The effects of CAFs on tumor organoid formation and growth. (A and B) Measuring the diameter of organoids under immunofluorescence and bright field vision (n = 6, 5 organoids for each well randomly were measured). (C) Mouse or human tumor organoids cultured with or without corresponding CAFs. (D) Diameters of mouse organoids cultured with or without mouse CAFs (n = 8 experimental settings with 3 biological replicates for each; 5 organoids for each well randomly were measured). (E) Number of mouse organoids cultured with or without mouse CAFs (n = 8 experimental settings with 3 biological replicates for each). (F) Diameters of human organoids cultured with or without human CAFs (n = 7 experimental settings with 3 biological replicates for each; 5 organoids for each well randomly were measured). (G) Number of human organoids cultured with or without mouse CAFs (n = 7 experimental settings with 3 biological replicates for each). (H and I) Diameters of formed organoids in mono- or co-cultures with different concentrations between organoids and CAFs (n = 6; 5 organoids for each well randomly were measured). (J and K) The number of formed organoids in mono- or co-cultures with different concentrations between organoids and CAFs (n = 6). (L) Ki67 staining for mouse organoid mono-culture (magnification, 400×). (M) Ki67 staining for mouse organoids and CAF co-culture (magnification, 400×). (N) Ki67 staining for human organoid mono-culture (magnification, 400×). (O) Ki67 staining for human organoids and CAF co-culture (magnification, 400×). (B and D–K) Data are expressed as means ± SD. Mann–Whitney U tests. ∗∗P < .01, ∗∗P < .05, ∗∗∗P < .001. hCAF, human cancer associated fibroblast; hOR, human organoid; mCAF, mouse cancer associated fibroblast; mOR, mouse organoid; RFP, red fluorescent protein.