Platelets Regulate Tumor-Induced Angiogenesis

The hypothesis that tumors require a vascular network to grow beyond a critical size is now widely accepted. There is also circumstantial evidence implicating the blood platelet in the process of angiogenesis. We have previously shown that the platelet sequesters pro- and anti-angiogenic proteins in discrete subsets of alpha granules and differentially releases them in vitro in response to PAR1 and PAR4 activating peptides. We have extended these studies by injecting two murine -- Lewis Lung Carcinoma (LLC), B16 F10 melanoma and a human renal cell carcinoma (RCC 786-O) into the perivitelline space of wild type and Fli1-GFP transgenic zebrafish embryos 48 hours post fertilization (hpf). One day after injecting 2000 tumor cells suspended in Matrigel, a network of new blood vessels had grown up toward the site of tumor injection. By 3 to 4 days post-injection (dpi) the vascular network had entered and enveloped the tumor mass. We then blocked the production of zebrafish thrombocytes, the fish equivalent of mammalian platelets, by injecting an anti-sense morpholino directed against c-mpl, the thrombopoietin receptor, into single cell embryos followed, at 48 hpf, by the injection of tumor cells. We have previously shown that this c-mpl morpholino inhibits thrombocyte production for 5–7 days. The initial wave of angiogenesis was normal in all of the c-mpl morphants but there was almost complete inhibition of subsequent angiogenesis and blood vessel extension into the LLC, B16 melanoma and RCC 1080 tumors. In contrast, control fish that had received a scrambled morpholino and had a normal number of circulating thrombocytes, had normal angiogenesis within the tumor mass. We then transplanted LLC and B16 F10 cells into CD41-GFP embryos that have fluorescent thrombocytes in their circulation. Three dpi we observed the arrest and adhesion of GFP⁺ thrombocytes to vessels within the tumor mass. We also noted that thrombocytes were present in tumor vessels before blood flow had been established. We conclude that platelets/thrombocytes play an important role in TIA and may serve as a vehicle for the delivery of angiogenic factors to human and murine tumors. Thrombocytes appear to be particularly important for the penetration of new vessels into the tumor mass. These studies raise the interesting possibility that inhibiting platelet production or granule secretion might help to inhibit TIA.