**4. Murine models of MM**

#### **4.1. Subcutaneous xenograft models**

The simplest way to generate an animal model of cancer consists in the injection of tumor cells into an immune-deficient mouse. This approach, known as the xenograft model, has been extensively employed for solid tumors [64,65] and then extended to MM. The xeno‐ graft model of MM consists in the subcutaneous injection of 1-2 x 107 human myeloma cells (from RPMI-8226, U266, ARH-77 or OPM-2 cell lines) into the flanks of Severe Combined Immune-Deficient (SCID), nonobese diabetic (NOD), SCID/NOD and SCID/beige, mice [66,67] (Fig.3A). The resulting plasmacytoma is palpable, and tumor burden measurable with a pair of caliper or, when lines are transduced with the eGFP-luc fusion gene, by biolu‐ minescence imaging [68]. After harvesting, tumor mass is suitable for histological examina‐ tion, allowing identification of vasculature and determination of cell proliferation/apoptosis. The model is currently used to assess the activity of new drugs on MM tumor growth and to establish the effective, minimally toxic, dose. As an example, this model has been employed to investigate the *in vivo* anti-myeloma effect induced by the mTOR inhibitor CCI-779 [69]. More recently, the efficacy of new inhibitors of the CXCR4/CXCL12 axis (AMD3100 and BKT140) [70], and of stressors of the endoplasmic reticulum (spicamycin analogue, KRN5500) [71], which inflict death of MM cells, have been demonstrated using the xenograft model. Besides mono-therapies, the model is suitable to evaluate the maximal effect, in terms of tumor volume reduction, obtainable with combined molecules [72].

While the xenograft model is extremely practical, particularly for drug testing, it still suffers from several limitations. In fact, it does not accurately mimic human disease, since myeloma cell lines do not behave as primary myeloma cells, more closely resembling the aggressive stage of plasma cell leukemia. More importantly, it fails to recapitulate the reciprocal interac‐ tions between MM cells and their microenvironment, which follow MM cell localization and retention inside the BM. As a result, drug efficacy can be over-estimated, lacking implanted MM cells the specific, proper human context of ECM and non-malignant accessory cells.

Murine models of MM, including the 5TMM model, contribute to overcome this latter limitation.

In an effort to reproduce *in vitro* the 3D specific microenvironment of the parental tissue, taking advantage of the rapid development of new technologies and tissue engineering techniques, an extremely wide variety of tissue models have been produced. The latter have already been successfully applied for investigating critical aspects of *in vivo* behaviour of a number of nor‐ mal and tumoral cells (reviewed and discussed in 26). On this basis, 3D culture systems have been proposed as the most physiologically relevant *in vitro* models to investigate tumor devel‐ opment and behaviour [60-62]. Recently, this experimental approach has been also exploited

Within this context, 3D *in vitro* (cell-based)/*ex-vivo* (tissue-based) human-derived culture systems represent important tools to generate new approaches to the understanding of the molecular mechanisms of MM progression, essential prerequisites for the development of

The simplest way to generate an animal model of cancer consists in the injection of tumor cells into an immune-deficient mouse. This approach, known as the xenograft model, has been extensively employed for solid tumors [64,65] and then extended to MM. The xeno‐

(from RPMI-8226, U266, ARH-77 or OPM-2 cell lines) into the flanks of Severe Combined Immune-Deficient (SCID), nonobese diabetic (NOD), SCID/NOD and SCID/beige, mice [66,67] (Fig.3A). The resulting plasmacytoma is palpable, and tumor burden measurable with a pair of caliper or, when lines are transduced with the eGFP-luc fusion gene, by biolu‐ minescence imaging [68]. After harvesting, tumor mass is suitable for histological examina‐ tion, allowing identification of vasculature and determination of cell proliferation/apoptosis. The model is currently used to assess the activity of new drugs on MM tumor growth and to establish the effective, minimally toxic, dose. As an example, this model has been employed to investigate the *in vivo* anti-myeloma effect induced by the mTOR inhibitor CCI-779 [69]. More recently, the efficacy of new inhibitors of the CXCR4/CXCL12 axis (AMD3100 and BKT140) [70], and of stressors of the endoplasmic reticulum (spicamycin analogue, KRN5500) [71], which inflict death of MM cells, have been demonstrated using the xenograft model. Besides mono-therapies, the model is suitable to evaluate the maximal effect, in

While the xenograft model is extremely practical, particularly for drug testing, it still suffers from several limitations. In fact, it does not accurately mimic human disease, since myeloma cell lines do not behave as primary myeloma cells, more closely resembling the aggressive stage of plasma cell leukemia. More importantly, it fails to recapitulate the reciprocal interac‐ tions between MM cells and their microenvironment, which follow MM cell localization and retention inside the BM. As a result, drug efficacy can be over-estimated, lacking implanted MM cells the specific, proper human context of ECM and non-malignant accessory cells.

human myeloma cells

for the study of MM-cell biology and sensitivity to therapeutic agents [63].

more effective interventional, diagnostic and prognostic strategies.

graft model of MM consists in the subcutaneous injection of 1-2 x 107

terms of tumor volume reduction, obtainable with combined molecules [72].

**4. Murine models of MM**

**4.1. Subcutaneous xenograft models**

46 Multiple Myeloma - A Quick Reflection on the Fast Progress


**Figure 3. Schematic representation of currently available MM animal models.** The major murine (m) and murinehuman(hu) models together with their main advantages and limitations are depicted. Synth = synthetic polymeric scaffold; BMSC= Bone Marrow Stromal Cells; SCID=severe combined immune deficient.

#### **4.2. 5TMM models**

The 5T model has been developed in the late seventies upon injection of mice with syngene‐ ic murine MM cells, spontaneously arising in elderly C57BL/KaLwRij mice [73,74]. The group of MM murine models collectively indicated as 5TMM mice comprises different types of mice, each bearing different tumor cells and having distinct characteristics (Figure 3B). The most commonly used, the 5T2MM and the 5T33MM models, display selective localiza‐ tion of cells in the BM, the presence of a serum M component and increased BM angiogene‐ sis. The first one is characterized by moderate growth and development of osteolytic lesions more closely reproducing the human disease, while the second one displays a more aggres‐ sive behaviour with rapid growth [75].

INA-6 cells in SCID-hu mice, but not in SCID mice, as well as their sensitivity to anti-myelo‐

Innovative Models to Assess Multiple Myeloma Biology and the Impact of Drugs

http://dx.doi.org/10.5772/54312

49

More recently, Pierfrancesco Tassone and Filippo Causa developed the so-called "SCIDsynth-hu" model (Figure 3D), based on the implantation of artificial bone scaffolds repopu‐ lated with human BMSC into SCID mice, followed by injection of purified MM cells from patients [82] (Fig. 3D). This model represents a further advancement over the previously de‐ scribed SCID-hu mouse (Fig. 3C). In fact, the use of 3D poly-β-caprolactone polymeric scaf‐ folds, closely reproducing the micro-architecture of a human bone, overcomes the restricted availability of human fetal bones for implant, and also allows to perform studies in the con‐

As for SCID-hu models, in SCID-synth-hu mice injected human MM cells were found to op‐ timally engraft the implanted "niche" and to interact with the human bone milieu, as dem‐ onstrated not only by histological and immunohistochemical analyses of the retrieved

Both systems thereby offer the possibility to investigate human MM cells-BM microenviron‐ ment interactions and to perform pre-clinical testing of anti-MM drugs in a clinically rele‐

MM cells accumulate a series of somatic mutations in the initiating and progressing phases of the disease [10], thus justifying development of genetically modified MM murine models, which recapitulate and explore the genetics of MM [83]. Recently, a model has been devel‐ oped based on the enforced B cell lineage-directed transgene expression of XBP-1s [84]. XBP-1 is a major regulator of the Unfolded Protein Response (UPR) and plasma cell differen‐ tiation. Moreover, XBP-1 over-expression has been implicated in human carcinogenesis and tumor growth in solid tumors and also in MM [84]. XBP-1 transgenic mice spontaneously develop MGUS which progresses to MM, exhibiting remarkable clinical features common to human MM. In particular, BM involvement with clonal MM cells, serum M spike, bone lytic

Another model exploited the deregulated expression of Myc. Myc activation occurs in postgerminal center malignancies, including Burkitt's lymphoma, and is a common feature in MM; in particular its over-expression is generally considered of prognostic significance [85]. Mice engineered to express c-Myc under the control of mouse immunoglobulin kappa (IgK) light-chain gene–regulatory elements (Vk-Myc mice) were developed [86]. Myc is a strong oncogene, and its constitutive expression in early B cells of Vk-Myc mice led to a very ag‐

To create a transgenic mouse model more closely resembling human MM, in their elegant work Chesi and co-workers selected the C57Bl6 strain, genetically predisposed to develop MGUS, and generated a vector (Vk\*Myc) containing a stop codon insertion in the human cmyc oncogene, which prevented its expression [87] (Fig. 3E). Myc could be then sporadically activated in post-germinal B cells as a result of somatic hypermutation, leading to the transi‐

implants, but also by demonstration of immungloglobulin production *in vivo* [82].

lesions and renal Ig deposition could be demonstrated [84].

gressive lymphoma, with extra-medullary localization [86].

ma agents, has also been documented [81].

text of an autologous setting [82].

vant context.

**4.4. Transgenic models**

Studies based on these models, substantially contributed by Karin Vanderkerken's group, have provided valuable insights into MM biology, and in particular on the mechanisms re‐ sponsible for bone disease, MM-associated neoangiogenesis, and MM cell homing to the BM [75]. Indeed, taking advantage from these models, it has been possible to dissect the single steps which participate to the homing process, including chemo-attraction, adhesion, transendothelial migration and invasion, and also to identify the molecular pairs involved [75]. Moreover, these models allow the assessment of the impact of drugs on MM cells inside their proper microenvironment. In particular, the 5T2MM model allowed to unravel the an‐ ti-tumor activity, in addition to prevention of bone resorption, of the amino-biphosphonate zolendronic acid [76]. More recently, the novel 'second-generation' pyrimidyl-hydroxamic acid-based histone deacetylase inhibitor JNJ-26481585 was found to reduce tumor burden and also to affect angiogenesis and osteolysis [77].

A major limitation of the model is represented by the limited availability of different 5T cell lines, which fails to recapitulate the high variability both in terms of genetics and of tumor behaviour which characterize MM developing in humans. Moreover, the results obtained with 5T models should be interpreted with caution, given the potential differences in the bi‐ ology of human vs murine myeloma.

#### **4.3. SCID-hu and SCID-synth-hu models**

In an attempt to "humanize" murine models, in 1997 Urashima established an *in vivo* model of human MM using SCID mice bilaterally implanted with human fetal bone grafts (SCIDhu mice) [78]. The purpose was to study the role of adhesion molecules which participate to human MM-BMSC interactions and regulate MM cell homing. The original experimental de‐ sign consisted in the injection of MM cell lines (ARH-77, OCI-My5, U-266 or RPMI-8226) (1x104 -105 ) into the BM cavity of the left bone implants in irradiated mice (Fig.3C). Human monoclonal MM cells grew within the human BM replacing the stroma and metastatized to the controlateral right bone implant, but not to murine bones or other murine organs [79], suggesting the existence of species-specific interactions. In myeloma-bearing mice, circulat‐ ing human Ig were detectable and mice developed tubular nephropathy, due to light chains deposition, closely mirroring MM clinical manifestations and physiopathology [79]. The model was successfully employed to study the efficacy of thalidomide as an anti-myeloma drug, disclosing its anti-angiogenic properties [80]. The engraftment of IL-6-dependent INA-6 cells in SCID-hu mice, but not in SCID mice, as well as their sensitivity to anti-myelo‐ ma agents, has also been documented [81].

More recently, Pierfrancesco Tassone and Filippo Causa developed the so-called "SCIDsynth-hu" model (Figure 3D), based on the implantation of artificial bone scaffolds repopu‐ lated with human BMSC into SCID mice, followed by injection of purified MM cells from patients [82] (Fig. 3D). This model represents a further advancement over the previously de‐ scribed SCID-hu mouse (Fig. 3C). In fact, the use of 3D poly-β-caprolactone polymeric scaf‐ folds, closely reproducing the micro-architecture of a human bone, overcomes the restricted availability of human fetal bones for implant, and also allows to perform studies in the con‐ text of an autologous setting [82].

As for SCID-hu models, in SCID-synth-hu mice injected human MM cells were found to op‐ timally engraft the implanted "niche" and to interact with the human bone milieu, as dem‐ onstrated not only by histological and immunohistochemical analyses of the retrieved implants, but also by demonstration of immungloglobulin production *in vivo* [82].

Both systems thereby offer the possibility to investigate human MM cells-BM microenviron‐ ment interactions and to perform pre-clinical testing of anti-MM drugs in a clinically rele‐ vant context.

#### **4.4. Transgenic models**

**4.2. 5TMM models**

sive behaviour with rapid growth [75].

48 Multiple Myeloma - A Quick Reflection on the Fast Progress

and also to affect angiogenesis and osteolysis [77].

ology of human vs murine myeloma.

(1x104


**4.3. SCID-hu and SCID-synth-hu models**

The 5T model has been developed in the late seventies upon injection of mice with syngene‐ ic murine MM cells, spontaneously arising in elderly C57BL/KaLwRij mice [73,74]. The group of MM murine models collectively indicated as 5TMM mice comprises different types of mice, each bearing different tumor cells and having distinct characteristics (Figure 3B). The most commonly used, the 5T2MM and the 5T33MM models, display selective localiza‐ tion of cells in the BM, the presence of a serum M component and increased BM angiogene‐ sis. The first one is characterized by moderate growth and development of osteolytic lesions more closely reproducing the human disease, while the second one displays a more aggres‐

Studies based on these models, substantially contributed by Karin Vanderkerken's group, have provided valuable insights into MM biology, and in particular on the mechanisms re‐ sponsible for bone disease, MM-associated neoangiogenesis, and MM cell homing to the BM [75]. Indeed, taking advantage from these models, it has been possible to dissect the single steps which participate to the homing process, including chemo-attraction, adhesion, transendothelial migration and invasion, and also to identify the molecular pairs involved [75]. Moreover, these models allow the assessment of the impact of drugs on MM cells inside their proper microenvironment. In particular, the 5T2MM model allowed to unravel the an‐ ti-tumor activity, in addition to prevention of bone resorption, of the amino-biphosphonate zolendronic acid [76]. More recently, the novel 'second-generation' pyrimidyl-hydroxamic acid-based histone deacetylase inhibitor JNJ-26481585 was found to reduce tumor burden

A major limitation of the model is represented by the limited availability of different 5T cell lines, which fails to recapitulate the high variability both in terms of genetics and of tumor behaviour which characterize MM developing in humans. Moreover, the results obtained with 5T models should be interpreted with caution, given the potential differences in the bi‐

In an attempt to "humanize" murine models, in 1997 Urashima established an *in vivo* model of human MM using SCID mice bilaterally implanted with human fetal bone grafts (SCIDhu mice) [78]. The purpose was to study the role of adhesion molecules which participate to human MM-BMSC interactions and regulate MM cell homing. The original experimental de‐ sign consisted in the injection of MM cell lines (ARH-77, OCI-My5, U-266 or RPMI-8226)

monoclonal MM cells grew within the human BM replacing the stroma and metastatized to the controlateral right bone implant, but not to murine bones or other murine organs [79], suggesting the existence of species-specific interactions. In myeloma-bearing mice, circulat‐ ing human Ig were detectable and mice developed tubular nephropathy, due to light chains deposition, closely mirroring MM clinical manifestations and physiopathology [79]. The model was successfully employed to study the efficacy of thalidomide as an anti-myeloma drug, disclosing its anti-angiogenic properties [80]. The engraftment of IL-6-dependent

) into the BM cavity of the left bone implants in irradiated mice (Fig.3C). Human

MM cells accumulate a series of somatic mutations in the initiating and progressing phases of the disease [10], thus justifying development of genetically modified MM murine models, which recapitulate and explore the genetics of MM [83]. Recently, a model has been devel‐ oped based on the enforced B cell lineage-directed transgene expression of XBP-1s [84]. XBP-1 is a major regulator of the Unfolded Protein Response (UPR) and plasma cell differen‐ tiation. Moreover, XBP-1 over-expression has been implicated in human carcinogenesis and tumor growth in solid tumors and also in MM [84]. XBP-1 transgenic mice spontaneously develop MGUS which progresses to MM, exhibiting remarkable clinical features common to human MM. In particular, BM involvement with clonal MM cells, serum M spike, bone lytic lesions and renal Ig deposition could be demonstrated [84].

Another model exploited the deregulated expression of Myc. Myc activation occurs in postgerminal center malignancies, including Burkitt's lymphoma, and is a common feature in MM; in particular its over-expression is generally considered of prognostic significance [85]. Mice engineered to express c-Myc under the control of mouse immunoglobulin kappa (IgK) light-chain gene–regulatory elements (Vk-Myc mice) were developed [86]. Myc is a strong oncogene, and its constitutive expression in early B cells of Vk-Myc mice led to a very ag‐ gressive lymphoma, with extra-medullary localization [86].

To create a transgenic mouse model more closely resembling human MM, in their elegant work Chesi and co-workers selected the C57Bl6 strain, genetically predisposed to develop MGUS, and generated a vector (Vk\*Myc) containing a stop codon insertion in the human cmyc oncogene, which prevented its expression [87] (Fig. 3E). Myc could be then sporadically activated in post-germinal B cells as a result of somatic hypermutation, leading to the transi‐ tion from the spontaneous monoclonal gammopathy to a disease that fully recapitulate the biological and clinical features of human MM. In fact, Vk\*Myc mice are characterized by the accumulation of slowly proliferating plasma cells exclusively inside the BM. Moreover, high levels of monoclonal antibody are detectable and end-organ damage develops, including anemia, kidney failure and lytic bone disease [87]. The model was found to be highly predic‐ tive of the activity of anti-myeloma drugs [88], including those that target microenviron‐ ment, and may potentially help to select new agents for evaluation in clinical trials.

On this basis, we successfully employed the microgravity-based RCCSTM technology for the generation (and long-term maintenance) of viable human-derived MM tissue explants and 3D cell constructs. Fig. 4 shows the culture chamber of the RCCS™ microgravity-based bio‐ reactor, and histo-morphological images of the *ex-vivo* models of human MM developed by our group. Isolated cells from the RPMI myeloma cell line, kept in Bioreactor, spontaneously self–aggregated forming spheroid-like structures which retained viability and were identifi‐

Innovative Models to Assess Multiple Myeloma Biology and the Impact of Drugs

http://dx.doi.org/10.5772/54312

51

**Figure 4. RCCS TM-based 3D** *ex-vivo* **models of MM developed by our group. A:** Detail of the culture chamber of the RCCS™ microgravity-based bioreactor; **B**: Monotypic 3D multi-cellular spheroids (RPMI cell line) cultured for 1 week in the RCCS™ bioreactor (H&E staining, left panels; CD38 staining, right panels); **C:** 3D tissue culture of skin biop‐ sies (1 week) showing intact architecture and identifiable blood and lymphatic (D2-40+) vessels **D**: MM tissue explants cultured for 3 days in the RCCS™ bioreactor (H&E staining), in the absence or presence of Bortezomib, the latter show‐

The suitability of our method for the culture of human tissue samples was, firstly, proved by using skin biopsies, which retained intact epidermal and dermal architecture, including ker‐ atin stratum and skin annexes. Moreover, both blood and lymphatic vasculature was identi‐ fiable and exhibited normal morphology, in particular patent lumen and complete endothelial lining (Fig.4C). The 3D culture of thick sections of human MM tissue explants fully preserved tissue architecture and microenvironment integrity (Fig.4D) for extended pe‐ riods of time. Moreover, the system was suitable for the assessment of drug sensitivity, not only of tumor compartment, but also of angiogenic vessels (Fig.4D). Indeed, quantification of MVD in treated specimens could represent a unique method to assess the anti-angiogenic effect of a drug in human samples *ex vivo*. Finally, specialized functions of both MM cells and their microenvironment, including beta-2 microglobulin and cytokine release and met‐

able with the specific anti-CD38 monoclonal antibody (Fig.4B).

ing plasma cells death. Arrows indicate bone lamellae.
