Tumor growth requires angiogenesis, the development of new blood vessels from existing blood vessels. Cancer cells release proangiogenic growth factors such as vascular endothelial growth factor (VEGF) to drive angiogenesis. Current cancer therapy that incorporate targeting agents that inhibit the production of new blood vessels have been only marginally successful. We hypothesize that intracellular signaling mechanisms downstream of proangiogenic factor receptors may differ in tumor-derived endothelial cells compared to normal endothelial cells, and may account for the limited effectiveness of antiangiogenic therapies in vivo. VEGF activates VEGF receptor-2 (VEGFR2) leading to elevated cytosolic calcium (Ca2+). In endothelial cells (ECs), increased cytosolic Ca2+ is required for VEGF-induced EC proliferation and migration, and is dependent on activation of phospholipase C-gamma (PLCg). Treatment with a PLCg blocker prevents the VEGFR2-induced increase in cytosolic Ca2+, but does not alter baseline Ca2+ levels. Here, we compared VEGF-induced Ca2+ influx in human umbilical vein endothelial cells (HUVECs) versus breast tumor ECs (TECs). TECs were purified from TG1-1 tumors grown in mammary fat pads of TIE2-GFP mice. To measure changes in cytosolic Ca2+, GFP-expressing TECs loaded with the Ca2+ indicator dye INDO-1 were imaged using two-photon laser scanning microscopy (TPLSM). For imaging, the cells were placed in a perfusion system that delivers reagents at a constant flow rate with constant aspiration for continuous fluid exchange. Changes in fluorescence intensity in response to VEGF were measured in individual GFP+ cells over time. In HUVECs, the VEGF-induced increase in intracellular Ca2+ is detected only after 18-hour serum deprivation (serum starved). In contrast, both serum starved and non-serum starved TECs respond to VEGF with increased intracellular Ca2+, but the magnitude of the response in both TEC groups is significantly lower than the serum-starved HUVECs. We hypothesized that changes in the VEGF receptor expression attenuated the response in TECs. By flow cytometry, VEGFR1 expression was elevated in TECs, while VEGFR2 expression was decreased compared to HUVEC. This change in receptor expression may account for the attenuated VEGF response because VEGFR1 has lower signaling capacity compared to VEGFR2. Furthermore, in nonstimulated, non-serum starved TECs the PLCg blocker U73122 altered baseline Ca2+, suggesting that constitutively active PLCg contributes to elevated baseline Ca2+ in TECs. These results demonstrate fundamental differences in VEGF signaling between HUVECs and TECs that may account for the limited success with antiangiogenic therapies that target VEGF or VEGFR2. With the powerful depth and resolution advantages of multiphoton microscopy, we will begin to determine if such abnormalities in intracellular signaling of TECs observed in vitro are also observed in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5298. doi:1538-7445.AM2012-5298