Presence Of anti-VEGF Antibody Research Articles

Role of Myeloid and Other Stromal Cell Types in Tumor Angiogenesis.

Abstract SCI-32Role of myeloid and other stromal cell types in tumor angiogenesis Napoleone Ferrara Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080, USA. Vascular endothelial growth factor (VEGF)-A is an important angiogenic factor, involved in both physiological and pathological growth of blood vessels. An anti-VEGF-A monoclonal antibodies and two small molecule VEGF RTK inhibitors have been approved by the FDA for cancer therapy. We have been investigating the mechanisms of refractoriness/resistance to anti-VEGF therapies. Our analysis points to the mobilization and recruitment of CD11b+ Gr1+myeloid cells from the bone marrow in the tumor as key mechanisms for VEGF-independent angiogenesis, at least in mouse models. Tumors that are refractory to anti-VEGF treatment result in increased peripheral mobilization and tumor homing of of CD11b+Gr1+ cells compared to sensitive tumors. Addition of CD11b+Gr1+ cells from resistant tumors was able to sustain the growth of sensitive tumors in the presence of anti-VEGF antibodies. In subsequent studies, we identified the secreted protein Bv8 as a mediator of myeloid cell-dependent angiogenesis. Blocking Bv8 using neutralizing antibodies inhibited the growth of several tumor cell lines transplanted in nude mice and also reduced the angiogenic switch in the RIP-Tag, a transgenic model of insulinoma. The expression of Bv8 by CD11b+ Gr1+myeloid was regulated by hematopoietic growth factor, especially G-CSF. Other studies have implicated tumor-associated fibroblasts as a source of alternative pro-angiogenic factors following VEGF blockade. We identified PDGF-C as one of such mediators. In summary, multiple stromal cell types and signaling pathways may contribute to inherent refractoriness and, potentially, acquired resistance to anti-VEGF therapies in tumors. DisclosuresFerrara:Genentech, Inc.: Employment.

PDGF-C Mediates the Angiogenic and Tumorigenic Properties of Fibroblasts Associated with Tumors Refractory to Anti-VEGF Treatment

Tumor- or cancer-associated fibroblasts (TAFs or CAFs) from different tumors exhibit distinct angiogenic and tumorigenic properties. Unlike normal skin fibroblasts or TAFs from TIB6 tumors that are sensitive to anti-VEGF treatment (TAF-TIB6), TAFs from resistant EL4 tumors (TAF-EL4) can stimulate TIB6 tumor growth even when VEGF is inhibited. We show that platelet-derived growth factor C (PDGF-C) is upregulated in TAFs from resistant tumors. PDGF-C-neutralizing antibodies blocked the angiogenesis induced by such TAFs in vivo, slowed the growth of EL4 and admixture (TAF-EL4 + TIB6) tumors, and exhibited additive effects with anti-VEGF-A antibodies. Hence, our data reveal an additional mechanism for TAF-mediated tumorigenesis and suggest that some tumors may overcome inhibition of VEGF-mediated angiogenesis through upregulation of PDGF-C.

A BRIEF COMMUNICATION

Previous studies have shown that dietary copper deficiency causes cardiac hypertrophy and depression of vascular epithelial growth factor (VEGF) expression in mouse model. Copper replenishment in the diet reverses cardiac hypertrophy and restores VEGF expression. The present study was undertaken to specifically determine the role of VEGF in copper effect on cell hypertrophy. Embryonic rat cardiac H9c2 cells were exposed to hydrogen peroxide to develop hypertrophy, determined by increases in cell size and total protein content. Copper addition at 5 microM in cultures suppressed cell hypertrophy. In the presence of anti-VEGF antibody, copper inhibitory effect on cell hypertrophy was blunted, and VEGF alone mimicked the inhibitory effect of copper. The results thus demonstrated that VEGF is critically involved in copper inhibition of cell hypertrophy induced by hydrogen peroxide in the H9c2 cells.

The Inhibitor of VEGF-Receptors Tyrosine Kinase PTK-787 Blocks the Invasive Potential of Adult T-Cell Leukemia Cells through the Endothelium.

Extravasation of tumor cells through the endothelial barrier is a critical step in cancer metastasis. HTLV-I associated adult T-cell leukemia/lymphoma (ATL) is an aggressive disease characterized by multiple organ invasion. We have shown that ATL-derived cells induce angiogenesis and communicate with endothelial cells through gap junctions. This results in induction of endothelial-derived metalloproteinases, sub-endothelial basement membrane degradation followed by endothelial cell retraction allowing neoplastic lymphocytes extravasation. In this study we have used the specific tyrosine kinase inhibitor of VEGF receptors PTK787, functional blocking antibodies against VEGF and the gap junction communication inhibitor18-a-G to dissect the respective role of VEGF signaling pathway and cell-to-cell communication in endothelial cell retraction and tumor cell extravasation. PTK787 has mild growth inhibitory and cytotoxic effects on both endothelial and tumor cells at a concentration of 50μM and significantly inhibited cell proliferation at 100μM. Hence, 20μM PTK787 which did not exhibit any cytotoxic or antiproliferative effects on both cell types was used in experiments that last 24 hours or longer (in vitro tube formation assay and invasion assay). Endothelial cells monolayers incubated with either ATL derived cells or their cell-free conditioned medium for 48 hours induced in vitro tube formation in Matrigel angiogenesis assay. This was inhibited by both anti-VEGF antibody and 18-a-G and to a greater extent and in a dose dependent manner by PTK787. Importantly, PTK787, and to a lesser extent anti-VEGF antibody and 18-alpha-G significantly attenuated the ability of ATL-derived cells to traverse endothelial cell monolayers in double compartment invasion assays. Signaling cascade that is activated upon VEGF stimulation of endothelial cells was investigated in confluent endothelial cells cocultured with ATL derived cells or cell-free conditioned medium in the absence or presence of PTK787 or anti-VEGF antibody, using phospho-specific antibodies against FAK Tyr 861, FAK Tyr 397, Src, ERK, PLCgamma, and Akt/PKB, known to be signal mediators in the Ras-Raf-MEKK-ERK signaling cascade. The addition of ATL derived cells or cell-free conditioned medium resulted in a transient activation of the above signal mediators with a peak increase at 15 minutes and gradual return to baseline after 60 minutes. This activation was down regulated in the presence of anti-VEGF antibody and to a greater extent in the presence of PTK787. This data establishes a novel role of VEGF signaling in tumor cell invasion and supports the potential use of PTK787 in the treatment of ATL and other hematological malignancies with angiogenic and invasive properties.

Vascular endothelial growth factor upregulates pigment epithelium-derived factor expression via VEGFR-1 in human retinal pigment epithelial cells

We previously demonstrated that differentiated retinal pigment epithelial (RPE) cells express high levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), and a critical balance between VEGF and PEDF is important to prevent the development of choroidal neovascularization. We report here that VEGF secreted by RPE cells upregulates PEDF expression via VEGFR-1 in an autocrine manner. PEDF mRNA and protein expression was downregulated by neutralizing antibody against VEGF in differentiated human RPE cells. VEGFR-1 neutralization decreased PEDF mRNA and protein expression whereas anti-VEGFR-2 antibody had no effect. Addition of placenta growth factor (PlGF) restored PEDF expression in the presence of anti-VEGF antibody. These results demonstrate a regulatory interaction between angiogenesis stimulators and inhibitors to maintain homeostasis in normal human retina.

Vascular endothelial growth factor enhances endothelial cell survival and tumor radioresistance.

Vascular endothelial growth factor (VEGF) is an important mediator of endothelial cell proliferation and survival. The purpose of the present studies was to investigate the role of VEGF in the tumor response to ionizing radiation. Two ras-transformed murine fibrosarcoma cell lines, VEGF+/+ and VEGF-/- were exposed to ionizing radiation (0, 1, 3, 5, 7 or 9 Gy) in vitro, and clonogenic survival was determined. VEGF+/+ and VEGF-/- xenografts were generated in athymic nude mice and then treated with ionizing radiation (ten 5-Gy fractions = 50 Gy). Mean fractional tumor volume was used to evaluate treatment efficacy. To determine whether VEGF enhances tumor radioresistance by targeting endothelial cells, we performed clonogenic survival assays with human umbilical vein endothelial cells. Surviving fractions were calculated after treatment with ionizing radiation (5 Gy) and recombinant hVEGF165 (0, 1, 10, and 100 ng/mL). To determine whether VEGF neutralization enhances tumor radiosensitivity, we employed anti-VEGF165 monoclonal antibody to treat human tumor xenografts. Tumors were exposed to ionizing radiation (four 5-Gy fractions = 20 Gy) and treated with anti-VEGF antibody (0, 5, and 25 microg/kg in four intraperitoneal doses). Mean fractional tumor volume was used to evaluate treatment efficacy. To elucidate the molecular mechanism contributing to the observed anti-VEGF/ionizing radiation interaction, we exposed human umbilical vein endothelial cells to ionizing radiation (5 Gy) in the presence of anti-VEGF antibody (1 microg/mL). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of cell lysates was probed for mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK1/MEK2). The in vitro radiosensitivities of the VEGF+/+ and VEGF-/- clones were equivalent (D0 = 146 vs 149). However, the VEGF+/+ xenografts were more resistant to the cytotoxic effects of ionizing radiation than the VEGF-/- xenografts. VEGF+/+ xenografts demonstrated a faster doubling time (4.5 vs 6.0 days) and a shorter growth delay (15 vs 23 days) than VEGF-/- xenografts. The surviving fraction of human umbilical vein endothelial cells after exposure to ionizing radiation was significantly enhanced in the presence of VEGF (6.4% vs 12.5%). Western blot analysis demonstrated that stimulation of MAPK and MEK1/MEK2 was abrogated after exposure to anti-VEGF antibody. These findings represent the first genetic evidence that factors other than inherent tumor cell radiosensitivity are important determinants of radiocurability. Antitumor strategies targeting VEGF and other endothelial cell survival mechanisms may be used to enhance the cytotoxic effects of radiotherapy.