Figure 6. Preadipocytes contribute to vitamin D₃-induced growth inhibition of mammary organoids.

VDR WT (A–G) and KO (H–N) organoids (isolated mammary ductal structures, cleared of surrounding adipose) cultured in Type I Collagen in the presence of vehicle (ethanol) (Day 0 (A & H) or Day 5 (B & I)), 25D₃ (100nM) (C & J), 1,25D₃ (100nM) (D & K). WT organoids (C) show a slight reduction in branching with 25D₃ (100nM) treatment, suggesting bioactivation of vitamin D₃ via the mammary ductal epithelial cells compared to KO organoids (J) that show no reduction in size of branching compared with the ethanol control groups (B & I, respectfully). Further reduction in WT organoid (D) size and branching was seen with 1,25D₃ treatment compared to KO organoid (K) and ethanol control groups (B & I). VDR wild type adipocytes (arrowheads, visible in F,G,M,N) cultured with VDR WT (E–G) or KO (L–N) organoids (arrows) embedded in Type I Collagen in the presence of vehicle (ethanol) (Day 5 (E & L)), 25D₃ (100nM) (F & M), 1,25D₃ (100nM) (G & N). Note the reduction in WT and KO organoid size and ductal branching when cultured with wild type adipocytes in the presence of 25D₃ (100nM) (F & M) compared with the ethanol control groups (E & L) and without adipocyte co-culture (C & J), suggesting bioactivation via wild type adipocytes. There was also a similar reduction in WT and KO organoid growth when treated with 1,25D₃ (G & N), suggesting VDR signaling and growth inhibitory secretion via WT adipocytes that results in growth inhibition of VDR KO organoids. n=3–5 organoids/genotype/treatment and n>5 branches measured/organoid.