Fig. 2

Clonal barcoding reveals heterogeneity in response to ICB. a Experimental design for clonal barcoding. We first transduced the parental CT26 line with the high-diversity ClonTracer barcode library at a low MOI (0.01). We selected and expanded the transduced cells (containing ~ 2000 distinct barcodes), and transplanted into syngeneic recipients, with 250,000 barcoded cells each flank site, two sites per recipient. We then treated the recipient mice with control IgG (N = 10), anti-PD-1 (N = 15), or anti-CTLA-4 (N = 10). After treatment, we harvested tumor for barcode quantification. b Anti-PD-1 or anti-CTLA-4 treatment (syringes) significantly reduces tumor growth compared to control IgG treatment (mean ± SD; *P < 0.05, ***P < 0.001; two-way ANOVA with Bonferroni post-test multiple comparison). c Distribution of relative tumor size (lower panel, normalized to the median value in each group) and its intra-group variance (upper panel) for the control IgG, anti-PD-1, and anti-CTLA-4 groups along the treatment course. ICB treatment led to significantly higher intra-group variance (*P < 0.05, ***P < 0.001; F test of equality of variance). d Clonality of barcode distribution in the anti-PD1 or anti-CTLA4 group is significantly higher than that in the control IgG treatment group (mean ± SD; *P < 0.05; one-way ANOVA with Bonferroni post-test multiple comparison). e The enrichment/depletion of barcodes between anti-PD1 and anti-CTLA4 is positively correlated