Figure EV5. Monitoring of immune cell infiltration after ICB and effect of late-stage deletion of Zeb1 on ICB.

(A) IHC-based quantification of immune cell infiltration and PD-L1 expression in tumors from Fib^Ctrl and Fib^ΔZeb1 mice after orthotopic transplantation of AKP^re organoids and intraperitoneal injection of a-PD-L1 antibodies or control IgGs. Numbers of experimental mice per condition are indicated (CD4, CD8, FOXP3, B220, PD-L1: P = 0.0032, 0.0076, 0.6442, 0.0027, >0.9999 (Fib^Ctrl-ICB vs Fib^ΔZeb1-ICB), p = 0.1568, 0.0525, 0.0304, 0.2150, 0.0004 (Fib^ΔZeb1-IgG vs Fib^ΔZeb1-ICB), Šídák’s multiple comparisons test). IgG-treated mice contributed to initial AKP^re analysis (Fig. 1). (B) Kaplan–Meier analysis showing tumor-free survival of Fib^Ctrl and Fib^ΔZeb1 mice after orthotopic transplantation of AKP^re organoids and intraperitoneal injection of a-PD-L1 antibodies or control IgGs, as indicated by red arrows. Recombination of Zeb1 was induced by tamoxifen food starting from day (d) 14. Mice were considered tumor-free if no tumor was detected by palpation. Numbers of experimental mice per condition are indicated (P = 0.7583, Mantel–Cox test). (C) Quantification of tumor volumes after orthotopic transplantation of AKP^re tumor organoids and ICB with late recombination of Zeb1. Only tumors collected after d28 were included in this analysis (n = 8 independent mice, P = 0.6276, Student’s t test). (D) IF-based quantification of stromal cells expressing ZEB1 (n = 8/8 independent mice for Fib^Ctrl/Fib^ΔZeb1, P = 0.0002, Student’s t test). (E) IHC-based quantification of immune cell infiltration and PD-L1 expression in AOM/DSS tumors from Fib^Ctrl and Fib^ΔZeb1 mice receiving ICB (2 intraperitoneal injections of a-PD-L1 and a-CTLA-4 antibodies or control IgGs per week starting at d70 of AOM/DSS tumorigenesis). Numbers of experimental mice per condition are indicated (CD4, CD8, FOXP3, B220, PD-L1: P = 0.9902, 0.0802, <0.0001, 0.0070, 0.0003 (Fib^ΔZeb1-IgG vs Fib^ΔZeb1-ICB), P = 0.6241, 0.7615, 0.0070, 0.3163, 0.0144 (Fib^Ctrl-IgG vs Fib^ΔZeb1-IgG), Šídák’s multiple comparisons test). Data information: Data are presented as mean ± SD (A, C–E). Source data are available online for this figure.