Angiogenesis has been acknowledged as a common attribute for growth and metastasis of tumors and a growing list of animal models and human cancer studies documented the ‘angiogenic switch’ as a secondary rate-limiting event in carcinogenesis (Hanahan and Weinberg, 2000). Indeed, complete or partial suppression of vascular growth by a number of different strategies has been consistently associated with suppression of tumor expansion and even reduction of tumor burden. Given the key functions in tumor angiogenesis, inhibiting vascular endothelial growth factor (VEGF) or blocking the kinase activity of VEGF receptors (VEGFR) have been a major focus in therapeutic exploitations. Current VEGF targeted therapies include the neutralizing anti-VEGF antibody bevacizumab and the receptor tyrosine kinase inhibitors (RTKIs) sunitinib (Sutent, Pfizer), sorafenib (Nexavar, Bayer) and pazopanib (Votrient, GlaxoSmithKilne) targeting VEGFR. These have been approved for treating an increasing number of human cancer types: bevacizumab for treating late-stage colon cancer, non-small-cell lung cancer and breast cancer, sorafenib for treating advanced renal cells carcinoma (RCC) and hepatocellular carcinoma, pazopanib for treating RCC and, sunitinib for treating RCC and gastrointestinal stromal tumors. While monotherapy demonstrated rather modest activities, when given in combination with conventional chemotherapeutic regimens they showed increased overall survival of patients.
While there have been demonstrable clinical benefits in the application of VEGF inhibitors and its signaling blockers, the effect is not yet complete. The reasons for this outcome are multiple and many parameters may be involved in the effectiveness of VEGF targeted therapies. To name a few, these include the necessity of VEGF signaling within the tumor, mobilization of bone marrow-derived pro-angiogenic cells, vessel co-option, etc. In this paper, Helfrich et al. (2010) now present results that pericyte coverage is a determinant to the effectiveness to anti-VEGF therapy. The authors demonstrate that, by using spontaneously developing murine melanoma model, tumor vessels covered with mature pericytes do not regress by anti-VEGF treatment. Consequently, the treatment does not affect the volume of growing tumors. Parallel findings were observed in human melanoma metastases taken at clinical relapse in patients undergoing adjuvant bevacizumab treatment.
As a component of blood vessels, mural cells associate with and cover the endothelial cells. Mural cells are subdivided into vascular smooth muscle cells and pericytes. Vascular smooth muscle cells are associated with arteries and veins. Pericytes are associated with capillaries, arterioles, and venules. Prevailing view is that mural cells attachment to endothelial cells regulates blood vessel stabilization and maturation (Benjamin et al., 1998). Helfrich et al. hypothesized that the level of vessel maturation is critically involved in the efficacy of angiogenesis inhibitors. To do this, the authors used a murine melanoma model containing qualitatively different angiogenic vessels. Helfrich et al. first showed that two different tumor vascular beds co-existed in a spontaneously growing melanoma of MT/ret transgenic mice: a high angiogenic phenotype with a mean microvascular vessel density of 250/mm2 and a low vessel density phenotype with a mean vessel density of 40 mm2. In addition, the low vessel density phenotype showed significantly increased vessel diameter (10-fold higher) and lumen size in comparison to high angiogenic phenotype. The relative abundance of the differential vascular beds was different as well: a mean distribution of 83% high angiogenic to 17% low angiogenic tumors. However, the differential vascular phenotype did not affect vessel perfusion (measured by FITC-lection perfusion). In addition, although there was correlation between the tumor growth kinetics and tumor angiogenesis during the first 3 weeks (i.e., increased tumor growth kinetics with high angiogenic vessels), the vessel phenotype did not affect the size of tumor volume nor tumor location.
The authors further analyzed pericyte coverage in both vascular phenotypes. NG-2, Desmin and PDGFR-β were used for early, immature pericyte markers and α smooth muscle actin (SMA) for a maker of mature pericyte and smooth muscle cells. Surprisingly, while the percentage of vessels that were covered by immature pericyte was comparable in both high- and low-angiogenic vessel phenotypes, there was a significant difference in the percentage of the vessels that were covered by mature pericyte and smooth muscle cells: 98% (in low-angiogenic vessels) versus 2% (in high-angiogenic vessels), suggesting that the low angiogenic tumors carry mature vessels. Levels of basement membrane investment were also different: most of the endothelial cell layers of low-angiogenic vessels were underlined by a well-constructed basal lamina but endothelial cells of high angiogenic vessels were covered with partly developed basal lamina which leads to direct interaction of tumor cells with endothelial cells. Consequently, excessive vessel leakiness was observed in tumors of high vessel density.
Searching for a candidate molecule that might account for the better pericyte coverage in low angiogenic tumors revealed that the level of Angiopoietin-1 (Ang-1) was almost equal in both high- and low-vascular beds, whereas the levels of pro-angiogenic factors VEGF-A, VEGFR-2, and Ang-2 were significantly reduced in low angiogenic vessels. Since Ang-1 is predominantly expressed by pericytes and Ang-1/Tie2 signaling axis is involved in mural cells attachment to endothelial cells and vessel maturation (Suri et al., 1996), this may partially explain the presence of mature vessels in low angiogenic tumors.
The authors further examined the role of pericyte coverage in the susceptibility of tumor vasculature to anti-VEGF therapy. The drug selected in this study was PTK/ZK, RTKIs shown to inhibit VEGF signaling by blocking autophosphorylation of VEGFR-1, -2, and -3. In both prevention and intervention trials, PTK/ZK treatment of MT/ret transgenic mice resulted in decrease in tumor numbers or inhibition of the development of novel tumors, without affecting the tumor volumes, compared with vehicle controls. Interestingly, PTK/ZK treatment resulted in vessel regression in high-angiogenic tumors but the low-angiogenic vessels were resistant to PTK/ZK treatment.
The authors extended their findings into human melanoma metastases, which had developed during the UK adjuvant bevacizumab (neutralizing anti-VEGF) trial in stage III cutaneous melanoma. Tumor-associated blood vessels of bevacizumab resistant metastases showed increased vessel diameter (17-fold higher compared to melanoma metastases observed from patients without therapy) with a better pericyte coverage, suggesting a role of mural cell recruitment and vessel maturation for the susceptibility to anti-VEGF therapy.
In conclusion, these new studies illustrate a regulatory role of pericyte through its attachment to endothelial cells in indifference of tumor vessels to anti-VEGF therapy (Figure 1). The effectiveness of anti-angiogenesis drug may now be predicted by the examination and the qualitative assessment of tumor vessels in the context of vessel density, vessel diameter and the level of pericyte coverage or basement membrane investment. Further, the Helfrich et al. present us not only with a mechanism whereby melanomas evade anti-VEGF therapy but also with an agenda for the design of anti-angiogenesis therapy based on the distinctive vascular phenotypes. Previous studies have shown the benefits of combinatorial targeting of both pericytes and endothelial cells in the tumor vasculature (Bergers et al., 2003). To this end, the nature of tumor endothelial cells, pericytes and their inter-signaling mechanisms needs to be better understood. Further investigation and identification of new biomarkers, the differential expression of growth factors other than Ang-1 and receptors in the two vascular beds, which might be associated with the resistance to anti-VEGF therapy are warranted. In light of this, it should also be mentioned that while kinases inhibitors are increasingly recognized as therapeutic interventions, development of highly selective kinase inhibitors is a main challenge. Due to the presence of the highly conserved catalytic domain, kinase inhibitors have shared features of binding to their targets. In addition, individual structural determinants for target binding remain mostly unclear. Consequently, these tyrosine kinase inhibitors have broad spectrum of activity or off-target kinase interactions. Taking this into consideration, Karaman et al. recently presented the most comprehensive study and methodology to quantitatively analyze kinase inhibitor selectivity (Karaman et al., 2008). We are now able to measure ‘selectivity score’ by calculating the binding interactions of known kinases and kinase inhibitors and by calculating the fold-difference between the affinities for each primary target and off-target, systemically. Advancement in designing safer, selective, and effective therapeutics is anticipated and promising.