Fig 2: Impact of O2 tension during tumor collection and processing on signaling networks.(A) Schematic view of experimental design. (B) Heatmap shows elevated protein levels under physioxia compared to ambient air. (C) Heatmap shows the top 40 differentially expressed phosphoproteins in EpCAM+ PyMT tumor cells under physioxia compared to ambient air. (D) STRING network analysis revealed that phosphoproteins described in (C) are part of RNA splicing machinery. (E) Wnt signaling network showed lower activity under physioxia compared to ambient air (n = 3). (F) Levels of total YAP1 protein and its downstream target CYR61 were lower in EpCAM+ PyMT tumor cells under physioxia compared to ambient air (n = 3). (G) Nuclear and cytoplasmic extracts were analyzed for the levels of active and total YAP1. Poly(adenosine diphosphate–ribose) polymerase 1/2 (PARP1/2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as loading controls for nuclear (NP) and cytoplasmic (CP) extracts, respectively. (H) EpCAM+ tumor cells processed under ambient air contained higher levels of NRF2 but lower levels of its degrader, KEAP1. PARP1/2 and GAPDH were used as loading controls for nuclear and cytoplasmic extracts, respectively (n = 4). (I) Nuclear extracts of PyMT tumor cells processed under ambient air displayed higher levels of NRF2 activity compared to physioxia (n = 4). (J) PyMT tumor cells processed and grown under ambient air displayed a higher proliferation rate compared to physioxia (n = 6). ***P < 0.001 by ANOVA.

Fig 3: 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 4: Unbiased clustering of fibroblast single cell transcriptomic data reveals four populations. a Schematic representation of negative selection strategy removing CD31+, CD45+, NG2+, and EPCAM+ cells. b Gating strategy and quantification of flow cytometry for single cell sequencing. After gating out doublets and DAPI+ dead cells, EpCAM−CD31−CD45−NG2− CAFs made up 2.5% of the cells. FSC forward scatter, SSC side scatter. For single marker staining see also Supplementary Figure 1. c Violin plot of detected genes in 784 sorted fibroblasts. d t-SNE layout of CAFs (n = 716) by RPKM-normalized transcriptomic data. Colors represent clusters assigned by DBSCAN. e Expression plots on t-SNE layout. log2(RPKM + 1) levels of CAF marker genes in individual cells. f Cell size and granularity as determined by forward-scattered light (FSC) and side-scattered light (SSC) of different CAF populations

Fig 5: Phenotypical plasticity of Lgr5+ cells that reside in Porcupine+ niches in lung, pancreatic and colon tumoursa, Immunofluorescence for GFP (green) and EpCAM (red) in a subcutaneous transplant established from a single-cell clone of KrasG12D/+; Trp53?/?; Lgr5CreER/+ cell line. Scale bar: 1 mm. b, Immunofluorescence for GFP (green) and Porcupine (red) in a subcutaneous transplant established from a single-cell clone of KrasG12D/+; Trp53?/?; Lgr5CreER/+ cell line. (a, b) are representative data from four replicate experiments, each with a different KrasG12D/+; Trp53?/?; Lgr5CreER/+ cell line. c, ISH for Lgr5 mRNA (purple) in KP; Pdx1::Cre pancreatic ductal adenocarcinoma (PDAC). Scale bars: 100 µm (top) and 10 µm (bottom). Representative data from 3 PDAC tumors analysed. d, Immunofluorescence staining for GFP (green) in a tdTomato+ (red) autochthonous KP; Lgr5GFP-CreER/+; Rosa26tdTomato/+; Pdx1::Cre PDAC. Scale bars: 100 µm (top) and 10 µm (bottom). e, Quantification of primary spheroids containing EdU+ cells per 100 Lgr5+/tdTomato+ or Lgr5-/tdTomato+ primary mouse PDAC cells plated. N = 4 wells/group. *P < 0.05, Student’s two-sided t-test; error bars: +SD. f, Immunofluorescence staining for GFP (green) and Porcupine (red) in autochthonous KP; Lgr5GFP-CreER/+; Pdx1::Cre PDAC. Note juxtaposition of Lgr5+ and Porcupine+ cells in the tumours. Scale bars: 100 µm (top) and 10 µm (bottom). (d, f) Representative data from 6 KP; Lgr5GFP-CreER/+; Pdx1::Cre PDAC tumors analysed. g, Immunofluorescence staining for GFP (green) and Porcupine in an autochthonous Apc?/?; Lgr5GFP-CreER/+intestinal adenoma. Again, note juxtaposition of Lgr5+ and Porcupine+ cells in the tumours. Scale bar: 100 µm. N = 3 tumor samples. h, Immunohistochemistry for Porcupine (brown) in human colorectal adenocarcinoma. Scale bars: 100 µm (top) and 10 µm (bottom). 5 human colorectal adenocarcinoma samples were analysed.