Fig. 4

ICB treatment enriched ICB-resistant cancer clones in responders. a Design for isolation of ICB-resistant clone. Tumors (large blue dot) were dissociated and cancer cells (small blue dots) were plated into 96-well plates at limiting dilution. Single cell-occupied wells were selected by microscopy and then expanded to establish stable lines. We genotyped each established line by Sanger sequencing of the barcode and inferred its ICB sensitivity based on enrichment/depletion by anti-PD1 and/or anti-CTLA4. b Frequency of the clones representing line B04 or line B64 is higher in responders than non-responders of ICB (mean ± SD; **P < 0.01, ***P < 0.001; one-way ANOVA with Benjamini-Hochberg post-test multiple comparison). c Frequency of the barcodes representing line B04 or line B64 is higher in the anti-PD1- or anti-CTLA4-treated group than the control group (boxplot shows the minimum, first quartile, median, third quartile, and maximum values of each group; *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA with Benjamini-Hochberg post-test multiple comparison). d Response to combined ICB treatment confirmed the ICB resistance of lines B04 and B64. We transplanted 250,000 cells (parental, line B04, or line B64) into the syngeneic recipients and treated the tumor with control IgG or combinatorial anti-PD1 + anti-CTLA4 on days 4, 7, and 10 post-transplantation. Tumors derived from the parental line decreased in size after ICB treatment, whereas those derived from line B04 or line B64 persisted