An intradermal assay for quantification and kinetics studies of tumor angiogenesis in mice

A method is reported for the study of early phases of neovascularization in syngeneic murine tumors and human tumor xenografts in nude mice. Using this method, the effect of irradiation of tumor cells or tumor bed on tumor angiogenesis was studied. Tumor cells were injected intradermally in the abdominal skin flap, which was reopened at 2-day intervals to quantify newly formed blood vessels at the site of tumor cell injection. Both tumor cell injection and blood vessel counting were performed under a dissecting microscope. Using three syngeneic murine tumors and two clones of a human colonic adenocarcinoma, it was observed that new blood vessels started appearing within a few days after tumor cell injection and that this event preceded measurable tumor growth. The number of blood vessels increased exponentially for several days but then their further growth slowed. The extent of angiogenesis depended on the tumor type and the number of tumor cells injected. The exposure of the skin flap to ionizing radiation prior to tumor cell injection reduced neovascularization. We further observed that heavily irradiated tumor cells retained their ability to induce angiogenic response and that lymphoid cells (peritoneal exudate and spleen cells) could also elicit an angiogenic response, although it is weaker than the response elicited by tumor cells. Thus this method is suitable for quantification and kinetics of early phases of tumor angiogenesis in individual mice bearing transplants of syngeneic tumors or human tumor xenografts, and it can be useful for investigating various regulators of tumor angiogenesis.