A QUANTITATIVE ASSAY USING BASEMENT-MEMBRANE EXTRACTS TO STUDY TUMOR ANGIOGENESIS IN-VIVO

We describe a quantitative assay for assessing tumor angiogenesis in v ivo using basement membrane extracts (Matrigel). Nude mice were inject ed s.c. with liquid Matrigel mixed with HT1080 human fibrosarcoma cell s. Since Matrigel rapidly forms a solid gel at body temperature, the g el containing tumor cells can be removed immediately and then processe d for histological studies. Tumor angiogenesis was monitored quantitat ively by measuring both the number and the total area of neovessels pr esent in the gels using an image analyzer, which could be achieved app roximately 72 hr later. Furthermore, HT1080 cell-conditioned medium, w hich may contain various tumor-derived factors, promoted the basement membrane degradation, migration, proliferation and tube formation of e ndothelial cells in vitro, as did Matrigel, although to a lesser exten t. In addition, Northern blot analysis demonstrated that the amount of vascular endothelial growth factor (VEGF) mRNA in HT1080 cells was mu ch higher than that in human fibroblasts or NIH3T3 cells. Our results suggest that angiogenesis observed in our assay may be due to the syne rgic effects of tumor angiogenic factors such as VEGF, and Matrigel. T he advantages of our assay are: 1) it is possible to assess early angi ogenesis quantitatively; and 2) this assay may be applicable for scree ning anti-angiogenic therapeutic agents to be used against human neopl asms. (C) 1996 Wiley-Liss, Inc.