Fig 2: CD105pos and CD105neg fibroblasts are identified in normal and tumor-bearing tissues(A) MC analysis of in-vitro-expanded primary fibroblasts. Plots show PDPN and CD105 levels. LIN, EpCAM CD31 CD45.(B) UMAP projection of CAFs from KPC pancreatic (n = 4), KPN colorectal (n = 5), MMTV-PyMT mammary (n = 4), KP lung (n = 4), and BRAFV600E melanoma (n = 3) GEMMs. FlowSOM clusters are color coded. Total of 5 × 105 cell displayed.(C) Stacked bar graphs of GEMM CAF (GCAF) clusters displayed as a fraction of total CAFs. FlowSOM colors based on (B).(D) Heatmap of marker median mass intensities (MMIs) displayed as Z scores. Each GCAF FlowSOM cluster is grouped by unsupervised hierarchical clustering based on marker MMIs. Cell-type annotations based on canonical phenotypic markers are listed. Tumor type/s that the GCAF clusters predominantly arise from are listed.(E) UMAP projection from (B) displaying overlaid signal intensity of CD105 with annotated tumor types.(F) UMAP projection from (B) displaying overlaid signal intensity of example markers. The tumor types of origin are highlighted: Pa, pancreatic; Co, colorectal; Ma, mammary; Lu, lung; Me, melanoma.(G–I) Representative IHC analysis of human colorectal (n = 9), breast (n = 8), and lung adenocarcinoma (n = 6) tumor samples stained for pan-cytokeratin (PCK) (green), vimentin (VIM) (purple), CD105 (yellow), and DAPI (blue). Scale bar, 500 µm.See also Figure S8 and Table S7.

Fig 3: CD105 expression discriminates two distinct CAF populations in murine and human PDA(A and B) Immunohistochemistry (IHC) of human PDA tumor samples stained for pan-cytokeratin (PCK) (green), CD105 (yellow), DAPI (blue) and vimentin (VIM) (A) or podoplanin (PDPN) (B) (purple). Insert is magnified with arrows annotating vessels (right) (B). Representative images of n = 15 tumor samples. Scale bar = 500 µm..(C and D) Fluorescence-activated cell sorting plots (C) and in vitro cultures (D) of CD105pos and CD105neg CAFs. Representative of n = 6 independent experiments. Scale bar, 150 µm.(E–J) RNA-seq expression analysis of paired CD105pos (n = 6) and CD105neg (n = 6) PDA CAFs. Isolations from the same tumor sample are linked. Gene expression calculated as transcripts per kilobase million (TPM). Displaying Eng (the gene encoding CD105) (E), canonical fibroblast genes (F), canonical pericyte genes (G), myCAF- and iCAF-associated genes (H), and genes associated with fibroblast heterogeneity in other studies (I–J).(K) Principal-component (PC) analysis of differentially expressed genes between CD105pos (n = 6, yellow) and CD105neg (n = 6, purple) PDA CAFs. DEGs determined using DEseq2 as >2 fold-change and Benjamini-Hochberg adjusted p < 0.05. Paired CAFs from the same tumor are linked.(L) Ingenuity Pathway Analysis of CD105pos (Yellow) and CD105neg (purple) CAF DEGs, displaying upstream activators.(M) Heatmap of expression levels of all 1007 CAF DEGs, displayed as row Z scores. Example DEGs are highlighted.(N–P) CD105pos PDA CAF DEGs (N), CD105neg PDA CAF DEGs (O), and CD105neg CAF DEGs associated with mesothelial cell identity (P). Gene expression calculated as TPM. Isolations from the same tumor sample are linked.Samples are compared using paired t tests (D–I and M–O). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.See also Figure S4 and Table S3.

Fig 4: Differential signaling engagement of CD105pos and CD105neg PaFs(A) MC analysis of CD105pos and CD105neg PaFs signaling. Data are displayed as median mass intensities (MMI) and column Z scores. Specific phosphorylation sites are annotated in brackets.(B–D) RNA-seq analysis of CD105pos and CD105neg PaFs stimulated as displayed for 6 h (n = 3). DEGs were identified using DEseq2 with Benjamini-Hochberg adjusted p < 0.05. Data are displayed as Venn diagrams (top), with example genes listed (below). Unique DEGs of CD105pos PaFs in red, CD105neg PaFs in blue, and shared in purple. Numbers of significant DEGs are displayed in parenthesis.(E and F) Expression of myCAF (E) and iCAF (F) genes from CD105pos and CD105neg PaFs stimulated with TGF-ß1 or IL-1a (n = 4) for 3 days. Eng expression is also shown. Data displayed as mean ± SD.Samples were compared using unpaired t tests (E and F). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.See also Figure S6 and Table S5.

Fig 5: Phenotypic plasticity of mesenchymal marker expression(A) Flow cytometry analysis of PDPN and CD105 in purified and in-vitro-cultured CD105pos and CD105neg pancreatic fibroblasts (PaFs) after 1 and 7 weeks. Plots are representative of n = 4 experiments. Relative frequencies shown in relevant quadrants.(B) Normalized Eng mRNA expression in purified CD105pos (n = 4) and CD105neg PaFs (n = 4) treated with control (top) or KPC PDA conditioned medium (bottom). Data displayed as mean ± SD.(C) Representative flow cytometry analysis (n = 4) of CD105 on GFPposCD105pos and GFPposCD105neg PaFs in mono- or co-culture with RFPpos KPC PDA tumor cells.(D) Representative flow cytometry analysis (n = 3) of CD105 in isolated CD105pos and CD105neg human PaFs after >3 weeks of in vitro culture.(E and F) MC analysis of primary PaFs treated with the indicated ligands for 3 days. Representative plots displaying relative frequencies of CD105pos and CD105neg PaFs.(G and H) Heatmap of median marker intensity (MMI) displayed as column Z scores for each phenotypic marker on CD105pos (G) and CD105neg (H) PaFs after 3 days of treatment as indicated. Boxplots show MMI with upper and lower boundary of the interquartile range and whiskers denoting maximum and minimum values minus outliers, across all conditions.(I and J) Representative flow cytometry analysis (n = 3) of CD105pos (I) and CD105neg (J) PaFs with IFN-?, IFN-? + KPC PDA conditioned medium, or IFN-? + TGF-ß1 treatment.Samples are compared using unpaired t tests (B) (top and bottom). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.See also Figure S5 and Table S4.