (PCI)

multiforme; Phase II trials for melanoma, glioma, and SCCHN; Phase I trials for NSCLC

treatment of multiple sclerosis

FDA approved (1994) for use in percutaneous coronary intervention (PCI)

patients with acute coronary syndrome or undergoing PCI

Phase II trials in melanoma, pancreatic cancer, and NSCLC

and Crohn's disease

αvβ3 Vitaxin humanized antibody Phase II trials

The primary rationale for targeting integrin αvβ3 in cancer is to reduce primary tumor growth and metastasis via nutrient deprivation due to inhibition of tumor angiogenesis. Several αvβ3 antagonists have gone to clinical trials with the most notable being cilengitide. Cilengitide is a cyclic peptide containing the RGD integrin-binding motif. It inhibits both αvβ3 and αvβ5. Cilengitide produces both anti-angiogenic and anti-tumor effects through inhibition of VEGF stimulation and FAK-Src and Erk signaling, respectively [105]. *In vitro*, cilengitide reduces cell growth and survival and inhibits endothelial and tumor cell migration. In clinical trials, cilengitide has been evaluated as a single agent and in combination with radiation, DNAalkylating agents and gemcitibine. Importantly, cilengitide in combination with radiotherapy and temozolomide (a DNA-alkylating agent) has reached phase III trials in glioblastoma multiforme patients. Other small molecule antagonists are in development for noncancer indications.

#### **6.2. Integrin α4**

The integrin α4 subunit is predominantly expressed in lymphocytes and leukocytes and supports endothelial transmigration of these cells via binding to VCAM-1. Consequently, α4 is important for immune function and has been targeted in diseases such as multiple sclerosis (MS), Crohn's disease and asthma that are characterized by excessive inflammation or an improper immune response. Natalizumab, the only FDA approved α4 antagonist, is a humanized mouse monoclonal antibody that binds both α4 heterodimers. The use of natali‐ zumab was successful in clinical trials in MS [106, 107] and Crohn's disease [108] with the exception of rare cases of progressive multi-focal leukoencephalopathy (PML) caused by reactivation of latent JC virus associated with immunosuppression [109]. Unfortunately, this side effect was detrimental enough to lead to limitation of the use of natalizumab to patients who are unresponsive to other treatments. Other α4 antagonists under clinical evaluation include MLN-00002 (human α4β7 antibody), firategrast and IVL745 (small molecules: [104]). Though the rationale for the use of most α4 antagonists is to reduce excessive infiltration of immune cells, these therapies have the potential for use against cancer cells that exploit α4 for tumor cell extravasation. The success of targeting α4 in cancer will depend on the ability to minimize immunosuppression or to indirectly impair α4 function via downstream targets.

#### **6.3. Integrin αIIbβ3**

Integrin αIIbβ3 is also a frequently targeted integrin. This heterodimer is expressed selectively on platelets and megakaryocytes and is mostly known for its role in blood coagulation. Antagonists of this receptor are primarily employed in diseases such as stroke, sickle cell anemia and acute coronary syndromes [104].

#### **7. Summary and considerations**

Integrins are a unique group of receptors that provide anchorage, mediate cell migration and invasion, and signal via cell survival and proliferation pathways. Aptly named, integrins integrate extracellular cues with intracellular signaling and serve to regulate many cellular processes that are mediated by other receptors, such as receptor tyrosine kinases. The importance of integrins in cancer development of the nervous system is well establish‐ ed; it seems inevitable therefore that they play a major role in neuroblastoma progres‐ sion. In fact, integrin expression has been linked to malignancy in neuroblastoma, possibly due to alterations in invasiveness and the ability to evade cell death in foreign tissue environments. Aggressive disease may modulate integrin expression (i.e. N-Myc).

[2] Ingber D. Integrins as mechanochemical transducers. Curr Opin Cell Biol. 1991 Oct;

Neuroblastoma Integrins http://dx.doi.org/10.5772/55991 207

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Targeting integrins has shown great clinical promise. By inhibiting ligand binding, many antagonists successfully disrupt cellular connections to the extracellular environment and prosurvival pathways that are necessary for tumor progression. As we continue to learn more about the downstream signaling activity of integrin receptors, we can also explore more therapeutic avenues against these targets, attacking the problem from both sides. However, the logical use of integrin antagonists in complex, multi-agent regimens is lacking. Given the synergy of integrins with signaling through receptor tyrosine kinases and in the induction of susceptibility to apoptosis, this is where one would suspect that these relatively non-toxic agents would have their greatest impact.

Though clinical studies of integrin-targeted drugs in neuroblastoma have not been performed, *in vitro* antagonism has been shown to decrease cell survival, migration and invasion. Despite these characteristics, integrin-targeted drugs are well tolerated. Given that current treatment for neuroblastoma still has a significant failure rate, the addition of new, low toxicity adjuncts to current treatment regimens seems a logical step forward. In the future, an increased understanding of the roles of specific integrins in neuroblastoma has the potential to provide better prognostic information regarding disease course, while targeting integrins, perhaps in combination with other targeted therapies as a cocktail addition to standard chemotherapy approaches, may lead to increased effectiveness in managing this disease.

#### **Author details**

Shanique A. Young, Ryon Graf and Dwayne G. Stupack\*

\*Address all correspondence to: dstupack@ucsd.edu

Reproductive Medicine Department, UCSD Moores Cancer Center, La Jolla, California, USA

#### **References**

[1] Hynes RO. The extracellular matrix: not just pretty fibrils. Science. 2009;326(5957): 1216-9. PubMed PMID: 19965464. eng.

[2] Ingber D. Integrins as mechanochemical transducers. Curr Opin Cell Biol. 1991 Oct; 3(5):841-8. PubMed PMID: 1931084. eng.

integrins integrate extracellular cues with intracellular signaling and serve to regulate many cellular processes that are mediated by other receptors, such as receptor tyrosine kinases. The importance of integrins in cancer development of the nervous system is well establish‐ ed; it seems inevitable therefore that they play a major role in neuroblastoma progres‐ sion. In fact, integrin expression has been linked to malignancy in neuroblastoma, possibly due to alterations in invasiveness and the ability to evade cell death in foreign tissue

Targeting integrins has shown great clinical promise. By inhibiting ligand binding, many antagonists successfully disrupt cellular connections to the extracellular environment and prosurvival pathways that are necessary for tumor progression. As we continue to learn more about the downstream signaling activity of integrin receptors, we can also explore more therapeutic avenues against these targets, attacking the problem from both sides. However, the logical use of integrin antagonists in complex, multi-agent regimens is lacking. Given the synergy of integrins with signaling through receptor tyrosine kinases and in the induction of susceptibility to apoptosis, this is where one would suspect that these relatively non-toxic

Though clinical studies of integrin-targeted drugs in neuroblastoma have not been performed, *in vitro* antagonism has been shown to decrease cell survival, migration and invasion. Despite these characteristics, integrin-targeted drugs are well tolerated. Given that current treatment for neuroblastoma still has a significant failure rate, the addition of new, low toxicity adjuncts to current treatment regimens seems a logical step forward. In the future, an increased understanding of the roles of specific integrins in neuroblastoma has the potential to provide better prognostic information regarding disease course, while targeting integrins, perhaps in combination with other targeted therapies as a cocktail addition to standard chemotherapy

Reproductive Medicine Department, UCSD Moores Cancer Center, La Jolla, California, USA

[1] Hynes RO. The extracellular matrix: not just pretty fibrils. Science. 2009;326(5957):

approaches, may lead to increased effectiveness in managing this disease.

Shanique A. Young, Ryon Graf and Dwayne G. Stupack\*

\*Address all correspondence to: dstupack@ucsd.edu

1216-9. PubMed PMID: 19965464. eng.

environments. Aggressive disease may modulate integrin expression (i.e. N-Myc).

agents would have their greatest impact.

**Author details**

206 Neuroblastoma

**References**


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[106] Dalton CM, Miszkiel KA, Barker GJ, MacManus DG, Pepple TI, Panzara M, et al. Ef‐ fect of natalizumab on conversion of gadolinium enhancing lesions to T1 hypoin‐ tense lesions in relapsing multiple sclerosis. J Neurol. 2004 Apr;251(4):407-13.

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[87] Judware R, Culp LA. Concomitant down-regulation of expression of integrin subu‐ nits by N-myc in human neuroblastoma cells: differential regulation of alpha2, al‐ pha3 and beta1. Oncogene. 1997 Mar;14(11):1341-50. PubMed PMID: 9178894. eng.

[88] Judware R, Culp LA. N-myc over-expression downregulates alpha3beta1 integrin ex‐ pression in human Saos-2 osteosarcoma cells. Clin Exp Metastasis. 1997 May;15(3):

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[91] Teitz T, Lahti JM, Kidd VJ. Aggressive childhood neuroblastomas do not express cas‐ pase-8: an important component of programmed cell death. J Mol Med (Berl). 2001

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[93] Teitz T, Wei T, Valentine MB, Vanin EF, Grenet J, Valentine VA, et al. Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of

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214 Neuroblastoma

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Multiple Sclerosis (AFFIRM) study. Lancet Neurol. 2009 Mar;8(3):254-60. PubMed PMID: 19201654. eng.

**Section 4**

**Neuroblastoma, Biology - 3**


**Section 4**

**Neuroblastoma, Biology - 3**

Multiple Sclerosis (AFFIRM) study. Lancet Neurol. 2009 Mar;8(3):254-60. PubMed

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PMID: 19201654. eng.

216 Neuroblastoma

**Chapter 10**

**Novel Therapeutic Approaches for Neuroblastoma**

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor of childhood, and 45% of patients have high-risk tumors, nearly all of which are metastatic (stage 4) when diagnosed [1]. Patients with neuroblastoma are risk stratified based on presenting factors including age, stage and location of disease, and specific biologic molecular markers of the tumor, including NMYC status and ploidy [1-3]. Treatment given is tailored to whether a patient has low, intermediate or high-risk disease. The overall prognosis for those with high risk or relapsed disease remains poor despite the standard therapies of surgery, radiation, and high dose chemotherapy followed by stem cell rescue. Additionally, many patients who survive suffer from complications related to their treatment. In this chapter, we review the literature that provides a rationale for the use of novel targeted agents to improve the treatment and survival while lessening toxicity of patients with neuroblastoma who have failed standard

In particular, we focus our discussion on a few specific signaling pathways. The central role of the phosphatidylinositol 3-kinase-Akt-phosphatase and tensin homolog (PI3K-Akt-PTEN) axis and RAF-MEK-ERK as potential molecular targets to control downstream effectors of coordinated cell division, tumor growth, angiogenesis, apoptosis, invasion and cellular metabolism in the tumor and surrounding stromal compartments. The PI3K and RAF-MEK-ERK pathways have also been implicated in modulating p53, the hypoxia-inducible factor 1

NMYC is known to play a role in the tumorigenesis of certain high-risk neuroblastoma tumors and its control has many implications in targeting therapy. Additional pathways and targets explored in this chapter are the RAS/Raf/MEK/ERK pathway, specific angiogenesis inhibitors including VEGF, ALK 1 mutations and inhibitors, and control of apoptosis through caspase 8.

and reproduction in any medium, provided the original work is properly cited.

© 2013 Joshi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

Shweta Joshi, Alok R. Singh, Lisa L.R. Hartman,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55451

**1. Introduction**

therapies.

(HIF1α), mycN and others.

Muamera Zulcic, Hyunah Ahn and Donald L. Durden

## **Novel Therapeutic Approaches for Neuroblastoma**

Shweta Joshi, Alok R. Singh, Lisa L.R. Hartman, Muamera Zulcic, Hyunah Ahn and Donald L. Durden

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55451

#### **1. Introduction**

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor of childhood, and 45% of patients have high-risk tumors, nearly all of which are metastatic (stage 4) when diagnosed [1]. Patients with neuroblastoma are risk stratified based on presenting factors including age, stage and location of disease, and specific biologic molecular markers of the tumor, including NMYC status and ploidy [1-3]. Treatment given is tailored to whether a patient has low, intermediate or high-risk disease. The overall prognosis for those with high risk or relapsed disease remains poor despite the standard therapies of surgery, radiation, and high dose chemotherapy followed by stem cell rescue. Additionally, many patients who survive suffer from complications related to their treatment. In this chapter, we review the literature that provides a rationale for the use of novel targeted agents to improve the treatment and survival while lessening toxicity of patients with neuroblastoma who have failed standard therapies.

In particular, we focus our discussion on a few specific signaling pathways. The central role of the phosphatidylinositol 3-kinase-Akt-phosphatase and tensin homolog (PI3K-Akt-PTEN) axis and RAF-MEK-ERK as potential molecular targets to control downstream effectors of coordinated cell division, tumor growth, angiogenesis, apoptosis, invasion and cellular metabolism in the tumor and surrounding stromal compartments. The PI3K and RAF-MEK-ERK pathways have also been implicated in modulating p53, the hypoxia-inducible factor 1 (HIF1α), mycN and others.

NMYC is known to play a role in the tumorigenesis of certain high-risk neuroblastoma tumors and its control has many implications in targeting therapy. Additional pathways and targets explored in this chapter are the RAS/Raf/MEK/ERK pathway, specific angiogenesis inhibitors including VEGF, ALK 1 mutations and inhibitors, and control of apoptosis through caspase 8.

© 2013 Joshi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

We also discuss the idea of synthetic lethality and the concepts of sequential versus simulta‐ neous inhibition. We will discuss the emerging importance of genomic and metabolomic profiling in tumor interrogation with therapeutic considerations.

We will review the literature supporting a role for cancer stem cells (CSCs) in the pathogenesis of neuroblastoma and the signaling pathways that define the CSC phenotype. We discuss the role targeted therapies in CSC related therapeutics and the adaptive responses that such cells have when exposed to targeted therapeutic agents.

Lastly, the emerging role of immunotherapeutics into both standard and targeted therapies for neuroblastoma is explored. This includes areas of T cell and macrophage infiltration of tumors, interleukin and cytokine involvement, and anti-GD2 human and mouse monoclonal antibodies.

## **2. PTEN and PI-3 kinase and mycN signaling as targets for NB therapeutics — The intercept node hypothesis**

The idea that some signaling pathways are more central to tumorigenesis than others was suggested by our laboratory and others [4]. From connectivity map analysis, some signaling proteins appear connected to a large number of upstream and downstream effector pathways. These are considered central "intercept nodes" [4, 5] which provide coordinate control over the output of a large number of cell surface receptor input. The specificity of signaling downstream of such intercept nodes is generally fine tuned by more specialized signaling effector proteins e.g. Rac2, HIF1α, NFκB or mycN which encode more specific signaling content. Two such central pathways, PTEN-PI-3-AKT and Raf-MEK-ERK are critical for NB survival, proliferation, invasion and angiogenesis *in vivo* [6-9]. A large number of small and large pharmaceutical companies have developed small molecule inhibitors which block these two pathways. Considering the importance of mycN amplification in the pathogenesis of NB, and the role of PI-3K and MAP kinase in the GSK3β dependent regulation of mycN a number of investigators have determined the efficacy of PI-3 kinase inhibitors in NB models [9]. Despite evidence of efficacy no PI-3 kinase inhibitors have entered pediatric oncology clinical trials to date. One pan PI-3 kinase inhibitor, SF1126 is slated to enter pediatric oncology Phase I clinical trials in early 2013 [10]. Importantly, the tumor and stromal compartment share many of the same signaling pathways to regulate the process of tumorigenesis *in vivo*.

to regulate this response [11]. We and others have shown that PTEN a major tumor suppressor protein regulates angiogenesis and loss of PTEN results in deregulation of PTEN and multiple downstream signaling pathways shown in Fig. 1 and 2 which have all been implicated in the

**Figure 1. The PI3K–Akt–PTEN intercept node.** As shown, a large number of growth factor receptors (GFR) of which TrkB is an oncogene in NB would feed into the central node to activate PI-3 kinase, AKT and/or Raf-MEK-ERK path‐ ways. Downstream subnodes encode specificity e.g. GSK3b, MDM2, mycN, Rac2, etc. Major tumor suppressors like PTEN and p53 control output from these two central nodes. MDM2 regulates p53 in an AKT dependent manner; RAF-

Novel Therapeutic Approaches for Neuroblastoma

http://dx.doi.org/10.5772/55451

221

In general, angiogenesis plays an important role in the progression and metastasis of malignant tumors [14]. In neuroblastoma, tumor vascularity is correlated with an aggressive phenotype [15, 16]. Pro-angiogenic factors are differentially expressed in high-risk neuroblastoma [17, 18]. Vascular endothelial growth factor (VEGF) is a specific endothelial cell mitogen that stimulates angiogenesis and plays a crucial role in tumor growth [19]. Over expression of VEGF has been demonstrated in neuroblastoma, nephroblastoma, as well as in various other cancers [20-22]. Recent studies have validated inhibition of VEGF as an effective antiangiogenic therapy in some of these cancers [23-25]. Although several preliminary studies have demon‐ strated that expression of angiogenic growth factors, including VEGF, correlate with a highrisk phenotype in neuroblastoma, clinical data are still insufficient to draw conclusions [17, 21, 26, 27]. Therefore, further clinical studies, are needed to evaluate the possible significance of these factors for use in a routine clinical practice. Preclinical studies also suggest that antian‐ giogenic strategies may be effective in the treatment of neuroblastoma [28]. In addition, phase I clinical trials (COG study) using the human anti-VEGF antibody, bevacizumab, in pediatric patients with refractory solid tumors reported promising results [29]. Recently, Jakovljevic *et al.* has determined VEGF expression by immunohistochemistry using antiVEGF antibody in

literature to exert coordinate control of angiogenesis *in vivo* [4, 5, 12, 13].

MEK-ERK and AKT regulate GSK3b to control mycN stabilitity and transcriptional activity.

## **3. Role of angiogenesis in tumorigenicity of neuroblastoma / PI3 kinase and VEGF inhibitors in treatment of neuroblastoma**

Work from a number of laboratories indicates that the angiogenic response is coordinately and highly regulated physiologic response to hypoxia and inflammation. Hence it is not surprising to learn that central node in mammalian cells control output from many cell surface receptors

We also discuss the idea of synthetic lethality and the concepts of sequential versus simulta‐ neous inhibition. We will discuss the emerging importance of genomic and metabolomic

We will review the literature supporting a role for cancer stem cells (CSCs) in the pathogenesis of neuroblastoma and the signaling pathways that define the CSC phenotype. We discuss the role targeted therapies in CSC related therapeutics and the adaptive responses that such cells

Lastly, the emerging role of immunotherapeutics into both standard and targeted therapies for neuroblastoma is explored. This includes areas of T cell and macrophage infiltration of tumors, interleukin and cytokine involvement, and anti-GD2 human and mouse monoclonal

**2. PTEN and PI-3 kinase and mycN signaling as targets for NB therapeutics**

The idea that some signaling pathways are more central to tumorigenesis than others was suggested by our laboratory and others [4]. From connectivity map analysis, some signaling proteins appear connected to a large number of upstream and downstream effector pathways. These are considered central "intercept nodes" [4, 5] which provide coordinate control over the output of a large number of cell surface receptor input. The specificity of signaling downstream of such intercept nodes is generally fine tuned by more specialized signaling effector proteins e.g. Rac2, HIF1α, NFκB or mycN which encode more specific signaling content. Two such central pathways, PTEN-PI-3-AKT and Raf-MEK-ERK are critical for NB survival, proliferation, invasion and angiogenesis *in vivo* [6-9]. A large number of small and large pharmaceutical companies have developed small molecule inhibitors which block these two pathways. Considering the importance of mycN amplification in the pathogenesis of NB, and the role of PI-3K and MAP kinase in the GSK3β dependent regulation of mycN a number of investigators have determined the efficacy of PI-3 kinase inhibitors in NB models [9]. Despite evidence of efficacy no PI-3 kinase inhibitors have entered pediatric oncology clinical trials to date. One pan PI-3 kinase inhibitor, SF1126 is slated to enter pediatric oncology Phase I clinical trials in early 2013 [10]. Importantly, the tumor and stromal compartment share many of the

same signaling pathways to regulate the process of tumorigenesis *in vivo*.

**and VEGF inhibitors in treatment of neuroblastoma**

**3. Role of angiogenesis in tumorigenicity of neuroblastoma / PI3 kinase**

Work from a number of laboratories indicates that the angiogenic response is coordinately and highly regulated physiologic response to hypoxia and inflammation. Hence it is not surprising to learn that central node in mammalian cells control output from many cell surface receptors

profiling in tumor interrogation with therapeutic considerations.

have when exposed to targeted therapeutic agents.

**— The intercept node hypothesis**

antibodies.

220 Neuroblastoma

**Figure 1. The PI3K–Akt–PTEN intercept node.** As shown, a large number of growth factor receptors (GFR) of which TrkB is an oncogene in NB would feed into the central node to activate PI-3 kinase, AKT and/or Raf-MEK-ERK path‐ ways. Downstream subnodes encode specificity e.g. GSK3b, MDM2, mycN, Rac2, etc. Major tumor suppressors like PTEN and p53 control output from these two central nodes. MDM2 regulates p53 in an AKT dependent manner; RAF-MEK-ERK and AKT regulate GSK3b to control mycN stabilitity and transcriptional activity.

to regulate this response [11]. We and others have shown that PTEN a major tumor suppressor protein regulates angiogenesis and loss of PTEN results in deregulation of PTEN and multiple downstream signaling pathways shown in Fig. 1 and 2 which have all been implicated in the literature to exert coordinate control of angiogenesis *in vivo* [4, 5, 12, 13].

In general, angiogenesis plays an important role in the progression and metastasis of malignant tumors [14]. In neuroblastoma, tumor vascularity is correlated with an aggressive phenotype [15, 16]. Pro-angiogenic factors are differentially expressed in high-risk neuroblastoma [17, 18]. Vascular endothelial growth factor (VEGF) is a specific endothelial cell mitogen that stimulates angiogenesis and plays a crucial role in tumor growth [19]. Over expression of VEGF has been demonstrated in neuroblastoma, nephroblastoma, as well as in various other cancers [20-22]. Recent studies have validated inhibition of VEGF as an effective antiangiogenic therapy in some of these cancers [23-25]. Although several preliminary studies have demon‐ strated that expression of angiogenic growth factors, including VEGF, correlate with a highrisk phenotype in neuroblastoma, clinical data are still insufficient to draw conclusions [17, 21, 26, 27]. Therefore, further clinical studies, are needed to evaluate the possible significance of these factors for use in a routine clinical practice. Preclinical studies also suggest that antian‐ giogenic strategies may be effective in the treatment of neuroblastoma [28]. In addition, phase I clinical trials (COG study) using the human anti-VEGF antibody, bevacizumab, in pediatric patients with refractory solid tumors reported promising results [29]. Recently, Jakovljevic *et al.* has determined VEGF expression by immunohistochemistry using antiVEGF antibody in paraffin embedded primary tumor tissue from 56 neuroblastoma patients and reported that VEGF expression correlated with disease stage and survival in neuroblastoma patients [30]. Whether inhibition of angiogenesis is a realistic approach for preventing dissemination of neuroblastoma remains to be determined, but we can suggest that inhibitors of VEGF can be used in the treatment of neuroblastoma. Finally, we suggest that the more global inhibition of PI3 kinase or combined PI3K/MEK inhibition would provide a more potent antiangiogenic modality to block tumor induced angiogenesis in this disease.

#### **4. Cancer stem cells in neuroblastoma tumorigenicity**

The Cancer Stem Cell Theory postulates that tumors contain a subset of cells that are capable of increased self-renewal and differentiation, can propagate tumor growth and are resistant to apoptosis [31, 32]. These stem-like cancer cells are analogous to normal stem cells [33] but differentiate into diverse cancer cells that form the major portion of the tumor. Recent evidence suggests the presence of stem cells in various cancers including those of the blood [34], breast [35], prostrate [36] and brain [37].

Evidence for the presence of cancer stem cells in brain tumors first came from the observation that human medulloblastoma, astrocytomas, and ependymomas contain cells that express the neural stem cell marker CD133 [38] [39]. Singh et al. [37] have shown that human brain tumors contain CD133+ stem-like cells that are capable of growing tumors in immune-deficient mice. Cournoyer et al.[40] have shown that CD133 high neuroblastoma (NB) cells have high tumor initiating cell properties, and Coulon et al.[41] suggest that CD133, ABC transporter, Wnt and NOTCH genes are sphere markers in NB cells. Overall, 19–29 % of cells in glioblastomas and 6–21 % of cells of medulloblastomas are reported to be CD133+ and tumorigenic [33]. Recently, several groups have suggested that CD15 (stage specific embryonic antigen 1 or SSEA-1), which is expressed on neural progenitor and stem cells, may be a better marker than CD133 of tumor-initiating cells in MB, glioma, and ependymoma [42-44]. Hansford et al has recently identified tumor initiating cells from NB bone marrow metastases that have several properties of cancer stem cells including the expression of stem cell markers, the ability to self renew and the capability to form metastatic NB in immunodeficient animals with as few as 10 cells [45]. Kaplan's laboratory has further defined the NB tumor initiating cell (TIC) with stem cell like properties to express, CD133 and CD44. These cells isolated from NB bone marrow have tumor initiating activity and upon profiling display sensitivity to a number of targeted therapeutic agents.

A key aspect of the tumor stem cell (TSC) niche is the balance of signals received, and over recent years considerable attention has been directed towards understanding the role of signaling pathways, which are critical mediators of normal stem cell biology, in cancers. The embryonic signaling pathways most commonly implicated in tumorigenesis include Hedge‐ hog, Notch, and Wnt pathways. Sonic Hedgehog (SHH) signaling is important in embryonic cell development and proliferation and aberrant pathway activation can lead to tumor formation, tumor cell self-renewal and the development of metastatic disease [48].Similarly,

Notch plays a crucial role in biological functions of development and cell fate including cell differentiation and proliferation [49]. Constitutive activation of Notch can lead to tumorigen‐ esis and cell survival, and Notch activity is involved in tumor angiogenesis [50]. The Wnt

[47].

**Figure 2. Signaling and cellular pathways controlling tumorigenicity of Neuroblastoma.** In tumor compartment PI3K–Akt–PTEN intercept node is a central regulator of survival, proliferation, invasion and angiogenesis in Neuroblas‐ toma. PI3K controls PIP3 levels, thereby regulating lipid-associated second messenger output from upstream effectors. PI3K and Akt can be activated by many cell surface receptors. Akt becomes locked in an active conformation and phosphorylates numerous proteins involved in growth and survival, cellular metabolism, stress response and angio‐ genesis. Akt modulates phosphorylation of GSK3β and relieves tonic inhibition of c-Myc and cyclin D to promote cell survival [46]. Akt contributes to the Warburg effect by inducing HIF1α transcription and stimulating aerobic glycolysis. Intratumoral hypoxia also drives angiogenesis through transcription of proangiogenic genes including *VEGF* and *PDGF*. Tumor angiogenesis is promoted by Akt-mediated phosphorylation of MDM2. Activated MDM2 translocates from the cytoplasm to the nucleus, where it binds p53, targeting it for ubiquitination and degradation. This process prevents p53 from exerting its antiangiogenic effect. A more effective strategy might be to modulate tumor growth and angiogenesis by targeting major signaling nodes such as the p53–MDM2 or PI3K–Akt–PTEN nodes with agents such as Nutlin 3A or with PI3K inhibitors (e.g. PI-103, BEZ-235 or SF1126), respectively. Abbreviations: GSK3β, glycogen synthase kinase 3 β; HIF1α, hypoxia inducible factor 1α; MDM2, mammalian double minute 2; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; In stromal compartment, the major cellular pathways of the im‐ mune response which may have anti- or pro-tumor effects are shown. NK cells and CD8+ CTLs may directly target tu‐ mor cells for lysis; however this may be countered by decreased tumor expression of NKG2D ligands or MHC class I. Dendritic cells are important for priming an anti-tumor immune response, although immature DCs and IDO-express‐ ing DCs may instead lead to the induction of tolerance. Myeloid-derived suppressor cells and regulatory T cells (Treg) may also suppress the anti-tumor CTL response. TH1 cells and M1 macrophages produce proinflammatory cytokines which help to stimulate the anti-tumor immune response, whilst TH2 cells (and other cell types) produce IL-10 which may have a predominantly inhibitory effect on the anti-tumor response. Tumor-associated M2 macrophages may pro‐ mote tumor growth and metastases *via* a number of different mechanisms. Figure adopted from Morgenstern *et al*

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223

paraffin embedded primary tumor tissue from 56 neuroblastoma patients and reported that VEGF expression correlated with disease stage and survival in neuroblastoma patients [30]. Whether inhibition of angiogenesis is a realistic approach for preventing dissemination of neuroblastoma remains to be determined, but we can suggest that inhibitors of VEGF can be used in the treatment of neuroblastoma. Finally, we suggest that the more global inhibition of PI3 kinase or combined PI3K/MEK inhibition would provide a more potent antiangiogenic

The Cancer Stem Cell Theory postulates that tumors contain a subset of cells that are capable of increased self-renewal and differentiation, can propagate tumor growth and are resistant to apoptosis [31, 32]. These stem-like cancer cells are analogous to normal stem cells [33] but differentiate into diverse cancer cells that form the major portion of the tumor. Recent evidence suggests the presence of stem cells in various cancers including those of the blood [34], breast

Evidence for the presence of cancer stem cells in brain tumors first came from the observation that human medulloblastoma, astrocytomas, and ependymomas contain cells that express the neural stem cell marker CD133 [38] [39]. Singh et al. [37] have shown that human brain tumors contain CD133+ stem-like cells that are capable of growing tumors in immune-deficient mice. Cournoyer et al.[40] have shown that CD133 high neuroblastoma (NB) cells have high tumor initiating cell properties, and Coulon et al.[41] suggest that CD133, ABC transporter, Wnt and NOTCH genes are sphere markers in NB cells. Overall, 19–29 % of cells in glioblastomas and 6–21 % of cells of medulloblastomas are reported to be CD133+ and tumorigenic [33]. Recently, several groups have suggested that CD15 (stage specific embryonic antigen 1 or SSEA-1), which is expressed on neural progenitor and stem cells, may be a better marker than CD133 of tumor-initiating cells in MB, glioma, and ependymoma [42-44]. Hansford et al has recently identified tumor initiating cells from NB bone marrow metastases that have several properties of cancer stem cells including the expression of stem cell markers, the ability to self renew and the capability to form metastatic NB in immunodeficient animals with as few as 10 cells [45]. Kaplan's laboratory has further defined the NB tumor initiating cell (TIC) with stem cell like properties to express, CD133 and CD44. These cells isolated from NB bone marrow have tumor initiating activity and upon profiling display sensitivity to a number of targeted therapeutic

A key aspect of the tumor stem cell (TSC) niche is the balance of signals received, and over recent years considerable attention has been directed towards understanding the role of signaling pathways, which are critical mediators of normal stem cell biology, in cancers. The embryonic signaling pathways most commonly implicated in tumorigenesis include Hedge‐ hog, Notch, and Wnt pathways. Sonic Hedgehog (SHH) signaling is important in embryonic cell development and proliferation and aberrant pathway activation can lead to tumor formation, tumor cell self-renewal and the development of metastatic disease [48].Similarly,

modality to block tumor induced angiogenesis in this disease.

[35], prostrate [36] and brain [37].

agents.

222 Neuroblastoma

**4. Cancer stem cells in neuroblastoma tumorigenicity**

**Figure 2. Signaling and cellular pathways controlling tumorigenicity of Neuroblastoma.** In tumor compartment PI3K–Akt–PTEN intercept node is a central regulator of survival, proliferation, invasion and angiogenesis in Neuroblas‐ toma. PI3K controls PIP3 levels, thereby regulating lipid-associated second messenger output from upstream effectors. PI3K and Akt can be activated by many cell surface receptors. Akt becomes locked in an active conformation and phosphorylates numerous proteins involved in growth and survival, cellular metabolism, stress response and angio‐ genesis. Akt modulates phosphorylation of GSK3β and relieves tonic inhibition of c-Myc and cyclin D to promote cell survival [46]. Akt contributes to the Warburg effect by inducing HIF1α transcription and stimulating aerobic glycolysis. Intratumoral hypoxia also drives angiogenesis through transcription of proangiogenic genes including *VEGF* and *PDGF*. Tumor angiogenesis is promoted by Akt-mediated phosphorylation of MDM2. Activated MDM2 translocates from the cytoplasm to the nucleus, where it binds p53, targeting it for ubiquitination and degradation. This process prevents p53 from exerting its antiangiogenic effect. A more effective strategy might be to modulate tumor growth and angiogenesis by targeting major signaling nodes such as the p53–MDM2 or PI3K–Akt–PTEN nodes with agents such as Nutlin 3A or with PI3K inhibitors (e.g. PI-103, BEZ-235 or SF1126), respectively. Abbreviations: GSK3β, glycogen synthase kinase 3 β; HIF1α, hypoxia inducible factor 1α; MDM2, mammalian double minute 2; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; In stromal compartment, the major cellular pathways of the im‐ mune response which may have anti- or pro-tumor effects are shown. NK cells and CD8+ CTLs may directly target tu‐ mor cells for lysis; however this may be countered by decreased tumor expression of NKG2D ligands or MHC class I. Dendritic cells are important for priming an anti-tumor immune response, although immature DCs and IDO-express‐ ing DCs may instead lead to the induction of tolerance. Myeloid-derived suppressor cells and regulatory T cells (Treg) may also suppress the anti-tumor CTL response. TH1 cells and M1 macrophages produce proinflammatory cytokines which help to stimulate the anti-tumor immune response, whilst TH2 cells (and other cell types) produce IL-10 which may have a predominantly inhibitory effect on the anti-tumor response. Tumor-associated M2 macrophages may pro‐ mote tumor growth and metastases *via* a number of different mechanisms. Figure adopted from Morgenstern *et al* [47].

Notch plays a crucial role in biological functions of development and cell fate including cell differentiation and proliferation [49]. Constitutive activation of Notch can lead to tumorigen‐ esis and cell survival, and Notch activity is involved in tumor angiogenesis [50]. The Wnt family proteins help direct a wide range of developmental processes including cell fate, proliferation, motility, and polarity [51]. Dysregulation of the Wnt pathways has been implicated in tumor formation, proliferation, and maintenance [52]. All of the current pediatric studies demonstrating that progenitor and stem cells can respond to embryonic signaling have been in MB or primitive neuroectodermal tumors (PNET). Aberrant SHH signaling has been implicated in MB, and recently was used to define one of four distinct molecular variants of MB [53].

activation of two high affinity glutamine transporters: SLC38A5 (also called SN2) and SLC1A5 (ASCT2), the transporter required for glutamine-dependent mTORC1 activation [60, 63]. In addition to facilitating glutamine uptake, Myc promotes the metabolism of imported gluta‐ mine into glutamic acid and ultimately into lactic acid [60]. Whether the tendency of Myc to complement Ras and PI3K-Akt [64, 65] is related to the interdependence of glutamine and glucose metabolism in support of cell growth remains an open question. The work of C. Dang and other points to a potential important metabolic requirement for glutamine in c-myc and mycN driven tumors where glutamine can serve a role in promoting tumor growth [58, 66]. This might suggest a role of agents which deplete glutamine (glutaminases) as a therapeutic target for mycN driven malignancies like neuroblastoma and the SHH subtype of medullo‐

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**6. Role of tumor infiltrating immune cells in tumorigenicity of**

tion is a recognized enabling characteristic of cancers [72].

cells may prove beneficial in its treatment of NB.

**a.** Tumor infiltrating Lymphocytes

Solid tumors are composed of tumor stromal cells, blood vessels, infiltrating immune cells and tumor cells themselves. Over the last decade, a growing body of literature has highlighted the importance of the tumor microenvironment for the prognosis of different types of cancer [68]. The tumor microenvironment contains many resident cell types, such as adipocytes and fibroblasts, but it is also populated by migratory hematopoietic cells, including lymphoid cells, granulocytes, mast cells, dendritic cells, natural killer cells, neutrophils and macrophages. These haematopoietic cells have pivotal roles in the progression and metastasis of tumors [69, 70]. The significance of tumor stroma for the overall prognosis may be in part due to the fact that several components of the tumor-microenvironment have been shown to compromise immune effect functions against tumor cells [71]. The concept of tumor-promoting inflamma‐

The first evidence suggesting immune responses to neuroblastoma was provided in 1968 when blood leukocytes, which were 50–70% lymphocytes, were reported to inhibit colony formation by neuroblastoma cells [73]. These lymphocytes inhibited colony formation by both autologous and allogeneic neuroblastoma cells but did not affect growth of fibroblasts from the same donors. Plasma from these patients also was reported to inhibit tumor cell colony formation in the presence of complement. In this same time, primary tumors were reported to contain leukocytes [74, 75], and some localized and metastatic neuroblastomas were reported to regress spontaneously [76, 77]. Together, these studies suggest that the immune system could develop an anti-neuroblastoma response. In this section, we will highlight the role of tumor infiltrating immune cells in progression of this disease and how blocking the function of these infiltrating

Tumor-associated lymphocyte population includes CD8+ cytotoxic T cells, CD4+ T helper cells, regulatory T cells (Tregs), NKT or γδT cells. Tregs are immunosuppressive regulatory T cells. Tregs are able to suppress the activity of CTLs by direct cell-cell contact and also secrete

blastoma.

**neuroblastoma**

In order to identify pathways required for proliferation and cell survival characteristics of TIC in neuroblastoma, Grinshtein et al. has performed drug screen on bone marrow derived tumor initiating cells (TICs) with a unique collection of pharmacological inhibitors. They identified that PI3K (phosphoinositide 3 kinase)/AKT, PKC (protein kinase C), Aurora, ErbB2, Trk and Polo-like kinase 1 (PLK1) are the potential kinase targets for survival of TIC [54]. Their studies demonstrated that PLK1 inhibitors are an attractive candidate therapy for metastatic NB. Another group suggested that both PI-3 kinase as well as Ras-RAF-MEK-Erk signaling pathways promote the tumorigenicity of the glioblastoma cancer stem like cells, and combined treatment with MEK and PI-3 kinase inhibitors can block the differentiation of glioblastoma cancer stem like cell into non tumor initiating status [55].

The therapeutic resistance of cancer stem cell to current treatment modalities such as chemo‐ therapy and radiation make these cells clinically relevant irrespective of their origin. Resistance to chemotherapeutic agents has been demonstrated in neuroblastoma stem cells and sarcoma stem cells including Ewing's sarcoma and osteosarcoma. Recent work by Hambardzumyan suggests that the PI-3 kinase pathway activity promotes post-radiation survival in cancer stem cells in medulloblastoma [67]. Although lots of literatures are available on the cancer stem cell in neuroblastoma but yet the novel signaling pathways controlling the proliferation and survival of cancer stem cell and the mechanism behind resistance developed due to chemo‐ therapy needs to be investigated.

#### **5. Neuroblastoma and cancer metabolism**

It has been known from a long time that cancer cells take up and metabolize glucose and glutamine to a degree that far exceeds their needs for these molecules in anabolic macromo‐ lecular synthesis [56]. Commonly occurring oncogenic signal transduction pathways initiated by receptor tyrosine kinases or Ras engage PI3K-Akt signaling to directly stimulate glycolytic metabolism under aerobic conditions a condition termed the Warburg and Pasteur effects [56-58]. Myc-activation/amplification is one of the most common oncogenic events observed in cancer and is known to drive the progression of a certain subgroup of neuroblastoma [59]. The activation of mycN could occur through amplification of the mycN gene or through upstream activation of signaling pathways that would stabilize mycN e.g. trkB, IGF-1 or the activation of Raf and/or PI-3K-AKT stimulation. Oncogenic levels of Myc have recently been linked to increased glutaminolysis through a coordinated transcriptional program program [60-62]. Quantitative RTPCR and ChIP experiments support Myc's binding and transcriptional activation of two high affinity glutamine transporters: SLC38A5 (also called SN2) and SLC1A5 (ASCT2), the transporter required for glutamine-dependent mTORC1 activation [60, 63]. In addition to facilitating glutamine uptake, Myc promotes the metabolism of imported gluta‐ mine into glutamic acid and ultimately into lactic acid [60]. Whether the tendency of Myc to complement Ras and PI3K-Akt [64, 65] is related to the interdependence of glutamine and glucose metabolism in support of cell growth remains an open question. The work of C. Dang and other points to a potential important metabolic requirement for glutamine in c-myc and mycN driven tumors where glutamine can serve a role in promoting tumor growth [58, 66]. This might suggest a role of agents which deplete glutamine (glutaminases) as a therapeutic target for mycN driven malignancies like neuroblastoma and the SHH subtype of medullo‐ blastoma.

## **6. Role of tumor infiltrating immune cells in tumorigenicity of neuroblastoma**

Solid tumors are composed of tumor stromal cells, blood vessels, infiltrating immune cells and tumor cells themselves. Over the last decade, a growing body of literature has highlighted the importance of the tumor microenvironment for the prognosis of different types of cancer [68]. The tumor microenvironment contains many resident cell types, such as adipocytes and fibroblasts, but it is also populated by migratory hematopoietic cells, including lymphoid cells, granulocytes, mast cells, dendritic cells, natural killer cells, neutrophils and macrophages. These haematopoietic cells have pivotal roles in the progression and metastasis of tumors [69, 70]. The significance of tumor stroma for the overall prognosis may be in part due to the fact that several components of the tumor-microenvironment have been shown to compromise immune effect functions against tumor cells [71]. The concept of tumor-promoting inflamma‐ tion is a recognized enabling characteristic of cancers [72].

The first evidence suggesting immune responses to neuroblastoma was provided in 1968 when blood leukocytes, which were 50–70% lymphocytes, were reported to inhibit colony formation by neuroblastoma cells [73]. These lymphocytes inhibited colony formation by both autologous and allogeneic neuroblastoma cells but did not affect growth of fibroblasts from the same donors. Plasma from these patients also was reported to inhibit tumor cell colony formation in the presence of complement. In this same time, primary tumors were reported to contain leukocytes [74, 75], and some localized and metastatic neuroblastomas were reported to regress spontaneously [76, 77]. Together, these studies suggest that the immune system could develop an anti-neuroblastoma response. In this section, we will highlight the role of tumor infiltrating immune cells in progression of this disease and how blocking the function of these infiltrating cells may prove beneficial in its treatment of NB.

**a.** Tumor infiltrating Lymphocytes

family proteins help direct a wide range of developmental processes including cell fate, proliferation, motility, and polarity [51]. Dysregulation of the Wnt pathways has been implicated in tumor formation, proliferation, and maintenance [52]. All of the current pediatric studies demonstrating that progenitor and stem cells can respond to embryonic signaling have been in MB or primitive neuroectodermal tumors (PNET). Aberrant SHH signaling has been implicated in MB, and recently was used to define one of four distinct molecular variants of

In order to identify pathways required for proliferation and cell survival characteristics of TIC in neuroblastoma, Grinshtein et al. has performed drug screen on bone marrow derived tumor initiating cells (TICs) with a unique collection of pharmacological inhibitors. They identified that PI3K (phosphoinositide 3 kinase)/AKT, PKC (protein kinase C), Aurora, ErbB2, Trk and Polo-like kinase 1 (PLK1) are the potential kinase targets for survival of TIC [54]. Their studies demonstrated that PLK1 inhibitors are an attractive candidate therapy for metastatic NB. Another group suggested that both PI-3 kinase as well as Ras-RAF-MEK-Erk signaling pathways promote the tumorigenicity of the glioblastoma cancer stem like cells, and combined treatment with MEK and PI-3 kinase inhibitors can block the differentiation of glioblastoma

The therapeutic resistance of cancer stem cell to current treatment modalities such as chemo‐ therapy and radiation make these cells clinically relevant irrespective of their origin. Resistance to chemotherapeutic agents has been demonstrated in neuroblastoma stem cells and sarcoma stem cells including Ewing's sarcoma and osteosarcoma. Recent work by Hambardzumyan suggests that the PI-3 kinase pathway activity promotes post-radiation survival in cancer stem cells in medulloblastoma [67]. Although lots of literatures are available on the cancer stem cell in neuroblastoma but yet the novel signaling pathways controlling the proliferation and survival of cancer stem cell and the mechanism behind resistance developed due to chemo‐

It has been known from a long time that cancer cells take up and metabolize glucose and glutamine to a degree that far exceeds their needs for these molecules in anabolic macromo‐ lecular synthesis [56]. Commonly occurring oncogenic signal transduction pathways initiated by receptor tyrosine kinases or Ras engage PI3K-Akt signaling to directly stimulate glycolytic metabolism under aerobic conditions a condition termed the Warburg and Pasteur effects [56-58]. Myc-activation/amplification is one of the most common oncogenic events observed in cancer and is known to drive the progression of a certain subgroup of neuroblastoma [59]. The activation of mycN could occur through amplification of the mycN gene or through upstream activation of signaling pathways that would stabilize mycN e.g. trkB, IGF-1 or the activation of Raf and/or PI-3K-AKT stimulation. Oncogenic levels of Myc have recently been linked to increased glutaminolysis through a coordinated transcriptional program program [60-62]. Quantitative RTPCR and ChIP experiments support Myc's binding and transcriptional

cancer stem like cell into non tumor initiating status [55].

**5. Neuroblastoma and cancer metabolism**

therapy needs to be investigated.

MB [53].

224 Neuroblastoma

Tumor-associated lymphocyte population includes CD8+ cytotoxic T cells, CD4+ T helper cells, regulatory T cells (Tregs), NKT or γδT cells. Tregs are immunosuppressive regulatory T cells. Tregs are able to suppress the activity of CTLs by direct cell-cell contact and also secrete immunoregulatory cytokines such as transforming growth factor β (TGF-β) and interleukin-10 (IL-10). However, the role of Tregs is much less clear and to our knowledge there are no published data on the presence (or otherwise) of Tregs in pediatric tumors.

innate and adaptive immune responses to infection and malignancy [92]. Two main subtypes of NKT cell are recognised, with Type I NKT cells expressing an invariant α-TCR chain and being implicated in antitumor immunity, whilst Type II NKT cells express a variety of TCR molecules (in addition to CD1d) and appear to have a more immune inhibitory role [91]. The presence of these immune effector cells within tumors has been examined in a number of different malignancies, including, neuroblastoma. Type I NKT cells were found in 53% of 98 untreated primary stage 4 neuroblastoma samples [93] and their infiltration correlated with favorable outcome, with expression of the chemokine CCL2 and with absence of MYCN amplification (indicating less aggressive disease). Subsequent investigations have confirmed that expression of CCL2 is repressed in MYCN amplified tumors, leading to a failure of NKT

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Recent studies have suggested anti-tumor role of NK cells in high risk neuroblastoma NK cells are activated to be cytotoxic and secrete IFNγ by IL-2. IL-2 alone has been tested in phase I and II trials for patients with neuroblastoma, and, although immune effects were documented, no objective tumor responses were observed [95, 96]. Lenalidomide is an immune modulating drug that activates T cells to secrete IL-2, which in turn activates NK cell cytotoxicity and ADCC [97, 98]. Clinical trials in children and adults demonstrated increased numbers of NK cells and cytotoxicity, decreased T regulatory cells, and increased secretion of IL-2, IL-15, and GM-CSF after 21 days of lenalidomide treatment [99, 100]. Thus, lenalidomide may be useful for

Macrophages represent a further important cellular component of the tumor stroma. Far from being mere bystanders to tumor development, there is increasing evidence that tumorassociated macrophages (TAMs) promote and facilitate tumor growth [101, 102]. Of key importance is the concept of distinct macrophage phenotypes, mirroring the dichotomy between TH1 and TH2 T helper cells and type I and type II immune responses. Alternatively activated M2-macrophages are involved in polarized Th2 inflammatory reactions and characterized by expression of arginase-1 and mannose and scavenger receptors [103, 104]. On the other extreme, classically activated M1 macrophages are IL-12 high, IL-23 high, IL-10 low; produce high levels of inducible nitric oxide synthetase (iNOS); secrete inflammatory cyto‐ kines such as IL-1β, IL-6, and TNF; and are inducer and effector cells in Th1 type inflammatory responses [105]. It has been suggested that tumor-associated macrophages (TAMs) display an

TAMs are recruited to tumors when stimulated by growth factors and chemokines, produced by the tumor cells [107, 108]. The conventional wisdom about TAM function is that they are recruited to reject the tumor, which has been recognized as foreign because tumors express unique antigens. However, there is a growing body of evidence that the tumor microenviron‐ ment is immunosuppressive [109], perhaps as a result of selection for such an environment a process recently termed 'immunoediting. Recent data indicate that TGF-β1 has an important role in suppressing these local responses and that inhibiting this molecule can result in tumor rejection [110, 111]. It is noteworthy that TAMs can both produce TGF-β1 and process latent

cell infiltration and potentially contributing to tumor immune escape [94].

activating NK cells to enhance mAb immunotherapy of neuroblastoma.

**c.** Role of tumor associated macrophages

M2-like phenotype [106].

CD8+ cytotoxic T lymphocytes (CTL) are a primary source of anti-tumor activity in the immune system [1, 3]. In many adult cancers the presence of significant numbers of tumor-infiltrating lymphocytes, potentially represents the host immune response against the tumor and is associated with improved prognosis [78-80]. In neuroblastoma, Martin *et al.* [81] suggested a correlation between lymphocyte infiltration and improved survival, although these data are confounded by tumor grade since lymphocytic infiltrates were seen more frequently in low grade, differentiating tumors. In a separate examination of 26 high-risk neuroblastoma tumor samples, there was minimal or undetectable infiltration of CD8+ or CD4+ T cells, CD20+ B cells or CD56+ NK cells within tumor nests [82], although in most patients CD8+ or CD4+ lympho‐ cytes were present within the peritumoral stroma. Interestingly, the majority of patients had evidence of small numbers of circulating cytotoxic T cells against the tumor antigen survivin (expressed by all of the tumors in this study) and these CTLs were highly functional in *in vitro* assays [82]. The experiments conducted by another group in NXS2 murine neuroblastoma model have shown that oral vaccination with a survivin DNA minigene was associated with increased target cell lysis, increased presence of CD8(+) T-cells at the primary tumor site, and enhanced production of pro-inflammatory cytokines [83]. Another pre-clinical study have demonstrated that tyrosine hydroxylase and MYCN proteins, which are relatively specific for neuroblastoma cells compared to normal cells, include peptides that can be targets for CTL. Vaccination of mice with tyrosine hydroxylase DNA minigenes can induce CTLs, eradicate established primary NXS2 neuroblastoma tumors, and inhibit spontaneous metastases without induction of autoimmunity [84, 85].

However, despite these cellular responses to NB, the presence of tumor-infiltrating CTL is rare, suggesting a block in T cell trafficking that may protect the tumor from CTL-mediated cytotoxicity. Therefore, strategies aiming to generate CTLs must take into account mechanisms by which neuroblastoma cells may avoid immune elimination. These include decreased expression of peptide presenting HLA class I molecules by tumor cells, which can impair target peptide recognition by CTLs [82, 86, 87]. Also, neuroblastoma cells express low levels of antigen processing genes, including LMP-2, LMP-7, and TAP-1, which are necessary for preparation of peptides from proteins for presentation by HLA class I molecules to CTLs [88, 89]. Neuroblastoma cells also induce monocytes to release HLA-G, which suppresses both CTL and NK mediated cytotoxicity by interacting with inhibitory receptors or inducing apoptosis via CD8 ligation or the Fas-FasL pathway [90]. Thus, effective CTL anti-tumor responses require that these escape mechanisms be evaluated and, if present, be overcome.

#### **b.** Natural Killer Cells

Natural Killer (NK) cells represent a particular subset of T lymphocytes, which express both T cell markers, such as the αβ T-cell receptor (TCR) and associated CD3 complex, and NK cell markers, such as NK1.1[91]. These cells recognize glycolipids presented by the MHC class Ilike molecule CD1d and are believed to play an important role at the interface between the innate and adaptive immune responses to infection and malignancy [92]. Two main subtypes of NKT cell are recognised, with Type I NKT cells expressing an invariant α-TCR chain and being implicated in antitumor immunity, whilst Type II NKT cells express a variety of TCR molecules (in addition to CD1d) and appear to have a more immune inhibitory role [91]. The presence of these immune effector cells within tumors has been examined in a number of different malignancies, including, neuroblastoma. Type I NKT cells were found in 53% of 98 untreated primary stage 4 neuroblastoma samples [93] and their infiltration correlated with favorable outcome, with expression of the chemokine CCL2 and with absence of MYCN amplification (indicating less aggressive disease). Subsequent investigations have confirmed that expression of CCL2 is repressed in MYCN amplified tumors, leading to a failure of NKT cell infiltration and potentially contributing to tumor immune escape [94].

Recent studies have suggested anti-tumor role of NK cells in high risk neuroblastoma NK cells are activated to be cytotoxic and secrete IFNγ by IL-2. IL-2 alone has been tested in phase I and II trials for patients with neuroblastoma, and, although immune effects were documented, no objective tumor responses were observed [95, 96]. Lenalidomide is an immune modulating drug that activates T cells to secrete IL-2, which in turn activates NK cell cytotoxicity and ADCC [97, 98]. Clinical trials in children and adults demonstrated increased numbers of NK cells and cytotoxicity, decreased T regulatory cells, and increased secretion of IL-2, IL-15, and GM-CSF after 21 days of lenalidomide treatment [99, 100]. Thus, lenalidomide may be useful for activating NK cells to enhance mAb immunotherapy of neuroblastoma.

**c.** Role of tumor associated macrophages

immunoregulatory cytokines such as transforming growth factor β (TGF-β) and interleukin-10 (IL-10). However, the role of Tregs is much less clear and to our knowledge there are no

However, despite these cellular responses to NB, the presence of tumor-infiltrating CTL is rare, suggesting a block in T cell trafficking that may protect the tumor from CTL-mediated cytotoxicity. Therefore, strategies aiming to generate CTLs must take into account mechanisms by which neuroblastoma cells may avoid immune elimination. These include decreased expression of peptide presenting HLA class I molecules by tumor cells, which can impair target peptide recognition by CTLs [82, 86, 87]. Also, neuroblastoma cells express low levels of antigen processing genes, including LMP-2, LMP-7, and TAP-1, which are necessary for preparation of peptides from proteins for presentation by HLA class I molecules to CTLs [88, 89]. Neuroblastoma cells also induce monocytes to release HLA-G, which suppresses both CTL and NK mediated cytotoxicity by interacting with inhibitory receptors or inducing apoptosis via CD8 ligation or the Fas-FasL pathway [90]. Thus, effective CTL anti-tumor responses

require that these escape mechanisms be evaluated and, if present, be overcome.

Natural Killer (NK) cells represent a particular subset of T lymphocytes, which express both T cell markers, such as the αβ T-cell receptor (TCR) and associated CD3 complex, and NK cell markers, such as NK1.1[91]. These cells recognize glycolipids presented by the MHC class Ilike molecule CD1d and are believed to play an important role at the interface between the

 cytotoxic T lymphocytes (CTL) are a primary source of anti-tumor activity in the immune system [1, 3]. In many adult cancers the presence of significant numbers of tumor-infiltrating lymphocytes, potentially represents the host immune response against the tumor and is associated with improved prognosis [78-80]. In neuroblastoma, Martin *et al.* [81] suggested a correlation between lymphocyte infiltration and improved survival, although these data are confounded by tumor grade since lymphocytic infiltrates were seen more frequently in low grade, differentiating tumors. In a separate examination of 26 high-risk neuroblastoma tumor samples, there was minimal or undetectable infiltration of CD8+ or CD4+ T cells, CD20+ B cells or CD56+ NK cells within tumor nests [82], although in most patients CD8+ or CD4+ lympho‐ cytes were present within the peritumoral stroma. Interestingly, the majority of patients had evidence of small numbers of circulating cytotoxic T cells against the tumor antigen survivin (expressed by all of the tumors in this study) and these CTLs were highly functional in *in vitro* assays [82]. The experiments conducted by another group in NXS2 murine neuroblastoma model have shown that oral vaccination with a survivin DNA minigene was associated with increased target cell lysis, increased presence of CD8(+) T-cells at the primary tumor site, and enhanced production of pro-inflammatory cytokines [83]. Another pre-clinical study have demonstrated that tyrosine hydroxylase and MYCN proteins, which are relatively specific for neuroblastoma cells compared to normal cells, include peptides that can be targets for CTL. Vaccination of mice with tyrosine hydroxylase DNA minigenes can induce CTLs, eradicate established primary NXS2 neuroblastoma tumors, and inhibit spontaneous metastases

published data on the presence (or otherwise) of Tregs in pediatric tumors.

without induction of autoimmunity [84, 85].

**b.** Natural Killer Cells

CD8+

226 Neuroblastoma

Macrophages represent a further important cellular component of the tumor stroma. Far from being mere bystanders to tumor development, there is increasing evidence that tumorassociated macrophages (TAMs) promote and facilitate tumor growth [101, 102]. Of key importance is the concept of distinct macrophage phenotypes, mirroring the dichotomy between TH1 and TH2 T helper cells and type I and type II immune responses. Alternatively activated M2-macrophages are involved in polarized Th2 inflammatory reactions and characterized by expression of arginase-1 and mannose and scavenger receptors [103, 104]. On the other extreme, classically activated M1 macrophages are IL-12 high, IL-23 high, IL-10 low; produce high levels of inducible nitric oxide synthetase (iNOS); secrete inflammatory cyto‐ kines such as IL-1β, IL-6, and TNF; and are inducer and effector cells in Th1 type inflammatory responses [105]. It has been suggested that tumor-associated macrophages (TAMs) display an M2-like phenotype [106].

TAMs are recruited to tumors when stimulated by growth factors and chemokines, produced by the tumor cells [107, 108]. The conventional wisdom about TAM function is that they are recruited to reject the tumor, which has been recognized as foreign because tumors express unique antigens. However, there is a growing body of evidence that the tumor microenviron‐ ment is immunosuppressive [109], perhaps as a result of selection for such an environment a process recently termed 'immunoediting. Recent data indicate that TGF-β1 has an important role in suppressing these local responses and that inhibiting this molecule can result in tumor rejection [110, 111]. It is noteworthy that TAMs can both produce TGF-β1 and process latent TGF-βs to produce their active forms[111]. In addition, the local cytokine milieu in the tumor tends to block the immunological functions of these newly recruited mononuclear phagocytes such as antigen presentation and cytotoxicity towards tumors, and diverts them towards specialized TAMs that are immunosuppressed and trophic [112]. A principal component of this cytokine mixture is CSF-1, which locally blocks the maturation of dendritic cells so that they are unable to present antigens and promotes the development of immunosuppressed trophic TAMs. TAMs promote tumor growth by affecting angiogenesis, immune suppression, invasion and metastasis [101, 102]. Existing literature suggests that tumor associated macro‐ phages secrete several genes including matrix metalloproteinases-9 (MMP-9) [113], urokinasetype plasminogen activator (uPA) [114], vascular endothelial growth factor (VEGF) [115], and cyclooxygenase-2 (Cox-2] [116] which promotes tumor growth by breaking down extracellular matrix. The role of TAMs in tumor growth and progression is highlighted in Figure 3.

is usually associated with advanced tumor progression and metastasis in most of the cancers. However the prognostic significance of tumor associated inflammatory cells in metastatic

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Recent reports suggest that interaction between tumor and inflammatory cells contribute to the clinical metastatic neuroblastoma phenotype [120]. It has been reported that metastatic neuro‐ blastomas had higher infiltration of TAMs than loco regional tumors, and metastatic tumors diagnosed in patients at age ≥ 18 months had higher expression of inflammation related genes than those in patients diagnosed at age < 18 months. They identified 14 genes, out of which nine weretumorcellrelatedandfivewereinflammationrelatedthatcomprisesaprognosticsignature forneuroblastoma.Expressionofinflammationrelatedgenes representingTAMs (*CD33*/*CD16*/ *IL6R*/*IL10*/*FCGR3*) contributed to 25% of the accuracy of a novel 14-gene tumor classification score[120].Another studybySongetal.,demonstratedthatCD1d+TAMspromoteneuroblasto‐ ma growth via IL-6 production and that expression of monocyte/macrophage markers, CD14/ CD16,andIL-6orIL-6Rinverselycorrelateswithlong-termdisease-freesurvivalinpatientswith stage 4 *MYCN*–non-amplified neuroblastoma [121]. They suggested that cotransfer of human monocytes and NKTs to tumor-bearing NOD/SCID mice decreased monocyte number at the tumorsiteandsuppressedtumorgrowthcomparedwithmicetransferredwithmonocytesalone. Thus killing of TAMs can be suggested as a novel mechanism of NKT antitumor activity that relates to the disease outcome. Although less is known about the role of stromal compartment in tumorigenecity in neuroblastoma and other childhood tumors but recent reports suggesting infiltration of macrophages in metastatic neuroblastoma opened new opportunities to target tumor associated immune system cells in childhood cancer. It is unclear whether these TAMs represent M2 macrophages and the mechanisms that control macrophage differentiation along

**7. Multiple 'Omics' analysis an emerging concept in treatment of**

The "Omics" is a neologism widely adopted by scientists to refer to large scale analysis of genes (genomic),proteins (proteomics) andlatelysmallmetabolites (metabolomics).Modernmolecu‐ lar achievements over the last decade have seen the increase and implementation of multiple 'omicstechnologiesinoncologythatpromisestoprovideforadeepercomprehensionofcomplex tumor pathways. It is believed that an integration of multiple "omics" technologies is likely to provide even further insight into the holistic view of the biology networks [122]. The studies of global expression profiles of both mRNA and protein are necessary to reveal the important pathways for an enigmatic disease such as neuroblastoma. During the past several years many studies utilized microarray-based high throughput technologies to investigate gene expres‐ sion profiles and DNA copy number alterations in neuroblastoma [123, 124]. Guo *et al*. has performedexonarrayprofilingtoinvestigateglobalalternative splicingpatternof47neuroblas‐ toma samples in stage 1 and stage 4 with normal or amplified MYCN copy number (stage 1-, 4 and 4+) Their results demonstrated a significant role of alternative splicing in high stage neuroblastoma and suggested a MYCN-associated splicing regulation pathway in stage 4+

disease and in childhood cancers is mostly unknown.

the M1 vs the M2 lineage in tumor biology.

**neuroblastoma**

**Figure 3. The role of TAMs in tumor growth, invasion and metastasis:** Tumor-derived chemokines, cytokines and vascu‐ lar endothelial growth factor (VEGF), actively recruit circulating blood monocytes at the tumor site In the tumor micro-envi‐ ronment monocytes differentiate into tumor-associated macrophages (TAM), where they promote tumor growth and metastasis and establish a symbiotic relationship with tumor cells. The above tumor-derived factors positively modulate TAM survival. TAMs also secrete growth factors, which promote tumor cell proliferation and survival, regulate matrix depo‐ sition and remodeling and activate neo-angiogenesis. Figure modified and adapted from Sica *et al.* [106]

Clinical studies have, on balance, shown a correlation between an abundance of TAMs and poor prognosis [108]. These data are particularly strong for breast, prostate, pancreatic, ovarian and cervical cancers; the data for stomach and lung cancers are contradictory [108, 117, 118], and in a small study in colorectal cancer, their presence was associated with good prognosis [119]. However, taking all reports into account regardless of method and sample number more than 80% show a significant correlation between TAM density and poor prognosis, whereas less than 10% associate TAM density with a good prognosis [108]. So, increased TAM density is usually associated with advanced tumor progression and metastasis in most of the cancers. However the prognostic significance of tumor associated inflammatory cells in metastatic disease and in childhood cancers is mostly unknown.

TGF-βs to produce their active forms[111]. In addition, the local cytokine milieu in the tumor tends to block the immunological functions of these newly recruited mononuclear phagocytes such as antigen presentation and cytotoxicity towards tumors, and diverts them towards specialized TAMs that are immunosuppressed and trophic [112]. A principal component of this cytokine mixture is CSF-1, which locally blocks the maturation of dendritic cells so that they are unable to present antigens and promotes the development of immunosuppressed trophic TAMs. TAMs promote tumor growth by affecting angiogenesis, immune suppression, invasion and metastasis [101, 102]. Existing literature suggests that tumor associated macro‐ phages secrete several genes including matrix metalloproteinases-9 (MMP-9) [113], urokinasetype plasminogen activator (uPA) [114], vascular endothelial growth factor (VEGF) [115], and cyclooxygenase-2 (Cox-2] [116] which promotes tumor growth by breaking down extracellular matrix. The role of TAMs in tumor growth and progression is highlighted in Figure 3.

228 Neuroblastoma

**Figure 3. The role of TAMs in tumor growth, invasion and metastasis:** Tumor-derived chemokines, cytokines and vascu‐ lar endothelial growth factor (VEGF), actively recruit circulating blood monocytes at the tumor site In the tumor micro-envi‐ ronment monocytes differentiate into tumor-associated macrophages (TAM), where they promote tumor growth and metastasis and establish a symbiotic relationship with tumor cells. The above tumor-derived factors positively modulate TAM survival. TAMs also secrete growth factors, which promote tumor cell proliferation and survival, regulate matrix depo‐

Clinical studies have, on balance, shown a correlation between an abundance of TAMs and poor prognosis [108]. These data are particularly strong for breast, prostate, pancreatic, ovarian and cervical cancers; the data for stomach and lung cancers are contradictory [108, 117, 118], and in a small study in colorectal cancer, their presence was associated with good prognosis [119]. However, taking all reports into account regardless of method and sample number more than 80% show a significant correlation between TAM density and poor prognosis, whereas less than 10% associate TAM density with a good prognosis [108]. So, increased TAM density

sition and remodeling and activate neo-angiogenesis. Figure modified and adapted from Sica *et al.* [106]

Recent reports suggest that interaction between tumor and inflammatory cells contribute to the clinical metastatic neuroblastoma phenotype [120]. It has been reported that metastatic neuro‐ blastomas had higher infiltration of TAMs than loco regional tumors, and metastatic tumors diagnosed in patients at age ≥ 18 months had higher expression of inflammation related genes than those in patients diagnosed at age < 18 months. They identified 14 genes, out of which nine weretumorcellrelatedandfivewereinflammationrelatedthatcomprisesaprognosticsignature forneuroblastoma.Expressionofinflammationrelatedgenes representingTAMs (*CD33*/*CD16*/ *IL6R*/*IL10*/*FCGR3*) contributed to 25% of the accuracy of a novel 14-gene tumor classification score[120].Another studybySongetal.,demonstratedthatCD1d+TAMspromoteneuroblasto‐ ma growth via IL-6 production and that expression of monocyte/macrophage markers, CD14/ CD16,andIL-6orIL-6Rinverselycorrelateswithlong-termdisease-freesurvivalinpatientswith stage 4 *MYCN*–non-amplified neuroblastoma [121]. They suggested that cotransfer of human monocytes and NKTs to tumor-bearing NOD/SCID mice decreased monocyte number at the tumorsiteandsuppressedtumorgrowthcomparedwithmicetransferredwithmonocytesalone. Thus killing of TAMs can be suggested as a novel mechanism of NKT antitumor activity that relates to the disease outcome. Although less is known about the role of stromal compartment in tumorigenecity in neuroblastoma and other childhood tumors but recent reports suggesting infiltration of macrophages in metastatic neuroblastoma opened new opportunities to target tumor associated immune system cells in childhood cancer. It is unclear whether these TAMs represent M2 macrophages and the mechanisms that control macrophage differentiation along the M1 vs the M2 lineage in tumor biology.

## **7. Multiple 'Omics' analysis an emerging concept in treatment of neuroblastoma**

The "Omics" is a neologism widely adopted by scientists to refer to large scale analysis of genes (genomic),proteins (proteomics) andlatelysmallmetabolites (metabolomics).Modernmolecu‐ lar achievements over the last decade have seen the increase and implementation of multiple 'omicstechnologiesinoncologythatpromisestoprovideforadeepercomprehensionofcomplex tumor pathways. It is believed that an integration of multiple "omics" technologies is likely to provide even further insight into the holistic view of the biology networks [122]. The studies of global expression profiles of both mRNA and protein are necessary to reveal the important pathways for an enigmatic disease such as neuroblastoma. During the past several years many studies utilized microarray-based high throughput technologies to investigate gene expres‐ sion profiles and DNA copy number alterations in neuroblastoma [123, 124]. Guo *et al*. has performedexonarrayprofilingtoinvestigateglobalalternative splicingpatternof47neuroblas‐ toma samples in stage 1 and stage 4 with normal or amplified MYCN copy number (stage 1-, 4 and 4+) Their results demonstrated a significant role of alternative splicing in high stage neuroblastoma and suggested a MYCN-associated splicing regulation pathway in stage 4+

tumors [125]. Studies from other group has measured copy number alterations in a representa‐ tive setof82diagnostic tumorsonacustomizedhigh-resolutionBACarraybasedCGHplatform supplemented with additional clones across 1p36, 2p24, 3p21-22, 11q14-24, and 16p12-13, and integrated these data with RNA expression data[126]. They used an unbiased statistical method to define a set of minimal common regions (MCRs) of aberration and on the basis of unsuper‐ vised hierarchal clustering they identified four distinct genomic subclasses. These genomic subsets were highly correlated with patient outcome, and individual MCRs remained prognos‐ tic in a multivariable model. These studies mentioned above identified prognostic markers and genomic alterations specific to high-risk neuroblastoma, and showed the capability of identify‐ ing signatures which predict patient outcome. Since mRNA expression is not always indica‐ tive of corresponding protein expression because the abundance of specific proteins can be controlledbypost-transcriptionaltranslationandpost-translationalmodifications,thereforethe use of proteomics will help in detecting directly the actual biological effector molecules and should provide more accurate functional information about biological systems. With this idea, Chen *et al*. has performed parallel global protein and mRNA expression profiling on NB tumors of stage4 *MYCN*-amplified (4+) and stage1 *MYCN*-not-amplified (1-) using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray respectively [127]. Pathway analy‐ sis of the differentially expressed proteins conducted by this group showed the enrichment of glycolysis, DNA replication and cell cycle processes in the upregulated proteins and cell adhesion, nervous system development and cell differentiation processes in the down-regulat‐ ed proteins in 4+ tumors; suggesting a less mature neural and a more invasive phenotype of 4+ tumor.

Their studies suggested that NB patients younger than 12 months contained a higher level of acetate and lysine. Conversely, higher amounts of glutathione, glutamate, myoinositol glycine,

Novel Therapeutic Approaches for Neuroblastoma

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231

Overall, the emerging concept of analyzing NB-specific 'omics profiles to better understand and define the behavior of advanced-stage tumors along with providing direct and targeted

**8. Antibody dependent cellular toxicity (ADCC) / Role of ITAM and ITIM**

The Fcγ receptors (FcγRs) expressed on hematopoietic cells play a key role in immune defenses by linking humoral and cellular immunity [133]. FcγRs display coordinate and opposing roles in immune responses depending on their cytoplasmic region and/or their associated chains. Indeed, the activating receptors contain an immunoreceptor tyrosine-based activation motif (ITAM) and initiate inflammatory, cytolytic, and phagocytic activities of immune effector cells. In contrast, the inhibitory receptors that downmodulate the immune responses contain an immunoreceptor tyrosine-based inhibitory motif (ITIM) [134, 135]. There are numerous Fc receptors for IgG (FcγR) that are widely expressed on immune cells. The FcγR family consists of four classes of receptors, FcγRI, FcγRII, FcγRIII, and FcγRIV, that have been identified in both mice and humans. There are significant similarities in the functions of the FcγR receptors between mice and humans, but there is limited homology in receptors themselves [136]. To date, only one inhibitory FcγR, FcγRIIb, has been identified and is the only receptor to have complete homology between mice and humans [136]. FcγRs can be found on virtually all hematopoietic cells except T cells; in most cases, cells coexpress activating and inhibitory FcγR, allowing for the balance between activating and inhibitory receptors to dictate their response [136]. NK cells are an exception to this rule and express only the activating FcγRIIIa. NK cells

Antibodiesdirectedagainstneoplasticcellsprovidenewtherapeuticapproachesagainstvarious malignancies, including lymphoma, leukemia, melanoma, and breast and colorectal carcino‐ ma [137, 138] There is increasing evidence that the Fc portion of the anti-tumor IgG is a major component of their therapeutic activity, along with other mechanisms such as activation of apoptosis, blockade of signaling pathways, or masking of tumor antigens. Thus, by binding to activating FcγRs expressed by immune effector cells, such as macrophages, monocytes, neutrophils, or NK cells, tumor-specific antibodies trigger the destruction of malignant cells via

Because of their rapid and unopposed responses to mAb, NK cells play a major role in the antitumor response elicited by tumor-specific mAbs. Multiple clinically successful mAbs utilize NK-mediated ADCC as a mechanism of action. Rituximab (anti-CD20), Herceptin (anti-Her-2/ neu), Cetuximab (anti-EGFR), and the anti-GD2-mAbs 3F8 and ch14.18 are examples of tumorspecific mAbs whose clinical activity can be attributed, at least in part, to NK cells. Natural killer (NK) cells are powerful effector cells that can be directed to eliminate tumor cells through

antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis [139, 140].

serine and ascorbic acid were detected in NB samples belonging to younger children.

therapy may ultimately translate into improved outcomes for high-risk NB.

**signaling in neuroblastoma**

do not express the inhibitory FcγRIIb.

Metabolomics falls behind its predecessor genomics and proteomics, but represent a burgeon‐ ing field with potential to fill up the gap between genotype and phenotype [128] The high throughput nature of metabolomics makes it an attractive tool for scientists involved in the process of drug development. The reason for that lies in the principle that a patients response to drugs and toxicities do not depend only on a person genetic make-up, but it is rather a factorial outcome of interactions between intrinsic factors and environment [128]. Therefore, metabolomics technology is a powerful tool that can accurately measure the entire spectrum of biochemical changes and mapping these changes to metabolic pathways [128, 129]. In 1995, Florian et al. [130] determined the metabolic characteristics of three types of human brain and nervous system tumors by high-resolution in vitro MRS and chromatographic analysis. Signals from leucine, isoleucine, glycine, valine, threonine, lactate, acetate, glutamate, and cholinecontaining compounds were similarly detected in meningiomas, glioblastomas, and NB. In 2007, Peet et al. [131] reported the results of in vitro 1H high-resolution magic angle spinning NMR spectroscopy (HRMAS) investigations performed on cell suspension of 13 lines of NB possessing multiple genetic alterations. In their study, a specific metabolite profile associated with *MYCN*-amplified and non-amplified tumor subtypes was described. Phosphocholine and taurine concentration ratios relative to total choline were found to be significantly more elevated in the *MYCN*-amplified as compared to the *MYCN*-non-amplified cell lines, and suggested that choline and taurine molecular pathways could be potential therapeutic targets in NB[131]. Recently, Imperiale *et al.* has characterized the metabolic content of intact biopsy samples obtained from 12 patients suffering from neuroblastoma by using (HRMAS) [132]. Their studies suggested that NB patients younger than 12 months contained a higher level of acetate and lysine. Conversely, higher amounts of glutathione, glutamate, myoinositol glycine, serine and ascorbic acid were detected in NB samples belonging to younger children.

tumors [125]. Studies from other group has measured copy number alterations in a representa‐ tive setof82diagnostic tumorsonacustomizedhigh-resolutionBACarraybasedCGHplatform supplemented with additional clones across 1p36, 2p24, 3p21-22, 11q14-24, and 16p12-13, and integrated these data with RNA expression data[126]. They used an unbiased statistical method to define a set of minimal common regions (MCRs) of aberration and on the basis of unsuper‐ vised hierarchal clustering they identified four distinct genomic subclasses. These genomic subsets were highly correlated with patient outcome, and individual MCRs remained prognos‐ tic in a multivariable model. These studies mentioned above identified prognostic markers and genomic alterations specific to high-risk neuroblastoma, and showed the capability of identify‐ ing signatures which predict patient outcome. Since mRNA expression is not always indica‐ tive of corresponding protein expression because the abundance of specific proteins can be controlledbypost-transcriptionaltranslationandpost-translationalmodifications,thereforethe use of proteomics will help in detecting directly the actual biological effector molecules and should provide more accurate functional information about biological systems. With this idea, Chen *et al*. has performed parallel global protein and mRNA expression profiling on NB tumors of stage4 *MYCN*-amplified (4+) and stage1 *MYCN*-not-amplified (1-) using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray respectively [127]. Pathway analy‐ sis of the differentially expressed proteins conducted by this group showed the enrichment of glycolysis, DNA replication and cell cycle processes in the upregulated proteins and cell adhesion, nervous system development and cell differentiation processes in the down-regulat‐ ed proteins in 4+ tumors; suggesting a less mature neural and a more invasive phenotype of 4+

Metabolomics falls behind its predecessor genomics and proteomics, but represent a burgeon‐ ing field with potential to fill up the gap between genotype and phenotype [128] The high throughput nature of metabolomics makes it an attractive tool for scientists involved in the process of drug development. The reason for that lies in the principle that a patients response to drugs and toxicities do not depend only on a person genetic make-up, but it is rather a factorial outcome of interactions between intrinsic factors and environment [128]. Therefore, metabolomics technology is a powerful tool that can accurately measure the entire spectrum of biochemical changes and mapping these changes to metabolic pathways [128, 129]. In 1995, Florian et al. [130] determined the metabolic characteristics of three types of human brain and nervous system tumors by high-resolution in vitro MRS and chromatographic analysis. Signals from leucine, isoleucine, glycine, valine, threonine, lactate, acetate, glutamate, and cholinecontaining compounds were similarly detected in meningiomas, glioblastomas, and NB. In 2007, Peet et al. [131] reported the results of in vitro 1H high-resolution magic angle spinning NMR spectroscopy (HRMAS) investigations performed on cell suspension of 13 lines of NB possessing multiple genetic alterations. In their study, a specific metabolite profile associated with *MYCN*-amplified and non-amplified tumor subtypes was described. Phosphocholine and taurine concentration ratios relative to total choline were found to be significantly more elevated in the *MYCN*-amplified as compared to the *MYCN*-non-amplified cell lines, and suggested that choline and taurine molecular pathways could be potential therapeutic targets in NB[131]. Recently, Imperiale *et al.* has characterized the metabolic content of intact biopsy samples obtained from 12 patients suffering from neuroblastoma by using (HRMAS) [132].

tumor.

230 Neuroblastoma

Overall, the emerging concept of analyzing NB-specific 'omics profiles to better understand and define the behavior of advanced-stage tumors along with providing direct and targeted therapy may ultimately translate into improved outcomes for high-risk NB.

## **8. Antibody dependent cellular toxicity (ADCC) / Role of ITAM and ITIM signaling in neuroblastoma**

The Fcγ receptors (FcγRs) expressed on hematopoietic cells play a key role in immune defenses by linking humoral and cellular immunity [133]. FcγRs display coordinate and opposing roles in immune responses depending on their cytoplasmic region and/or their associated chains. Indeed, the activating receptors contain an immunoreceptor tyrosine-based activation motif (ITAM) and initiate inflammatory, cytolytic, and phagocytic activities of immune effector cells. In contrast, the inhibitory receptors that downmodulate the immune responses contain an immunoreceptor tyrosine-based inhibitory motif (ITIM) [134, 135]. There are numerous Fc receptors for IgG (FcγR) that are widely expressed on immune cells. The FcγR family consists of four classes of receptors, FcγRI, FcγRII, FcγRIII, and FcγRIV, that have been identified in both mice and humans. There are significant similarities in the functions of the FcγR receptors between mice and humans, but there is limited homology in receptors themselves [136]. To date, only one inhibitory FcγR, FcγRIIb, has been identified and is the only receptor to have complete homology between mice and humans [136]. FcγRs can be found on virtually all hematopoietic cells except T cells; in most cases, cells coexpress activating and inhibitory FcγR, allowing for the balance between activating and inhibitory receptors to dictate their response [136]. NK cells are an exception to this rule and express only the activating FcγRIIIa. NK cells do not express the inhibitory FcγRIIb.

Antibodiesdirectedagainstneoplasticcellsprovidenewtherapeuticapproachesagainstvarious malignancies, including lymphoma, leukemia, melanoma, and breast and colorectal carcino‐ ma [137, 138] There is increasing evidence that the Fc portion of the anti-tumor IgG is a major component of their therapeutic activity, along with other mechanisms such as activation of apoptosis, blockade of signaling pathways, or masking of tumor antigens. Thus, by binding to activating FcγRs expressed by immune effector cells, such as macrophages, monocytes, neutrophils, or NK cells, tumor-specific antibodies trigger the destruction of malignant cells via antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis [139, 140].

Because of their rapid and unopposed responses to mAb, NK cells play a major role in the antitumor response elicited by tumor-specific mAbs. Multiple clinically successful mAbs utilize NK-mediated ADCC as a mechanism of action. Rituximab (anti-CD20), Herceptin (anti-Her-2/ neu), Cetuximab (anti-EGFR), and the anti-GD2-mAbs 3F8 and ch14.18 are examples of tumorspecific mAbs whose clinical activity can be attributed, at least in part, to NK cells. Natural killer (NK) cells are powerful effector cells that can be directed to eliminate tumor cells through tumor-targeted monoclonal antibodies (mAbs). Some tumor-targeted mAbs have been successfully applied in the clinic and are included in the standard of care for certain malig‐ nancies. Strategies to augment the antitumor response by NK cells have led to an increased understanding of how to improve their effector responses. Next-generation reagents, such as molecularly modified mAbs and mAb-cytokine fusion proteins (immunocytokines, ICs) designed to augment NK-mediated killing, are showing promise in preclinical and some clinical settings. Continued research into the antitumor effects induced by NK cells and tumortargeted mAbs suggests that additional intrinsic and extrinsic factors may influence the antitumor response. Therefore more research is needed that focuses on evaluating which NK cell and tumor criteria are best predictive of a clinical response and which combination immunotherapy regimens to pursue for distinct clinical settings.

penia, infection, hypokalemia, hypotension, and capillary leak syndrome. Thus, a search for new agents to combine with ch14.18 to improve efficacy and decrease toxicity is justified.

Novel Therapeutic Approaches for Neuroblastoma

http://dx.doi.org/10.5772/55451

233

Immunocytokines commonly known as antibody-cytokine fusion proteins combine the targeting ability of antibodies with the functional activity of cytokines, and are known to improve antibody-based therapy by delivering cytokines to the microenvironment to both activate effector cells and modulate the microenvironment. To date, immunocytokine research has focused on ADCC mediated by NK cells and on induction of CTL. An anti-GD2/IL-2 immunocytokine eradicated hepatic metastases of neuroblastomas in SCID mice that had been reconstituted with human lymphokine (IL-2) activated killer cells [153, 154]. In contrast, the combination of monoclonal anti-GD2 antibody and IL-2 at doses equivalent to the immuno‐ cytokine only reduced tumor load. In a syngeneic murine model of GD2 expressing melanoma, targeting with an anti-GD2 antibody/IL-2 immunocytokine resulted in generation of CD8+ T lymphocytes that could eradicate tumor as well as prevent tumor growth [154]. Based upon these data, phase I and II studies have tested a humanized anti-GD2/IL-2 immunocytokine (hu14.18/IL-2) in patients with refractory or relapsed neuroblastoma. In the phase I study of 27 patients, treatment with hu14.18/IL2 caused elevated serum levels of soluble IL-2 receptor alpha (sIL2R\_) and lymphocytosis. There were no measurable complete or partial responses to hu14.18/IL2; however, three patients showed evidence of antitumor activity [155]. In the phase II study, 39 patients with recurrent or refractory neuroblastoma were enrolled (36 evaluable). No responses were seen for patients with disease measurable by standard radio‐ graphic criteria (stratum 1) (n = 13). Of 23 patients with disease evaluable only by 123Imetaiodobenzylguanidine (MIBG) scintigraphy and/or bone marrow histology (stratum 2), five patients (21.7%) responded; all had a complete response of 9, 13, 20, 30, and 35+ months duration. Grade 3 and 4 non-hematologic toxicities included capillary leak, hypoxia, pain, rash, allergic reaction, elevated transaminases, and hyperbilirubinemia, which were reversible within a few days of completing a treatment course. These results support further testing of

hu14.18/IL2 in children with non-bulky high-risk neuroblastoma [156].

Herein, we have reviewed a number of important areas of basic and translational research related to emerging novel therapies for the pediatric solid tumor, neuroblastoma. These include: 1) Signaling pathways within the tumor cell itself e.g. oncogenes and tumor suppres‐ sor proteins 2) Signaling pathways that regulate the tumor stromal compartment to control angiogenesis and the immune system and 3) Elements of cancer metabolism related to the

Future studies will tap into these areas of basic science investigation to illuminate new avenues for therapeutics. We hereby advocate the need to genotype and perform molecular profiling by multi "omic" analysis on the tumor and stromal cells within the tumor and metastatic sites. Moreover, we suggest that we should examine the adaptive responses to targeted therapeutic agents in mouse models and patients treated with these agents in search of most potent

**10. Summary**

oncogene addiction hypothesis.

#### **9. Tumor associated gangliosides / GD2 monoclonal antibodies**

Gangliosides (GD) are membrane-associated glycosphingolipids which have important regulatory roles during embryogenesis and have also been implicated in tumor development. Particular gangliosides, which show restricted patterns of expression in normal tissue, may be expressed at high levels by tumor cells (e.g. GD3 by melanoma) and are implicated both in tumorigenesis and as mediators of metastatic spread [141]. There is also evidence that gangliosides secreted by tumor cells can modulate the immune response and, in particular, act to inhibit dendritic cell differentiation and function. Neuroblastoma (and other neuroen‐ docrine) tumor cells ubiquitously express the ganglioside GD2, whilst expression in normal tissues is restricted to neurons. Thus, GD2 is an attractive antigen for neuroblastoma immu‐ notherapy strategies [142] including humanized anti-GD2 monoclonal antibodies such as ch14.18 [143], or GD2-directed cytotoxic lymphocytes. A chimeric human–murine anti-GD2 monoclonal antibody [144] called ch14.18 has shown activity against neuroblastoma in preclinical studies [145] and early-phase clinicaltrials[146, 147], this activity could be enhanced when ch14.18 is used in combination with granulocyte–macrophage colony-stimulating factor (GM-CSF) [148] or interleukin-2 [149, 150] to augment antibody-dependent cell-mediated cytotoxicity. The feasibility of administering ch14.18 in combination with GM-CSF, interleu‐ kin-2, and isotretinoin during the early post-transplantation period has been shown in two sequential pilot phase 1 studies [143, 151]. This progression of clinical trials culminated in the recently completed phase III randomized study of isotretinoin together with ch14.18, IL-2, and GMCSF vs. isotretinoin only for children with high-risk neuroblastoma who had a clinical response to induction therapy and myeloablative consolidation therapy/AHSCT. Immuno‐ therapy after consolidation significantly improve event free survival (EFS) (66 ±5% vs. 46 ±5% at 2 years, P = 0.01) and overall survival (86 ± 4% vs. 75 ± 5% at 2 years, P = 0.02). This was the first demonstration that antibody based therapy improves EFS and overall survival. Although EFS was improved by adding immunotherapy to isotretinoin, approximately 40% of patients still relapsed during or after this therapy [152]. Additionally, the combination of ch14.18 with IL-2 and GM-CSF has significant toxicities, including neuropathic pain, fever without neutro‐ penia, infection, hypokalemia, hypotension, and capillary leak syndrome. Thus, a search for new agents to combine with ch14.18 to improve efficacy and decrease toxicity is justified.

Immunocytokines commonly known as antibody-cytokine fusion proteins combine the targeting ability of antibodies with the functional activity of cytokines, and are known to improve antibody-based therapy by delivering cytokines to the microenvironment to both activate effector cells and modulate the microenvironment. To date, immunocytokine research has focused on ADCC mediated by NK cells and on induction of CTL. An anti-GD2/IL-2 immunocytokine eradicated hepatic metastases of neuroblastomas in SCID mice that had been reconstituted with human lymphokine (IL-2) activated killer cells [153, 154]. In contrast, the combination of monoclonal anti-GD2 antibody and IL-2 at doses equivalent to the immuno‐ cytokine only reduced tumor load. In a syngeneic murine model of GD2 expressing melanoma, targeting with an anti-GD2 antibody/IL-2 immunocytokine resulted in generation of CD8+ T lymphocytes that could eradicate tumor as well as prevent tumor growth [154]. Based upon these data, phase I and II studies have tested a humanized anti-GD2/IL-2 immunocytokine (hu14.18/IL-2) in patients with refractory or relapsed neuroblastoma. In the phase I study of 27 patients, treatment with hu14.18/IL2 caused elevated serum levels of soluble IL-2 receptor alpha (sIL2R\_) and lymphocytosis. There were no measurable complete or partial responses to hu14.18/IL2; however, three patients showed evidence of antitumor activity [155]. In the phase II study, 39 patients with recurrent or refractory neuroblastoma were enrolled (36 evaluable). No responses were seen for patients with disease measurable by standard radio‐ graphic criteria (stratum 1) (n = 13). Of 23 patients with disease evaluable only by 123Imetaiodobenzylguanidine (MIBG) scintigraphy and/or bone marrow histology (stratum 2), five patients (21.7%) responded; all had a complete response of 9, 13, 20, 30, and 35+ months duration. Grade 3 and 4 non-hematologic toxicities included capillary leak, hypoxia, pain, rash, allergic reaction, elevated transaminases, and hyperbilirubinemia, which were reversible within a few days of completing a treatment course. These results support further testing of hu14.18/IL2 in children with non-bulky high-risk neuroblastoma [156].

#### **10. Summary**

tumor-targeted monoclonal antibodies (mAbs). Some tumor-targeted mAbs have been successfully applied in the clinic and are included in the standard of care for certain malig‐ nancies. Strategies to augment the antitumor response by NK cells have led to an increased understanding of how to improve their effector responses. Next-generation reagents, such as molecularly modified mAbs and mAb-cytokine fusion proteins (immunocytokines, ICs) designed to augment NK-mediated killing, are showing promise in preclinical and some clinical settings. Continued research into the antitumor effects induced by NK cells and tumortargeted mAbs suggests that additional intrinsic and extrinsic factors may influence the antitumor response. Therefore more research is needed that focuses on evaluating which NK cell and tumor criteria are best predictive of a clinical response and which combination

immunotherapy regimens to pursue for distinct clinical settings.

232 Neuroblastoma

**9. Tumor associated gangliosides / GD2 monoclonal antibodies**

Gangliosides (GD) are membrane-associated glycosphingolipids which have important regulatory roles during embryogenesis and have also been implicated in tumor development. Particular gangliosides, which show restricted patterns of expression in normal tissue, may be expressed at high levels by tumor cells (e.g. GD3 by melanoma) and are implicated both in tumorigenesis and as mediators of metastatic spread [141]. There is also evidence that gangliosides secreted by tumor cells can modulate the immune response and, in particular, act to inhibit dendritic cell differentiation and function. Neuroblastoma (and other neuroen‐ docrine) tumor cells ubiquitously express the ganglioside GD2, whilst expression in normal tissues is restricted to neurons. Thus, GD2 is an attractive antigen for neuroblastoma immu‐ notherapy strategies [142] including humanized anti-GD2 monoclonal antibodies such as ch14.18 [143], or GD2-directed cytotoxic lymphocytes. A chimeric human–murine anti-GD2 monoclonal antibody [144] called ch14.18 has shown activity against neuroblastoma in preclinical studies [145] and early-phase clinicaltrials[146, 147], this activity could be enhanced when ch14.18 is used in combination with granulocyte–macrophage colony-stimulating factor (GM-CSF) [148] or interleukin-2 [149, 150] to augment antibody-dependent cell-mediated cytotoxicity. The feasibility of administering ch14.18 in combination with GM-CSF, interleu‐ kin-2, and isotretinoin during the early post-transplantation period has been shown in two sequential pilot phase 1 studies [143, 151]. This progression of clinical trials culminated in the recently completed phase III randomized study of isotretinoin together with ch14.18, IL-2, and GMCSF vs. isotretinoin only for children with high-risk neuroblastoma who had a clinical response to induction therapy and myeloablative consolidation therapy/AHSCT. Immuno‐ therapy after consolidation significantly improve event free survival (EFS) (66 ±5% vs. 46 ±5% at 2 years, P = 0.01) and overall survival (86 ± 4% vs. 75 ± 5% at 2 years, P = 0.02). This was the first demonstration that antibody based therapy improves EFS and overall survival. Although EFS was improved by adding immunotherapy to isotretinoin, approximately 40% of patients still relapsed during or after this therapy [152]. Additionally, the combination of ch14.18 with IL-2 and GM-CSF has significant toxicities, including neuropathic pain, fever without neutro‐

Herein, we have reviewed a number of important areas of basic and translational research related to emerging novel therapies for the pediatric solid tumor, neuroblastoma. These include: 1) Signaling pathways within the tumor cell itself e.g. oncogenes and tumor suppres‐ sor proteins 2) Signaling pathways that regulate the tumor stromal compartment to control angiogenesis and the immune system and 3) Elements of cancer metabolism related to the oncogene addiction hypothesis.

Future studies will tap into these areas of basic science investigation to illuminate new avenues for therapeutics. We hereby advocate the need to genotype and perform molecular profiling by multi "omic" analysis on the tumor and stromal cells within the tumor and metastatic sites. Moreover, we suggest that we should examine the adaptive responses to targeted therapeutic agents in mouse models and patients treated with these agents in search of most potent combinations and mechanisms for resistance. This will be required to affect a cure of this difficult to treat disease.

[7] Kang J, Rychahou PG, Ishola TA, Mourot JM, Evers BM, Chung DH. N-myc is a nov‐ el regulator of PI3K-mediated VEGF expression in neuroblastoma. Oncogene. 2008

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235

[8] Chanthery YH, Gustafson WC, Itsara M, Persson A, Hackett CS, Grimmer M, et al. Paracrine signaling through MYCN enhances tumor-vascular interactions in neuro‐

[9] Peirce SK, Findley HW, Prince C, Dasgupta A, Cooper T, Durden DL. The PI-3 kin‐ ase-Akt-MDM2-survivin signaling axis in high-risk neuroblastoma: a target for PI-3 kinase inhibitor intervention. Cancer Chemother Pharmacol. 2011 Aug;68(2):325-35.

[10] Garlich JR, De P, Dey N, Su JD, Peng X, Miller A, et al. A vascular targeted pan phos‐ phoinositide 3-kinase inhibitor prodrug, SF1126, with antitumor and antiangiogenic

[11] Wen S, Stolarov J, Myers MP, Su JD, Wigler MH, Tonks NK, et al. PTEN controls tu‐ mor-induced angiogenesis. Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4622-7. [12] Fang J, Ding M, Yang L, Liu LZ, Jiang BH. PI3K/PTEN/AKT signaling regulates pros‐

[13] Tian T, Nan KJ, Wang SH, Liang X, Lu CX, Guo H, et al. PTEN regulates angiogene‐ sis and VEGF expression through phosphatase-dependent and -independent mecha‐

[14] Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch

[15] Meitar D, Crawford SE, Rademaker AW, Cohn SL. Tumor angiogenesis correlates with metastatic disease, N-myc amplification, and poor outcome in human neuro‐

[16] Ribatti D, Vacca A, Nico B, De Falco G, Giuseppe Montaldo P, Ponzoni M. Angiogen‐ esis and anti-angiogenesis in neuroblastoma. Eur J Cancer. 2002 Apr;38(6):750-7. [17] Eggert A, Ikegaki N, Kwiatkowski J, Zhao H, Brodeur GM, Himelstein BP. High-lev‐ el expression of angiogenic factors is associated with advanced tumor stage in hu‐

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#### **Author details**

Shweta Joshi1 , Alok R. Singh1 , Lisa L.R. Hartman1,2, Muamera Zulcic1 , Hyunah Ahn2 and Donald L. Durden1

\*Address all correspondence to: ddurden@ucsd.edu

1 Department of Pediatrics, Moores Cancer Center, University of California San Diego, La Jolla, CA, USA

2 Department of Pediatrics, Rady Children's Hospital-San Diego/UCSD, MC San Diego CA, USA

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**Chapter 11**

**miRNAs as Essential Mediators of the Actions of Retinoic**

The discovery of microRNAs (miRNAs, miRs) led to a profound change on our vision about the regulation of gene expression in eukaryotes. MicroRNAs are an emerging class of small noncoding endogenous RNAs that participate on the fine tuning of gene expression at the posttranscriptional level. First discovered at the early 90s in the nematode *C. elegans* [1], microRNAs have been involved in multiple important biological processes both in animal as in plant cells. These regulatory RNAs are transcribed as primary longer transcripts, which are then processed into 19-23-nt mature miRNAs. One strand of the mature miRNA is then incorporated into the RNA-induced silencing complex (RISC) to regulate gene expression by targeting the 3'-untrans‐ lated region (3'UTR) of mRNAs with consequent translational repression and/or target mRNA degradation. This mode of action demonstrates the great regulatory potential of miRNAs, since a unique mRNA can be targeted by diverse miRNAs and conversely each miRNA may have hundreds of different target mRNAs. In recent years miRNAs have been established as impor‐ tant regulators of tumor development, progression and metastasis, and have demonstrated to be useful for tumor diagnosis and classification. Moreover, miRNA regulation might represent a

Neuroblastoma is the most common extracranial solid tumor in childhood and the most common tumor in infants, which originates from aberrant development of primordial neural crest cells. Several lines of evidence support the idea that microRNA deregulation could contribute to neuroblastoma pathogenesis and progression [2, 3], and the usefulness of miRNA profiles for neuroblastoma diagnostics, classification and prognosis has been recently reported [4]. Neuroblastoma cell lines can be induced to differentiate *in vitro* by several agents, including Retinoic Acid (RA) [5, 6], the biologically active form of vitamin A. RA treatments lead to

and reproduction in any medium, provided the original work is properly cited.

© 2013 Meseguer et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Acid in Neuroblastoma Cells**

Domingo Barettino

**1. Introduction**

http://dx.doi.org/10.5772/55444

Salvador Meseguer, Juan-Manuel Escamilla and

Additional information is available at the end of the chapter

new avenue for cancer treatment in a near future.

