Figure 4.

Systemic anti-PD-1 and anti-CTLA-4, combined with radiation at a distant tumour site, induces immune-mediated tumour control in the orthotopic setting. (a) Diagram showing that radiation was delivered to the subcutaneous tumour only. (b–e) Mice were implanted with subcutaneous and orthotopic 6694c2 tumours. On day 6 following inoculation, the subcutaneous tumour was irradiated (1 × 5 Gy). Mice were administered 200 µg each of anti-PD-1, anti-CTLA-4 or isotype controls intraperitoneally every 7 days starting on day 6. At day 14, orthotopic tumours were harvested, weighed and digested. Following tumour digestion, cells were stained for immune markers for spectral flow cytometry analysis. (f) In addition to harvesting orthotopic tumours, pancreatic draining lymph nodes were harvested from orthotopic tumour-bearing mice. Lymph nodes were mechanically digested, and cells were cocultured with 6694c2 cells or an unrelated cell line and analysed for IFNγ production by ELISpot. Spot number is reported with background subtracted. Data are shown as mean ± s.e.m. and p-values were calculated by Mann–Whitney test. n = 5 mice in the radiation αPD-1 αCTLA-4-treated group and n = 4 mice in the isotype-treated group; one mouse reached endpoint in the study prior to tumour harvest.