Figure 5. Subcutaneous and orthotopic KPAR tumours are responsive to immune checkpoint blockade.

(A) Growth of KPAR subcutaneous tumours from mice treated intraperitoneally with 200μg anti-PD-1 and/or 200μg anti-CTLA-4 or corresponding isotype control (IgG Ctl) on day 10, 14, 17 and 21. Data are mean tumour volumes ± SEM, n=6 mice per group. Two-way ANOVA; ** P≤0.01, *** P≤0.001.

(B) Kaplan-Meier survival of mice from (A). The black arrow indicates the time at which mice that previously rejected the primary tumour were re-challenged on the opposite flank. Log-rank (Mantel-Cox) test; ** P≤0.01.

(C-D) Flow cytometry analysis of the frequency of Foxp3⁺ Tregs (C), effector memory CD8⁺ T cells (D, left) and PD-1⁺ CD8⁺ T cells (D, right) in subcutaneous tumours after treatment as in (A). Treatment was on day 10, 14 and 17 and mice were culled on day 18. Data are mean ± SEM, n=5 mice per group. One-way ANOVA; ** P≤0.01, *** P≤0.001.

(E) Kaplan-Meier survival of mice treated with anti-PD-1 and/or anti-CTLA-4 after orthotopic transplantation of KPAR cells. Treatment was initiated once tumours were detectable by micro-CT and were administered twice weekly for a maximum of 3 weeks. n=6 mice (IgG Ctl, anti-PD1, anti-CTLA-4) or n=7 mice (anti-PD-1 + anti-CTLA-4). Log-rank (Mantel-Cox) test; * P≤0.05, ** P≤0.01.

(F-G) Quantification of immunohistochemistry staining for CD8 (F) and Foxp3 (G) in orthotopic KPAR lung tumours after treatment as in (E). Data are mean (large symbols) ± SEM, n=3 mice per group, small symbols represent individual tumours. One-way ANOVA; ns P>0.05, * P≤0.05, ** P≤0.01.