Extended Data Fig. 8: Cross-resistance to immunotherapy is cell intrinsic, acquired during resistance formation and specific to MAPK pathway inhibition.

a, Active response to RAFi in N^TT Braf/Pten tumours (7 doses) and resistance formation upon 27 doses (CTRL, n = 6; 7 doses n = 8, 27 doses n = 10 tumours). b, Characterization of suppressive myeloid cells, T cells and CD103⁺DCs in Braf/Pten tumours actively responding to RAFi (7 doses) and in relapsing tumours, fully resistant to RAFi (27 doses) (n = 8 tumours per group; except CD3⁺ 7 doses, n = 7; CD3⁺ 27 doses, n = 9; CD11b⁺ Gr-1, CD103⁺ 27 doses, n = 10). Experiment performed twice; representative example shown. P-value top row: ** 0.0011, ** 0.0085, ** 0.0014; bottom row: ns 0.08, ** 0.0018, *** 0.0003. c, Proliferation FC in Braf/Pten and Braf melanoma cell lines (made resistant to TT in vitro) 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, 100 nM, 300 nM, 1 µM, 3 µM RAFi). d, Proliferation FC of Braf melanoma cell lines after 72 h in indicated drug conditions of N^TT and N^{TT-Dacarbazine} cell lines (n = technical duplicates), (drug concentration: CTRL, 10 µg, 50 µg, 100 µg, 500 µg Dacarbazine). e, pERK status in CaTCH-isolated N^TT and R^TT Braf/Pten cell lines, 1-hour post drug exposure. Experiment performed twice; representative example shown. f, Treatment response to ACT in matched CaTCH isolated N^TT and R^TT Braf/Pten^OVA tumours (CTRL, n = 3 mice, ACT, n = 5 mice). Experiment performed twice; representative example shown. P-value: **** 3E-5, ns 0.9871. g, h PCA plot displaying top 500 most variable genes for (g) Braf/Pten and (h) Braf melanoma tumours. i, Expression of genes comprising the ccIES in sorted N^TT and R^TT Braf/Pten^OVA melanoma cells (left, tumours were not exposed to RAFi) (all groups n = 3 tumours) and in sorted RAFi/MEKi R^TT melanoma cells (all groups n = 3 tumours) (right). j, Overall survival stratified based on ccIES expression in TCGA melanoma patients (n = 469 patients). k, l Progression-free survival stratified based on ccIES expression in patients receiving (k) anti-PD-1/CTLA-4 combination therapy (n = 32 patients) or (l) anti-PD-1 monotherapy (n = 121 patients). m, Correlation of ccIES with CD103 score and T cell score in TCGA melanoma patients (n = 469 patients). Data in (a, b, c, d, f) displayed as mean ± SEM. Data analysis (b) two-tailed unpaired t–test with Welch correction for unequal variance or with Mann-Whitney-U-test if not normal distributed (f) two-way ANOVA. P-value in (j-l) derived from a Cox proportional hazards model using gene score as a continuous variable and analysis in (m) two-sided Pearson Correlation coefficient (PCC). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non-significant.

Source data