Figure 5.

CAF-conditioned media drives resistance to fulvestrant.A, cell viability of MCF7 cells grown in different patient-derived CAF-CM (#1-27) and treated with 500 nM fulvestrant for 72 h. PrestoBlue was used to assess cell viability. Data are presented as mean + SD. Each data point is the mean of one independent experiment. B, representative confocal images of control PDOs or PDO-CAF co-cultures stained for VIM1 and EdU. C and D, quantification of EdU+ (red) cells in control PDOs and PDO-CAF co-cultures (C) and PDOs grown in nonconditioned or CAF-CM media (D), and treated with 500 nM fulvestrant for 96 h. Proliferation was assessed by pulsing PDOs with EdU for 4 h and quantifying the ratio of EdU+ cells per total (DAPI+) number of cells in 20 PDOs. Each data point represents the percentage of EdU+ cells in one PDO, red line indicates the mean ratio of EdU+ cells per treatment group. E and F, heatmap of RNA-seq normalized enrichment scores (NES) for hormone receptor signaling (E) and cytokine signaling (F) pathways in PDOs grown in nonconditioned media or CAF-CM and treated with 500 nM fulvestrant for 96 h. Yellow indicates pathways that were most significantly upregulated and blue indicates downregulated pathways (scale: log2). Black boxes denote unavailable reads. Student’s t test was used to assess significance comparing each CAF-CM to NC sample, and control PDOs to PDO-CAF co-culture. The scale bar represents 40 μm. CAFs, cancer-associated fibroblasts; DAPI, 4′,6-diamidino-2-phenylindole; EdU, 5-ethynyl-2′-deoxyuridine; NC, nonconditioned; PDOs, patient-derived organoids; VIM1, vimentin.