Figure 4.

Stromal Cells Influence Organoid Branching Morphogenesis via Cell-Cell Contact and Express Unique Factors Compared with Non-prostate Fibroblasts

(A) Bright-field image on sequential days of stromal cell (boxed and zoom) taxis toward an organoid followed by distal bud formation (black arrow). Large scale bar, 200 μm; small scale bar, 50 μm. PrE-1 in co-culture with PrS-2.

(B) PrE-1 organoids grown in mono-culture (mono), Transwell co-culture (Trans), or direct co-culture (Co) with PrS mix 4 stromal cells. Circularity and maximum/minimum radius ratio measurements quantified from triplicate wells per condition; error bars show mean with 95% confidence interval for circularity and standard error of the mean for maximum/minimum radius ratio.

(C) Bright-field and fluorescent images of GFP-labeled PrS mix 3 cells in co-culture with PrE-1 organoids at days 3 and 10. Fluorescent images of GFP-labeled PrS mix 3 cells in co-culture with PrE-1 organoids at day 12 with phalloidin (red) in bottom panel. Scale bars, 200 μm.

(D) Fluorescent image of GFP-labeled PrS mix 3 cells in co-culture with PrE-1 epithelial organoid incorporation of an EdU analog (pink). Scale bar, 200 μm.

(E) Bright-field and fluorescent images of GFP-labeled NIH3T3 cells co-cultured with PrE-1 organoids. Circularity and maximum/minimum radius ratio measurements of organoids in mono-culture and co-culture with NIH3T3 cells. Triplicate wells per condition; error bars show mean with 95% confidence interval for circularity and standard error of the mean for maximum/minimum radius ratio. Scale bar, 200 μm.

(F) Comparative gene expression profiling of the human growth factors and TGF-β pathway by qPCR arrays in PrS mix 1 and NIH3T3 cells grown in 3D conditions. Array normalization details in Methods section.

(G) Circularity and maximum/minimum radius ratio measurements of organoids in mono-culture and co-culture treated with Noggin (NOG) or FGFR inhibitor (BGJ398). PrS mix 4 and PrE-1 organoids used in triplicate wells per condition; error bars show mean with 95% confidence interval for circularity and standard error of the mean for maximum/minimum radius ratio.

p value < 0.1 was considered statistically significant. *p < 0.1, **p < 0.05, ***p < 0.01, ****p < 0.001; ns, not significant.