Extended Data Fig. 6: The CT26 colon carcinoma model displays cross-resistance and an immune-evasive TME.

a, Treatment response of subcutaneously injected CT26 colon carcinoma (CTRL or MEKi, n = 8 tumours) continuously treated with TT; arrow indicating start of therapy. Experiment performed twice; representative example shown. b, Proliferation FC in CT26 colon carcinoma cell lines after 72 h at indicated drug conditions. Line indicating FC in proliferation of N^TT cells on lowest drug condition (n = technical triplicates) (drug concentration: DMSO CTRL, 10 nM, 30 nM, 100 nM, 300 nM MEKi). c, pERK status in N^TT and R^TT CT26 colon carcinoma cell lines, 1-hour post drug exposure. Experiment performed twice; representative example shown. d, Treatment response in mice bearing N^TT and R^TT CT26 (Kras^G12D/G12D Cdkn2a ^-/-) tumours (N^TT and R^TT CTRL, n = 6; N^TT and R^TT anti-PD-1, n = 14 tumours) treated with anti-PD-1; arrows indicate therapy administration. Experiment performed 5 times; representative example shown. P-value: **** 1.5E-8, ns 0.2838. e, MHC-I surface expression of N^TT and R^TT cell lines (baseline and 24 h post 10 ng/ml IFN-γ exposure). Experiment performed 3 times; representative example shown. f, Gene expression changes in N^TT and R^TT CT26 colon carcinoma cell lines treated with IFN-γ. Correlation between genes deregulated in N^TT (x-Axis) and R^TT (y-Axis) cell lines (P < 0.05), dots display individual genes. P-value: <1E-15. g, PCA plot displaying top 500 most variable genes for CT26 colon carcinoma cell lines treated with IFN-γ. h, i T cells in untreated N^TT and R^TT CT26 colon carcinoma tumours assessed by IF staining and (h) quantified separately at tumour margin and centre (n = 3 tumours per condition; all 15 ROI, except R^TT centre n = 16) and (i) displayed as a representative picture (scale bar 100 µm and 20 µm). Experiment performed twice. P-value: ** 0.0017, **** 3E-5. j, CD103⁺ DCs in untreated N^TT and R^TT CT26 colon carcinoma tumours assessed by IF staining and quantified separately at tumour margin and centre (n = 3 tumours per condition, all 15 ROI). P-value: *** 0.0002, * 0.046. k, CD103⁺ DC infiltration N^TT and R^TT tumours of CT26 colon carcinoma, assessed by flow cytometry (n = 16, 14 tumours each). Data represents pool of 2 independent experiments. P-value: * 0.016. l, CD103⁺ DC infiltration N^TT and R^TT tumours of CT26 colon carcinoma, alternative gating strategy displayed in Extended Data Fig. 4h (n = 5, 8 tumours). P-value: ** 0.0081. m, Suppressive myeloid cell infiltration in N^TT and R^TT tumours of CT26 colon carcinoma, assessed by flow cytometry (n = 16, 14 tumours each). Data represents pool of 2 independent experiments. P-value: **** 7E-6. n, MFI of indicated maturation markers on CD103⁺ DCs from N^TT and R^TT CT26 colon carcinoma (N^TT, n = 6; R^TT, n = 8 tumours) assessed by flow cytometry. P-value: * 0.0426, ns 0.1419, * 0.0293. Experiment performed once. Data in (b, d, h, j-n) displayed as mean ± SEM. Data analysis (d) two-way ANOVA (f) two-sided Pearson correlation (h, j-n) two-tailed unpaired t–test with Welch correction for unequal variance or with Mann-Whitney-U-test if not normal distributed. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non-significant.

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