Figure 4.

MCAs^TC/CAF have a higher invasive capacity than MCAs^TC. (A) Schematic diagram of MCA formation in hanging drops. (B) MCAs^TC/CAF and MCAs^TC morphology by fluorescence microscope. CAFs and SKOV3 cells were labeled with FM4-64 (red) and GFP (green), respectively. Scale bar, 50 µm. (C) Identification of primary mesothelial cells. Mesothelial cells are cytokeratin 8- and vimentin-positive. Scale bars, 50 µm. (D) Schematic diagram of mesothelial clearance assays. (E) Representative images of mesothelial clearance assays with SKOV3 MCAs^TC/CAF and MCAs^TC at 0, 2, 4 and 7 days. Mesothelial cells and SKOV3 cells were labeled with FM4-64 (red) and GFP (green), respectively. (F) Histograms show that SKOV3 MCAs^TC/CAF have a stronger mesothelial clearance ability than SKOV3 MCAs^TC. (G) In vitro adhesion assays. Histograms show that MCAs^TC/CAF have a stronger collagen adhesion capacity than MCAs^TC. (H) Schematic diagram of adhesion assays in vivo. MCAs were intraperitoneally injected into mice (N=3/group). After 4 h, the mice were sacrificed and the tumor cells bound to the peritoneum were evaluated under a microscope. (I) Representative images of adhesion assays in vivo. Tumor cell attachment to the mesentery, parietal peritoneum and omentum. (J) Histograms show the differences in tumor cell florescence intensity in the mesentery, parietal peritoneum, and omentum between groups. ^*P<0.05, ^**P<0.01 and ^***P<0.001. MCA, multicellular aggregate; CAFs, cancer-associated fibroblasts; TC, tumor cell; GFP, green fluorescent protein.