Fig 1: vCAF and mCAF marker genes can be used to trace back subpopulations in tissue sections. a Violin plots of selected vCAF differentially expressed genes in log2(RPKM + 1). Violin colors represent mean expression of each population. Genes were sorted based on gene ontology terms. b Immunohistochemistry (IHC) staining of desmin on MMTV-PyMT tumor sections (6 µm). Images were acquired from the leading edge and the tumor center. Yellow boxes (left) indicate 2× magnified area (right). c IF staining of Nidogen-2 (green) and CD31 (magenta) or PDGFRa (red) on MMTV-PyMT tumor sections (5 µm) from mice of age 8 weeks, 12 weeks, and 15 weeks (top to bottom). Nuclei were counterstained with DAPI. d Immunofluorescence (IF) staining of Nidogen-2 on human tumor tissue (5 µm). Nuclei were counterstained with DAPI. e Violin plots of selected mCAF differentially expressed genes in log2(RPKM + 1). f IHC staining of fibulin-1 and PDGFRa in MMTV-PyMT tumor sections (6 µm). Images were acquired from the leading edge and the tumor center. IHC staining of fibulin-1 (g) and PDGFRa (h) in human tumor tissue sections (6 µm). FACS-sort of MMTV-PyMT tumor (i) and mammary gland (j) tissue. Gating on single, living CD45-CD31-NG2-EPCAM- cells followed by gating on PDGFRa+ cells (blue box) or PDGFRa- cells (red box). k Violin plots of cell cycle gene expression in log2(RPKM + 1). l IF staining for Nidogen-2 (red) and Ki-67 (grey) on sections (5 µm) from PDGFRa-EGFP (green) reporter mice. Arrows indicate Nid2+Ki67+ cCAF. Scale bar: 50 µm
Fig 2: dCAF express the oncogenic driver gene and share gene expression with the tumor epithelium. a Violin plots of selected dCAF differentially expressed genes in log2(RPKM + 1). Violin colors represent mean expression of each population. Genes were sorted based on gene ontology terms. b IF staining of SCRG1 (green) and EPCAM (magenta) on MMTV-PyMT tumor sections (5 µm) from mice of age 8 weeks, 12 weeks, and 15 weeks (left to right). Nuclei were counter stained with DAPI (blue). Yellow boxes indicate the area magnified in the lower panel. c Expression plots based on t-SNE clustering. log2(RPKM + 1) levels of the virus-derived oncogenic driver gene in individual cells. d IF staining of Nidogen-2 (cyan) and SCRG1 (red) on MMTV-PyMT tumor tissue derived from PFGFRa-EGFP (green) reporter mice. Nuclei are stained with DAPI (blue). White arrowheads and stars indicate mCAF and dCAF, respectively. e IF staining of Nidogen-2 (cyan) and SCRG1 (red) on MMTV-PyMT tumor tissue derived from PFGFRa-EGFP (green) reporter mice. The image was acquired by 2-photon microscopy. Collagen fibers were detected by second harmonic generation (SHG, magenta). Dotted lines separate malignant tissue (T) from stroma (S). Scale bar: 50 µm
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