CAFs form extensive filopodial contacts with AGS cells and have up-regulated ROR2 expression on cytoneme tips. (A) pCAF cells (C) transfected with membrane GFP form an extensive and complex network of filopodia which engulf neighboring AGS cells (A) in 2D cell culture. The scale bar represents 10 µm. (B) primary gastric cancer–associated myofibroblasts (gCAM) (C) transfected with membrane GFP extend multiple filopodia in 3D culture that contact AGS cells (A) transfected with membrane mCherry, and transport multiple membrane-bound vesicles from producing to receiving cell (SI Appendix, Fig. S1A). The scale bar represents 10 µm. (C) mRNA levels of the indicated gene are shown as fold change away from AGS. Wnt5a is shown in red, with values on the left Y axis. ROR2 is shown in blue, with values on the right Y axis. gNM, primary gastric normal myofibroblasts; gCAM6, primary gastric cancer–associated myofibroblast patient 6; gCAM13, primary gastric cancer–associated myofibroblast patient 13; pCAF2, pancreatic CAF cell line 2. Values shown were calculated using the 2−ΔΔCt method and are plotted on a log10 scale. Comparison of 40-Ct values for all cells is shown in SI Appendix, Fig. S1 B and C. (D) Immunofluorescence on pCAF2 cells fixed using a previously described method to preserve filopodia. The left-hand column indicates WNT5A/B antibody staining, the center panel indicates ROR2 antibody staining, and the right-hand panel indicates WNT5A/B and ROR2 antibody staining (blue and red, respectively) merged with Phalloidin FITC shown in green. Three representative experiments are shown, and yellow arrowheads indicate WNT5A/ROR2 clusters. Antibody controls are shown in SI Appendix, Fig. S1 G and H. The scale bar represents 10 µm.
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