Figure 5.

Domino evaluation of intercellular interactions through VEGF-A with PDO-CAF co-culture validation. (A) Heatmap plotting mean normalized expression of each ligand for each group, demonstrating VEGF-A as a ligand originating in the epithelial populations with the CAFs receiving this signal from the Peng(10) dataset. (B) VEGF-A expression in human PDAC tissue shown first as H&E stain at 5X magnification with focused section at 40X magnification from H&E and VEGF-A IHC with hematoxylin nuclear stain. H&E and IHC completed on sequentially cut slides. (C) Differential expression by qPCR of VEGF-A in monoculture and co-culture CAFs and epithelial cells after 24 or 96 hours of co-culture. Plotted are the Fold Change values comparing our PDO co-culture to monoculture using GAPDH as an endogenous control. (D) VEGF concentration on ELISA of monoculture and co-culture. N=7 PDO-CAF cultures from 7 distinct patients. (E) Angiogenesis assay in HUVEC cells cultured in organoid, CAF, or co-culture conditioned media (CM). Top: transillumination at 4X magnification after 8 hours in culture. Bottom Left: Quantification of segment length of HUVEC networks following culture for 8 hours with increased segment length after culture in CAF CM with trend towards increased segment length in co-culture CM. Bottom Right: Quantification of Nodes in angiogenesis assay after 8 hours in culture. (F) Representative flow cytometry contour plots examining VEGFR1 and VEGFR2 surface expression in CAF (top) and PDO (bottom) monoculture or co-culture. (G) Quantification of CAF (top) VEGFR1 and VEGF2 percent positive, PDO (middle) percent positive VEGFR1 and VEGFR2 and PDO (bottom) Median Fluorescence Intensity expression in monoculture and co-culture across 5 patients. Colored circles on bar graphs correspond to distinct PDOs across experimental conditions. Comparisons of monoculture and co-culture conditions are statistically supported using the two-tailed students t-test with equal variance in PRISM (V9.2.0 [283]). Significance is measured as: ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant.