Two mutually exclusive subpopulations of CAFs with reversible features coexist in pancreatic cancer. (A and B) Flow cytometric analysis of αSMA and IL-6 in PSCs cultured alone or in either co-culture (A) or trans-well culture (B) with tumor organoids. Red frame indicates the gate defining myCAFs (αSMA^high IL-6^low) and black frame indicates the gate defining iCAFs (αSMA^low IL-6^high). Numbers indicate percentage of cells within marked gate. Graphs on the right are showing the fold change induction of myCAFs and iCAFs in co-culture, normalized to PSCs in monoculture. Results show mean ± SD of four (A) or two (B) biological replicates. *, P < 0.05; **, P < 0.01, unpaired Student’s t test. (C) Fluorescent RNA ISH of fixed and sectioned co-cultures for Il6 (red) and Krt18 (green), illustrating the spatial distribution of IL-6⁺ PSCs (iCAFs) with respect to KRT18⁺ tumor organoids (n = 2). Higher magnification on the right. Counterstain, DAPI (blue). Arrow indicates example of an iCAF. Bars, 50 µm. (D) qPCR analysis of interleukins (Il6 and Il11) and markers of fibroblast (Pdgfra, Pdgfrb, Acta2, and Fap), epithelial (Krt19) and macrophage (CD11b) lineages in samples of primary cells sorted from KPC mouse tumors. Sorting was performed using three markers: PDGFRα (CD140a) for fibroblasts (n = 3), EpCAM for epithelial cells (n = 3) and CD45 for immune cells (n = 2). Results show mean ± SD of two to three biological replicates. All gene expression changes are statistically significant when compared with the reference population, P < 0.01, unpaired Student’s t test. (E) Representative image of sequential IHC for IL-6 (purple) and PDGFRβ (brown) in a KPC mouse tumor (n = 3). Arrows indicate double positive cells. T, tumor gland. Bars, 50 µm. (F, left) Representative image of sequential IHC for PDGFRβ (gray), IL-6 (brown), and Ki67 (purple) in a KPC mouse tumor (n = 3). Arrows indicate examples of triple positive cells. Bar, 50 µm. (right) Quantification of Ki67 staining in PDGFRβ⁺/IL-6⁺ cells (iCAFs), total of 593 cells were counted. (G) Representative image of sequential IHC for IL-6 (brown) and PDGFRβ (purple) in a human PDA (n = 6). Arrowheads indicate double positive cells. T, tumor gland. Bars, 50 µm. (H) Representative image of RNA ISH for Acta2 (blue) and Il6 (red) in KPC mouse tumors (n = 4). Arrows indicate examples of Acta2-positive cells in the periglandular area, arrowheads indicate examples of Il6-positive cells further away from neoplastic cells. Bar, 50 µm. T, tumor glands. (I) Representative IF image for αSMA (green) and IL-6 (red) in a KPC mouse tumor (n = 3). Counterstain, DAPI (blue). Arrowheads indicate examples of αSMA-positive cells in the periglandular area; * indicates examples of IL-6–positive cells further away from neoplastic cells. Bar, 50 µm. T, tumor glands. (J) qPCR analysis of Il6, Il11, Lif, and Acta2 transcript levels in two PSC lines (PSC4 and PSC5) first grown as monocultures in Matrigel (quiescent PSCs), then transferred to trans-well cultures with tumor organoids (iCAFs), and finally plated as monolayer cultures (myofibroblasts). Results show mean ± SD of two technical replicates for each PSC line. **, P < 0.01; ***, P < 0.001, unpaired Student’s t test.