Figure 2.

Metformin shifts tumor, regional and systemic immunity when combined with ICIs. Male mice were inoculated subcutaneously with 1×10⁶ mEER cells; tumors were allowed to develop for 18 days to an average size of 65 mm² prior to treatment. Beginning on day 18 mice received five consecutive daily doses of metformin (40 mg/kg) delivered i.p. with (n=8) or without (n=6) immune checkpoint inhibitors (red line indicates time period of administration). Anti-PD-1 (250 μg/dose) and anti-CTLA-4 (100 μg/dose) checkpoint inhibitors were delivered on days 19 and 22 i.p. All mice were euthanized at day 23 post-tumor inoculation when tumors were all still approximately equal in size (~100 mm²). Tumor, spleen and tumor draining lymph nodes (tdLN) were harvested, processed and stained for flow cytometric analysis (A). tumor growth curves were generated from the available tumor measurements over the time period analyzed (B). Experiment performed once. DC, dendritic cell; ICIs, i.p., intraperitoneally.