**11.3 Role of stromal cells**

Preclinical data suggest that cells in the tumour stroma, including fibroblasts, neutrophils, macrophages and myeloid progenitors, mediate resistance to VEGFtargeted agents (**Figure 4C**) [124]. For example, tumour-derived granulocytecolony stimulating factor (G-CSF) mobilises myeloid cells from bone marrow, and is believed to promote pro-angiogenic Bv8 signalling by myeloid cells, which in tumours may confer resistance to anti-VEGF treatment [125]. Immunohistochemistry studies in human tumours showed expression of Bv8 in tumour-infiltrating neutrophils, which were seen in around 15% of breast carcinomas [126].

### **11.4 Tumour cell adaptation to stress**

It is presumed that the inhibition of tumour vascularisation by anti-angiogenic agents will lead to a reduction in oxygen and nutrients available to the tumour thus causing retardation of tumour growth. However, tumours may develop a number of survival mechanisms enabling them to adapt to such hostile conditions (**Figure 4D**).

#### *11.4.1 Metabolism*

Some studies have suggested that anti-angiogenic therapy leads to metabolic reprogramming of tumour cells, allowing them to adapt to reduced vascular supply. Preclinical studies have demonstrated that treatment with anti-VEGF antibodies can lead to tumour cells relying on anaerobic metabolism and the glycolytic pathway for ATP [127]. Furthermore, the withdrawal of anti-angiogenic therapy has been shown to cause an increase in lipid metabolism, leading to a rebound in tumour growth [127].

#### *11.4.2 Autophagy*

Tumours treated with anti-angiogenic agents may also adapt to survive by activation of autophagy. Autophagy can occur in response to treatment related stressors such as hypoxia and occurs when organelles and proteins in the cell are degraded and recycled by lysosomes [128]. Autophagy-mediating molecules such as BNIP3 have been identified in GBM tumour cells after exposure (a) to hypoxic conditions in vitro, (b) to bevacizumab therapy in vivo or (c) to bevacizumab therapy in human tumours [129]. Furthermore, a recent study has reported that when MDA-MB-231 breast cancer cells were treated with an agent that induced autophagy, they exhibited increased invasiveness [130].

#### *11.4.3 Cancer stem cells (CSCs)*

It is becoming clear that many solid tumours contain relatively rare subpopulations of cancer stem cells. These are clones of tumour cells that are able to sustain self-renewal and can tolerate hostile environments [131]**.** Furthermore, it has been proposed that hypoxia induced by anti-angiogenic therapy can (a) select for CSCs, and (b) maintain the niche that supports the survival of CSCs [132]. Conceivably, these persistent clones of CSCs may render the tumour more invasive and metastatic and may also lead to antiangiogenic therapy resistance [133].

#### *11.4.4 Enhanced tumour aggressiveness*

Anti-angiogenic therapy has been proposed to induce hypoxic tumour microenvironments, enhancing the aggressiveness of tumour cells (**Figure 4F**) [134]. This

**59**

*DOI: http://dx.doi.org/10.5772/intechopen.89667*

increase in tumour invasiveness [136].

by VEGF-targeted therapies.

*11.5.1 Intussusception*

*11.5.2 Vascular mimicry*

tic detail is lacking.

*11.5.3 Vessel co-option*

**11.5 Alternative vascularisation mechanisms**

*Angiogenesis and Its Role in the Tumour Microenvironment: A Target for Cancer Therapy*

may help explain why the response to anti-angiogenic therapy is often transient as anti-angiogenic agents can cause initial reductions in tumour burden and a prolonged PFS, but with minimal or no improvement in OS [118]. Anti-angiogenic agents have demonstrated an ability to select for more aggressive cancer cells and enhance tumour cell invasion, growth and metastasis [135]. Moreover, it is now well accepted that some GBM patients with tumours treated with bevacizumab show an

Despite the dogma that tumours primarily employ VEGF-dependent sprouting angiogenesis, emerging evidence now exists for alternative tumour vascularisation mechanisms, including: intussusceptive microvascular growth (IMG) (sometimes known merely as 'intussusception'), glomeruloid angiogenesis, vascular mimicry (also sometimes called 'vasculogenic mimicry'), looping angiogenesis, and vessel co-option (also sometimes called 'vascular co-option') (**Figure 4E**) [21]. These mechanisms may occur by alternative signalling pathways that may not be inhibited

Intussusception is a mechanism whereby pre-existing vessels split into two daughter vessels without the need for endothelial cell proliferation and sprouting (**Figure 4E**). It has been observed in embryonic development and within experimental tumours recovering from anti-angiogenic therapy and radiotherapy [137]. The molecular mechanisms that control this process are still not well understood.

Vascular mimicry (VM) is a process observed in clinical and preclinical studies whereby tumour cells differentiate into vascular-like structures themselves [138] (**Figure 4E**). It has been shown that basic fibroblast growth factor (bFGF) and VEGF, are incapable of inducing VM channels and networks in poorly aggressive melanoma cell lines, suggesting that VM channel formation maybe be independent of these classical pro-angiogenic growth factors [139]. However, further mechanis-

Vessel co-option is the process whereby, when a tumour invades, existing local vessels become directly incorporated into the tumour (**Figure 4E**). Histopathological studies have indicated that colorectal and breast cancer liver

Vessel co-option has been shown to mediate resistance to VEGF inhibitors in mouse models of melanoma metastasis to the brain and in mouse models of glioblastoma multiforme, and has been observed in glioblastoma patients who have progressed on anti-VEGF therapy [142–144]. Recently, it has been demonstrated that vessel co-option plays a role in mediating resistance to anti-angiogenic therapy

In tumour samples obtained from primary lung cancer patients, gene expression

arrays have been utilised to identify pathways differentially expressed between angiogenic tumours and vessel co-opting tumours [146]. Stromal expression of thrombospondin-1 appeared to be up regulated in angiogenic tumours, whilst in

metastases may utilise vessel co-option [140, 141].

in colorectal cancer liver metastases [145].

*DOI: http://dx.doi.org/10.5772/intechopen.89667 Angiogenesis and Its Role in the Tumour Microenvironment: A Target for Cancer Therapy*

may help explain why the response to anti-angiogenic therapy is often transient as anti-angiogenic agents can cause initial reductions in tumour burden and a prolonged PFS, but with minimal or no improvement in OS [118]. Anti-angiogenic agents have demonstrated an ability to select for more aggressive cancer cells and enhance tumour cell invasion, growth and metastasis [135]. Moreover, it is now well accepted that some GBM patients with tumours treated with bevacizumab show an increase in tumour invasiveness [136].

#### **11.5 Alternative vascularisation mechanisms**

Despite the dogma that tumours primarily employ VEGF-dependent sprouting angiogenesis, emerging evidence now exists for alternative tumour vascularisation mechanisms, including: intussusceptive microvascular growth (IMG) (sometimes known merely as 'intussusception'), glomeruloid angiogenesis, vascular mimicry (also sometimes called 'vasculogenic mimicry'), looping angiogenesis, and vessel co-option (also sometimes called 'vascular co-option') (**Figure 4E**) [21]. These mechanisms may occur by alternative signalling pathways that may not be inhibited by VEGF-targeted therapies.

#### *11.5.1 Intussusception*

*Current Cancer Treatment*

**11.3 Role of stromal cells**

*11.4.1 Metabolism*

*11.4.2 Autophagy*

*11.4.3 Cancer stem cells (CSCs)*

*11.4.4 Enhanced tumour aggressiveness*

**11.4 Tumour cell adaptation to stress**

Preclinical data suggest that cells in the tumour stroma, including fibroblasts, neutrophils, macrophages and myeloid progenitors, mediate resistance to VEGFtargeted agents (**Figure 4C**) [124]. For example, tumour-derived granulocytecolony stimulating factor (G-CSF) mobilises myeloid cells from bone marrow, and is believed to promote pro-angiogenic Bv8 signalling by myeloid cells, which in tumours may confer resistance to anti-VEGF treatment [125]. Immunohistochemistry studies in human tumours showed expression of Bv8 in tumour-infiltrating neutro-

It is presumed that the inhibition of tumour vascularisation by anti-angiogenic agents will lead to a reduction in oxygen and nutrients available to the tumour thus causing retardation of tumour growth. However, tumours may develop a number of survival mechanisms enabling them to adapt to such hostile conditions (**Figure 4D**).

Some studies have suggested that anti-angiogenic therapy leads to metabolic reprogramming of tumour cells, allowing them to adapt to reduced vascular supply. Preclinical studies have demonstrated that treatment with anti-VEGF antibodies can lead to tumour cells relying on anaerobic metabolism and the glycolytic pathway for ATP [127]. Furthermore, the withdrawal of anti-angiogenic therapy has been shown to cause an increase in lipid metabolism, leading to a rebound in tumour growth [127].

Tumours treated with anti-angiogenic agents may also adapt to survive by activation of autophagy. Autophagy can occur in response to treatment related stressors such as hypoxia and occurs when organelles and proteins in the cell are degraded and recycled by lysosomes [128]. Autophagy-mediating molecules such as BNIP3 have been identified in GBM tumour cells after exposure (a) to hypoxic conditions in vitro, (b) to bevacizumab therapy in vivo or (c) to bevacizumab therapy in human tumours [129]. Furthermore, a recent study has reported that when MDA-MB-231 breast cancer cells were treated with an agent that induced

It is becoming clear that many solid tumours contain relatively rare subpopulations of cancer stem cells. These are clones of tumour cells that are able to sustain self-renewal and can tolerate hostile environments [131]**.** Furthermore, it has been proposed that hypoxia induced by anti-angiogenic therapy can (a) select for CSCs, and (b) maintain the niche that supports the survival of CSCs [132]. Conceivably, these persistent clones of CSCs may render the tumour more invasive and meta-

Anti-angiogenic therapy has been proposed to induce hypoxic tumour microenvironments, enhancing the aggressiveness of tumour cells (**Figure 4F**) [134]. This

autophagy, they exhibited increased invasiveness [130].

static and may also lead to antiangiogenic therapy resistance [133].

phils, which were seen in around 15% of breast carcinomas [126].

**58**

Intussusception is a mechanism whereby pre-existing vessels split into two daughter vessels without the need for endothelial cell proliferation and sprouting (**Figure 4E**). It has been observed in embryonic development and within experimental tumours recovering from anti-angiogenic therapy and radiotherapy [137]. The molecular mechanisms that control this process are still not well understood.

#### *11.5.2 Vascular mimicry*

Vascular mimicry (VM) is a process observed in clinical and preclinical studies whereby tumour cells differentiate into vascular-like structures themselves [138] (**Figure 4E**). It has been shown that basic fibroblast growth factor (bFGF) and VEGF, are incapable of inducing VM channels and networks in poorly aggressive melanoma cell lines, suggesting that VM channel formation maybe be independent of these classical pro-angiogenic growth factors [139]. However, further mechanistic detail is lacking.

#### *11.5.3 Vessel co-option*

Vessel co-option is the process whereby, when a tumour invades, existing local vessels become directly incorporated into the tumour (**Figure 4E**). Histopathological studies have indicated that colorectal and breast cancer liver metastases may utilise vessel co-option [140, 141].

Vessel co-option has been shown to mediate resistance to VEGF inhibitors in mouse models of melanoma metastasis to the brain and in mouse models of glioblastoma multiforme, and has been observed in glioblastoma patients who have progressed on anti-VEGF therapy [142–144]. Recently, it has been demonstrated that vessel co-option plays a role in mediating resistance to anti-angiogenic therapy in colorectal cancer liver metastases [145].

In tumour samples obtained from primary lung cancer patients, gene expression arrays have been utilised to identify pathways differentially expressed between angiogenic tumours and vessel co-opting tumours [146]. Stromal expression of thrombospondin-1 appeared to be up regulated in angiogenic tumours, whilst in

vessel co-option tumours, there was increased expression of genes involved in oxidative phosphorylation in primary [146]. Surprisingly, no differences in classic hypoxia or angiogenesis related genes were found between angiogenic and nonangiogenic tumours.

In a glioma rat model of breast cancer brain and lung metastasis, co-opted blood vessels were seen in early-stage tumours and these vessels were found to overexpress angiopoietin-2, a natural antagonist of angiopoietin-1 [147]. As these tumours grew to become more hypoxic, VEGF was upregulated at the hypoxic tumour periphery and stimulated angiogenesis [147]. These observations suggest that a transition from vessel co-option to angiogenesis, or vice versa, may be dependent on the relative expression of pro-angiogenic growth factors (angiopoeitin-1, VEGF) and anti-angiogenic factors (angiopoeitin-2).

Cell adhesion molecules have been implicated in facilitating the process of vessel co-option. In a preclinical brain metastasis model, Carbonell et al. demonstrated that the β1 integrin subunit in breast cancer and lymphoma cells facilitates (a) tumour cell adhesion to the vascular basement membrane of existing brain vessels, (b) tumour cell invasion and (c) the process of vessel co-option [148]. When the function of the β1 integrin subunit was blocked, adhesion to vessels was attenuated and brain metastasis colonies failed to become established and grow [148].

Furthermore, the L1 cell adhesion molecule (L1CAM) has been shown to be involved in vessel co-option in the brain [149]. The ability of cancer cells to coopt blood vessels was suppressed when L1CAM expression was depleted using shRNA. Conversely, when L1CAM was overexpressed, tumour cells demonstrated enhanced adherence to the outer surface of vessels and tumour growth alongside them. Although such mechanisms are likely to be more specific for vessel co-option in the brain, similar mechanisms may be at work during vessel co-option at other anatomical sites.
