Extended Data Fig. 5: Spatial distribution of IL-1β⁺ TAMs.

a, Left panels. Selected regions of interest showing signal intensity (IF staining) of KRT19 (tumor cells), F4/80 (macrophages), PDGFR-α (fibroblasts) and IL-1β in mouse PDAC (orthotopic KPC, end-stage). Right panels. Quantification of macrophages (cells/mm²) in stromal or tumor areas (n = 2 mice, n = 4 sections/mouse, n = 10 ROI/area). ****p < 0.0001 (unpaired student’s two-tailed t test). b, Left panels. Selected regions of interest showing signal intensity (IF staining) of FOLR2, F4/80, IL-1β, and DAPI in control pancreas (day 0), mouse PDAC (orthotopic KPC), or normal adjacent tissue (NAT) at the indicated time points. Right panels. Quantification of IL-1β⁺ and FOLR2⁺ macrophages (cells/mm²) in stromal, tumor areas or NAT areas. Ctrl pancreas, n = 2 mice, n = 2 sections/mouse, n = 10 ROI/section; Day 15 PDAC, n = 4 mice, n = 2 sections/mouse, n = 5 ROI/areas; Day 30 PDAC, n = 5 mice, n = 2 sections/mouse, n = 10 ROI/areas. c, Selected regions of interest with inset magnifications showing signal intensity (IF staining) of KRT8-18, CD163, NLRP3, and DAPI (middle), or CD163, FOLR2 and DAPI (right) on consecutive sections of human PDAC samples. d, Annotation (see Methods) of tumor or stromal areas in spatial transcriptomics data (Visium) for the indicated tissue sections of mouse PDAC (orthotopic KPC, end-stage). e, Signal intensity (IF staining) of KRT19, F4/80, PDGFR-α and DAPI for the indicated tissue sections of mouse PDAC (orthotopic KPC, end-stage). f, Percentages (DestVI deconvolution) of tumor cells (left), macrophages (middle) and fibroblasts (right) in spatial transcriptomics data (Visium) for the indicated tissue sections of mouse PDAC (orthotopic KPC, end-stage). g, Percentage of spots with concordant annotation as stroma or tumor by spatial transcriptomics (Visium) and IF staining for the indicated tissue sections of mouse PDAC (orthotopic KPC, end-stage). h, Correlation values (red, positive; blue, negative; white, non-significant) between mean gene expression (Visium) of marker genes of the indicated TAM subsets (DestVI generative model, see Methods) and spatial principal components (sPC) coordinates for spots of a selected tissue section (A1) of mouse PDAC (orthotopic KPC, end-stage). i, Coordinates of spatial Principal Component 1 (sPC1) of macrophage-enriched spots (DestVI deconvolution) in spatial transcriptomics data (Visium) for the indicated tissue sections of mouse PDAC (orthotopic KPC, end-stage). j, Scaled mean expression of marker genes of IL-1β⁺ TAMs (DestVI generative model) in macrophage-enriched spots (DestVI deconvolution) in spatial transcriptomics data (Visium) for the indicated tissue sections of mouse PDAC (orthotopic KPC, end-stage). k, Enrichment (PAGE) of expression of marker genes of IL-1β⁺ TAMs (left) or FOLR2⁺ TAMs (right) in macrophage-enriched spots (DestVI deconvolution) in spatial transcriptomics data (Visium) for the indicated tissue sections of mouse PDAC (orthotopic KPC, end-stage). Colors indicate macrophage-enriched ST spots with significance (estimated on all spots) of p < 0.001. l, Enrichment (PAGE) of expression of PGE₂ + TNF-α synergized genes in macrophage-enriched spots (DestVI deconvolution) in spatial transcriptomics data (Visium) for the indicated tissue sections of mouse PDAC (orthotopic KPC, end-stage). Colors indicate macrophage-enriched ST spots with significance (estimated on all spots) of p < 0.001. m, Louvain clustering of spots in spatial transcriptomics data (Visium) for a selected tissue section (A1) of mouse PDAC (orthotopic KPC, end-stage). n, Enrichment (PAGE) of expression of genes belonging to the indicated GO BPs in spatial transcriptomics data (Visium) for the indicated tissue sections of mouse PDAC (orthotopic KPC, end-stage). GSEA was performed on genes ranked by log₂FC between cluster 4 versus other spots. Colors indicate spots with significance (estimated on all spots) of p < 0.001. o, Signal intensity (IF staining) of F4/80, IL-1β, and DAPI (left), or of CD31, VEGFR2 and KRT19 (middle) in consecutive sections of mouse PDAC (orthotopic KPC, end-stage). Arrows indicate IL-1β⁺ F4/80⁺ cells (left) and CD31⁺ VEGFR2⁺ cells (middle). Quantification of IL-1β⁺ F4/80⁺ cells in areas with high and low density of CD31⁺ VEGFR2⁺ cells (right, n = 5 mice, n = 2 sections/mouse). ***p < 0.001 (unpaired student’s two tailed t test). p, Signal intensity (IF staining) of F4/80, IL-1β and KRT19 (left), or of anti-Hypoxyprobe (middle) in consecutive sections of mouse PDAC (orthotopic KPC, end-stage). Quantification of IL-1β⁺ F4/80⁺ cells in hypoxic and non-hypoxic areas (right, n = 3 mice, n = 2 sections/mouse). *p < 0.05 (unpaired student’s two tailed t test).

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