Characterization of abnormal angiogenesis in B16BL6 melanoma

Abstract

[Aim] Since the progression and metastasis of malignant tumors depend on their local microcirculation, we sought to characterize the tumor angiogenesis in a highly metastatic mouse melanoma model, B16BL6 (B16), injected with Matrigel into syngeneic C57BL/6 mice. [Materials & Methods] B16 cells in Matrigel were injected into the dosal subcutis of mice. After various times, the tumor baring mice were injected FITC‐conjugated tomato lectin intravenously to label all blood vessels 10 min before being perfused with 4% PFA. The tumor tissues were removed and either frozen or epon‐embedded for subsequent analyses. For the 3‐dimentional imaging by the confocal laser‐scanning microscopy and transmission electron microscopy, cryosections were made and the imunofluorescent staining for various markers was performed. [Results & Discussion] We found that B16 with Matrigel grew significantly faster than B16 alone and was accompanied by altered tumor angiogenesis. The tumor vessels grew vigorously in the opposite direction to the tumor without invading into the tumor mass until at least day 10. In addition, the vascular branching did not result from sprouting as was seen in B16 alone without Matrigel, but resulted from vascular splitting due to either compression from outside of the vessels or septum formation by endothelial cells. This phenomenon was characteristic of B16 cells, but not of other metastatic tumor cells tested, such as a Lewis lung carcinoma cell line and a hybridoma cell line (ASH‐1). The reduction of various angiogenic factors from Matrigel did not affect the angiogenic patterns and tumor growth. However, the denaturation of Matrigel by repeated freezing and thawing did abolish the peculiar angiogenic patterns of B16 with intact Matrigel. We conclude that the tumor vessels may vigorously alter their angiogenic patterns depending on the local microenvironment.