Extended Data Fig. 3: Related to Fig. 2. Kupffer cells engulf tumour cells.

a- Selected pathways (see Methods) upregulated in RNA-seq analysis of KCs from C57BL/6j mice 2 weeks after intra-portal injection of 1 × 10⁶ KPC-1 cells, in comparison to no injection, n = 3 mice per group, (Supplementary data T 2). b- Scatterplot of differentially expressed genes (adj. p-values are obtained using Benjamini and Hochberg method for multiple testing and considered significant when adj. p < 0.05) in KCs from tumour-bearing mice, from the RNA-seq analysis in (a). Selected genes are indicated in blue (lectins and chemokines) and in pink (cytokeratins, from engulfed tumour cells). c- Expression of activating and inhibitory receptors by KCs from RNAseq data (Supplementary data  2 and 3) and RTqPCR data (*). Second to 4th column indicates if gene expression is up or down in metastatic liver, fold changes, and adj. p value. Fifth to 7^th column ‘Clec4f^CreId3^f/f’ indicates gene which expression is up or down regulated in Clec4f^CreId3^f/f mice, fold changes, and adj. p value. Column 8 indicates the presence of ITIM or ITAM motifs. d-Heatmap represents the top 100 up and down DEG genes in Tim4⁺CD206⁺ KC cells and Tim4⁺CD206^hi KC respectively, in RNA-seq analysis from (a). e- Representative immunofluorescence staining for CK19, F4/80, Tim4 on frozen liver section from C57BL/6 J mice received 1 × 10⁶ KPC-1 cells through intra-portal injection for 2 weeks. n = 3 mice per group. f- Representative whole mount immunofluorescence imaging and quantification of tdT⁺% cells in Tim4⁺ KCs from metastatic liver, from C57BL/6 J mice which received 2 × 10⁵ KPC-2-luci-tdT cells through pancreatic orthotopic injection and are analysed weekly for 2 months. n = 3 mice per group. g- Representative immunofluorescence staining for CK19, Tim4, F4/80, quantification of CK19 relative MFI in Tim4⁺ KCs on liver samples from KPC mice (p48^Cre Kras^LSL-G12Dp53^LSL-R172H) and control mice (Kras^LSL-G12Dp53^LSL-R172H). Dots represent individual KCs from 3 mice per group. h- Analysis of livers from mice receiving phosphatidylserine blockade D89E or PBS 6 h before intra-portal injection of 1 × 10⁶ KPC-1-tdT cells and analysed after 24 h. Left: Quantification by Immunofluorescence of Tim4⁺ KCs stained with tdT, n = 4/group; right: quantification of CD45⁻GFP⁺ tumour cells by flow cytometry (right). PBS n = 4, D89E n = 4. Statistics: Kruskal-Wallis test (g), unpaired two-tailed t test (h), Dots represent individual mice (f,h). mean ± sd. ns, not significant.

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