Fig. 4. Malignant transformation of epithelial MMH-R and fibroblastoid MMH-RT cells analyzed in vitro and in vivo. (A) Phase contrast images depicting the typical colonies formed in vitro by anchorage-independent growth of MMH-R and MMH-RT cells in soft agar. (B) Kinetics of tumor formation in vivo after subcutaneous injection of MMH-R (circles) and MMH-RT cells (squares) into immunocompromized SCID/BALB/c recipient mice. (C) Visualization of endothelial cells in histological sections of tumors by immunological staining with anti-von Willebrand Factor. Insets represent lower magnifications (10x) of histological sections. (D) Dedifferentiation of epithelial MMH-R and fibroblastoid MMH-RT cells after tumor formation in vivo. Histological sections of tumors give rise to poorly differentiated cell carcinomas as shown by immunological staining with ZO-1. The cytoplasmic distribution of ZO-1 appears to be very weak in vascularized MMH-RT-derived tumors, and cell boundary staining is exclusively displayed by endothelial cells (white arrow). (E) Assessment of invasive properties in vitro. The ability of epithelial MMH-R and fibroblastoid MMH-RT cells to migrate through Matrigel matrices as reconstituted basement membranes is shown. Invaded cells on lower surfaces of membranes were visualized by immunofluorescence microscopy of GFP-positive MMH-R and MMH-RT cells.