Supplementary Figure 1. Metformin has indirect effects on immunity in the tumor microenvironment. (a) mRNA expression of Slc22a1 was quantified from direct ex vivo sorted CD8+ LN and TIL, in vitro activated CD8 T cells, CD45- direct ex vivo B16, or in vitro cultured MC38. Slc22a1 expression is graphed as fold change over naïve T cell expression scaled to a endogenous control gene (PPIB) using the ΔΔCt method. (b) Oxygen consumption rate trace (left) or extracellular acidification rate trace (right) of in vitro OT-1 activated T cells cultured for 3 days in the presence of the indicated metformin concentrations. (c) Quantification of ex vivo 2NBDG glucose tracer uptake in CD8+ T cells from B16-bearing mice treated with metformin or vehicle for 3 days. (d) Histogram (left) and quantification (right) of PDL1 expression on CD45+ CD11b+ cells from B16-bearing mice treated as in c. (e) Histogram (left) and quantification (right) of PDL1 expression on CD45+ CD11b+ CD11c+ cells from B16-bearing mice treated as in c. (f) Quantification of % CD11b+ Gr1+ MDSCs from CD45+ Ly6C– cells from B16-bearing mice treated as in c. (g) Immunoblot (left) and quantification (right) of Hif1α and actin in CD8+ T cells from B16-bearing mice treated as in c. * p < 0.05 by unpaired t-test. Results are representative (a, b) or represent the mean (c-g) of 2 (a-c, g) or 3 (d-f) independent experiments.
Supplementary Figure 2. Metformin in the drinking water enhances PD-1 therapy in B16 melanoma. Tumor measurements of C57/BL6 mice inoculated with B16 melanoma. Mice began receiving treatment on d5, receiving 0.2mg anti-PD1 or its isotype control every 4 days. Mice receiving anti-PD1 also got 1g/L metformin drinking water. Number of partial responders (PR) and complete responders (CR) of the total inoculated mice is reported, as well as average tumor growth curve for all mice (far right). ** p <0.01 by or two-way ANOVA with repeated-measures. Results represent 4 independent experiments.
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