High expression of αSMA is a distinctive property of periglandular CAFs in mouse and human PDA. (A, left) Representative immunofluorescence (IF) co-staining of FAP (green) and αSMA (red) in a well-differentiated human PDA (n = 4). Counterstain, DAPI (blue). (right) Higher magnification illustrating the distribution and co-localization of FAP and αSMA. Bars, 50 µm. T, tumor glands. (B) Representative image of RNA ISH for Cytokeratin 18 (KRT18, blue) and αSMA (ACTA2, red) transcripts in a well-differentiated human PDA (n = 3). Bar, 50 µm. T, tumor gland. (C, left) Representative image of RNA ISH for Fap (blue) and Acta2 (red) in a KPC mouse tumor (n = 3). (right) Higher magnification. Bars, 25 µm. T, tumor glands. (D, top) Representative image of fluorescent RNA ISH for Fap (green) and Acta2 (red) in a KPC mouse tumor (n = 3), showing transcript distribution across three cell layers of the stroma, starting from the first layer adjacent to the tumor gland (T) and moving outwards. Counterstain, DAPI (blue). Bar, 50 µm. (bottom) Quantification of Fap and Acta2 fluorescence intensity in the three cell layers. Results show mean ± SD of three tumor glands. Data are normalized to layer 1. ***, P < 0.001, unpaired Student’s t test. (E) Representative images of IHC of αSMA and YFP in sequential tissue sections from KPCY mice, with either preinvasive Pancreatic Intraepithelial Neoplasia (PanIN) or invasive cancer (n = 2). Arrows indicate areas of myCAFs. Bar, 50 µm.