Expression of a functional VEGFR-1 in tumor cells is a major determinant of anti-PlGF antibodies efficacy.

PlGF, one of the ligands for VEGFR-1, has been implicated in tumor angiogenesis. However, more recent studies indicate that genetic or pharmacological inhibition of PlGF signaling does not result in reduction of microvascular density in a variety of tumor models. Here we screened 12 human tumor cell lines and identified 3 that are growth inhibited by anti-PlGF antibodies in vivo. We found that efficacy of anti-PlGF treatment strongly correlates with VEGFR-1 expression in tumor cells, but not with antiangiogenesis. In addition, PlGF induced VEGFR-1 signaling and biological responses in tumor cell lines sensitive to anti-PlGF, but not in refractory tumor cell lines or in endothelial cells. Also, genetic ablation of VEGFR-1 signaling in the host did not affect the efficacy of PlGF blockade. Collectively, these findings suggest that the role of PlGF in tumorigenesis largely consists of promoting autocrine/paracrine growth of tumor cells expressing a functional VEGFR-1 rather than stimulation of angiogenesis.

Antiangiogenic therapy decreases integrin expression in normalized tumor blood vessels.

Tumor blood vessels normalized by antiangiogenic therapy may provide improved delivery of chemotherapeutic agents during a window of time but it is unknown how protein expression in tumor vascular endothelial cells changes. We evaluated the distribution of RGD-4C phage, which binds alpha(v)beta(3), alpha(v)beta(5), and alpha(5)beta(1) integrins on tumor blood vessels before and after antiangiogenic therapy. Unlike the control phage, fd-tet, RGD-4C phage homed to vascular endothelial cells in spontaneous tumors in RIP-Tag2 transgenic mice in a dose-dependent fashion. The distribution of phage was similar to alpha(v)beta(3) and alpha(5)beta(1) integrin expression. Blood vessels that survived treatment with AG-013736, a small molecule inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptors, had only 4% as much binding of RGD-4C phage compared with vessels in untreated tumors. Cellular distribution of RGD-4C phage in surviving tumor vessels matched the alpha(5)beta(1) integrin expression. The reduction in integrin expression on tumor vessels after antiangiogenic therapy raises the possibility that integrin-targeted delivery of diagnostics or therapeutics may be compromised. Efficacious delivery of drugs may benefit from identification by in vivo phage display of targeting peptides that bind to tumor blood vessels normalized by antiangiogenic agents.

Rapid access of antibodies to alpha5beta1 integrin overexpressed on the luminal surface of tumor blood vessels.

Integrin alpha(5)beta(1) is overexpressed on endothelial cells of tumor vessels and is uniformly and rapidly accessible to antibodies in the bloodstream. Here, we determined whether antibodies rapidly gain access to integrin overexpressed on the abluminal (basolateral) surface of endothelial cells through vascular leakiness or whether the rapid accessibility results instead because the integrin is overexpressed on the luminal (apical) surface of endothelial cells due to loss of cell polarity. Using tumors in RIP-Tag2 transgenic mice as a model, we first compared the binding pattern of intravascular anti-alpha(5)beta(1) integrin antibody with the leakage pattern of nonspecific IgG. The distributions did not match: anti-alpha(5)beta(1) integrin antibody uniformly labeled the tumor vasculature, but IgG was located in patchy sites of leakage. We next injected an antibody to fibrinogen/fibrin, which resulted in patchy labeling of tumors that matched the leakage of IgG and the overall distribution of fibrin in tumors. Similarly, injected antibodies to the basement membrane protein fibronectin, a ligand of alpha(5)beta(1) integrin, or type IV collagen produced patchy sites of leakage instead of uniform labeling of vascular basement membrane. Differences in the kinetics of labeling, which for alpha(5)beta(1) integrin antibody was near maximal by 10 minutes but for the other antibodies gradually increased over 6 hours, indicated differences in accessibility of their respective targets. Isosurface rendering of confocal microscopic images was consistent with antibody binding to alpha(5)beta(1) integrin on the luminal surface of endothelial cells. Together, these findings indicate that the rapid accessibility of alpha(5)beta(1) integrin in RIP-Tag2 tumors results from overexpression of the integrin on the luminal surface of tumor vessels.

A hierarchy of self-renewing tumor-initiating cell types in glioblastoma.

The neural stem cell marker CD133 is reported to identify cells within glioblastoma (GBM) that can initiate neurosphere growth and tumor formation; however, instances of CD133(-) cells exhibiting similar properties have also been reported. Here, we show that some PTEN-deficient GBM tumors produce a series of CD133(+) and CD133(-) self-renewing tumor-initiating cell types and provide evidence that these cell types constitute a lineage hierarchy. Our results show that the capacities for self-renewal and tumor initiation in GBM need not be restricted to a uniform population of stemlike cells, but can be shared by a lineage of self-renewing cell types expressing a range of markers of forebrain lineage.

Inhibition of VEGF-A prevents the angiogenic switch and results in increased survival of Apc+/min mice.

Anti-VEGF-A monoclonal antibodies, in combination with chemotherapy, result in a survival benefit in patients with metastatic colorectal and non-small cell lung cancer, but little is known regarding the impact of anti-VEGF-A therapy on benign or premalignant tumors. The Apc+/min mice have been widely used as a model recapitulating early intestinal adenoma formation. To investigate whether tumor growth in Apc+/min mice is mediated by VEGF-A-dependent angiogenesis, we used two independent approaches to inhibit VEGF-A: monotherapy with a monoclonal antibody (Mab) targeting VEGF-A and genetic deletion of VEGF-A selectively in intestinal epithelial cells. Short-term (3 or 6 weeks) treatment with anti-VEGF-A Mab G6-31 resulted in a nearly complete suppression of adenoma growth throughout the small intestine. Growth inhibition by Mab G6-31 was associated with a decrease in vascular density. Long-term (up to 52 weeks) treatment with Mab G6-31 led to a substantial increase in median survival. Deletion of VEGF-A in intestinal epithelial cells of Apc+/min mice yielded a significant inhibition of tumor growth, albeit of lesser magnitude than that resulting from Mab G6-31 administration. These results establish that inhibition of VEGF-A signaling is sufficient for tumor growth cessation and confers a long-term survival benefit in an intestinal adenoma model. Therefore, VEGF-A inhibition may be a previously uncharacterized strategy for the prevention of the angiogenic switch and growth in intestinal adenomas.

Uniform overexpression and rapid accessibility of alpha5beta1 integrin on blood vessels in tumors.

Integrin alpha5beta1 is among the proteins overexpressed on tumor vessels and is a potential target for diagnostics and therapeutics. Here, we mapped the distribution of alpha5beta1 integrin in three murine tumor models and identified sites of expression that are rapidly accessible to intravascular antibodies. When examined by conventional immunohistochemistry, alpha5beta1 integrin expression was strong on most blood vessels in RIP-Tag2 transgenic mouse tumors, adenomatous polyposis coli (apc) mouse adenomas, and implanted MCa-IV mammary carcinomas. Expression increased during malignant progression in RIP-Tag2 mice. However, immunoreactivity was also strong in normal pancreatic ducts, intestinal smooth muscle, and several other sites. To determine which sites of expression were rapidly accessible from the bloodstream, we intravenously injected anti-alpha5beta1 integrin antibody and 10 minutes to 24 hours later examined the amount and distribution of labeling. The injected antibody strongly labeled tumor vessels at all time points but did not label most normal blood vessels or gain access to pancreatic ducts or intestinal smooth muscle. Intense vascular labeling by anti-alpha5beta1 integrin antibody co-localized with the uniform CD31 immunoreactivity of tumor vessels and contrasted sharply with the patchy accumulation of nonspecific IgG at sites of leakage. This strategy of injecting antibodies revealed the uniform overexpression and rapid accessibility of alpha5beta1 integrin on tumor vessels and may prove useful in assessing other potential therapeutic targets in cancer.

Angiogenesis with pericyte abnormalities in a transgenic model of prostate carcinoma.

BACKGROUND: Previous studies of the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model vasculature suggest that, as tumors develop, vessels invade the glandular epithelium. However, changes in the vasculature are difficult to study in conventional thin tissue sections. The authors used a new approach to characterize morphologic and architectural changes of blood vessels and pericytes during tumor development in TRAMP mice. METHODS: Eighty-micron cryostat sections of normal prostate and three histopathologic stages of TRAMP tumor sections, classified by epithelial cell E-cadherin immunoreactivity, were immunostained with vascular endothelial cell and pericyte receptor antibodies and evaluated by confocal microscopy. RESULTS: In the normal mouse prostate, capillaries were most abundant in the fibromuscular tunica between the epithelium and smooth muscle of the ductules. In the prostatic intraepithelial neoplasia (PIN) stage, vessels accompanied epithelial cell protrusions into the ductule lumen but remained in the connective tissue at the basal side of the epithelium. Well differentiated tissues had extensive angiogenesis with five times the normal mean vascularity outside ductules. Vessels were of variable diameter, were associated with an increased number of pericytes, and some had endothelial sprouts. Angiogenic blood vessels from poorly differentiated adenocarcinomas were tortuous, variable in caliber, and lacked the normal hierarchy. Pericytes on these vessels had an abnormal phenotype manifested by alpha-smooth muscle actin expression and loose association with endothelial cells. Angiogenesis and loss of vascular hierarchy were also found in human prostate carcinoma. CONCLUSIONS: Vascular abnormalities, which begin at the PIN stage and intensify in well differentiated and poorly differentiated tumors, may be useful readouts for early detection and treatment assessment in prostate carcinoma.