Article Figures & Data
Figures
- Figure 1.
Tumor hypoxia is variable between tumor types and inhibits T-cell function. A, OCR trace of B16 and MC38 cells (50,000 cells/well) interrogated for mitochondrial activity in the Seahorse instrument. B, ECAR trace of B16 and MC38 cells interrogated for glycolytic activity in the Seahorse instrument. C, Hypoxyprobe staining of T cells isolated from B16 and MC38 tumors. Results are tabulated to the right. LN, lymph node. D, CellTrace Violet (CTV) dye dilution showing proliferation of OT-I T cells activated with peptide in ambient normoxia (20%) or hypoxic (1.5%) conditions. Shaded histogram, unstimulated cells. E, Cytokine production of CD8+ T cell stimulated as in D overnight. NS, no stimulation. F, Cytotoxicity [propidium iodide (PI) staining] of parental or OVA-expressing B16 tumor cells incubated with previously activated, effector OT-I T cells overnight under conditions of normoxia or hypoxia. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by unpaired t test (C and E) or two-way ANOVA with repeated measures (F). Results represent three independent experiments.
- Figure 2.
Metformin treatment acts as an inhibitor of tumor oxygen consumption. A, OCR of B16 or MC38 in vitro cultured cells (50,000 cells/well) treated overnight in the presence or absence of 10 mmol/L metformin. B, OCR of B16 tumor cells (CD45-depleted) plated directly ex vivo from mice bearing small tumors treated with PBS or metformin (Met; 50 mg/kg) for 3 days. C, Pimonidazole staining of full tumor sections (stitched from 300–500 individual panels) from mice bearing B16 tumors receiving PBS or metformin treatment for 3 days as in B. Tabulated results quantify the internal hypoxyprobe signal from a set threshold normalized for each day of imaging. Scale bar, 1 mm. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by unpaired t test. Data represent the mean (A–C tabulation) or are representative (C, images) of at least three independent experiments.
- Figure 3.
Metformin treatment reduces intratumoral T-cell hypoxia. A, OCR (top) or ECAR (bottom) from B16-bearing mice treated with metformin or vehicle for 3 days. CD45+CD8+ T cells were sorted by flow cytometry and assayed directly ex vivo, whereas B16 tumor cells were CD45-depleted before assaying. LN, lymph node. B, Flow cytogram (left) and tabulation (right) of pimonidazole staining in T cells from B16-bearing mice treated with metformin or vehicle for 3 days. C, Tumor area at 21 days for mice treated during tumor progression with PBS or metformin (Met). D and E, Quantification of CD44hi CD8+ T cells (D) from B16-bearing mice treated with metformin or vehicle for 3 days (E) PD-1 and Tim-3 expression in CD8+ T cells from mice treated as in D. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by unpaired t test. Results are representative of three (A, B, D, and E) or four (C) independent experiments.
- Figure 4.
Metabolic remodeling synergizes with checkpoint blockade to affect antitumor immunity. A, Tumor measurements of C57/BL6 mice inoculated with B16 melanoma. Mice began receiving treatment on day 5 as indicated, receiving 0.2 mg anti-PD-1 or its isotype control every 4 days, and either metformin (met) or vehicle. Number of mice tumor-free of the total inoculated is reported. B, Representative flow cytogram depicting IFNγ and TNFα production from CD8+ tumor-infiltrating T cells from B16-bearing mice treated as in A. C, Tabulated IFNγ staining from multiple mice; each dot represents one animal. D, Ki67 expression in CD8+ T cells from mice treated as in A as indicated. E, As in A, but mice were inoculated with MC38. **, P < 0.01; ***, P < 0.001 by unpaired t test. Results represent the mean (A, C, D, and E) or are representative of (B) three to five independent experiments.
Additional Files
Supplementary Data
- Supplementary Figure Legends - Supplementary figure legends.
- Supplementary Figure 1 - Metformin has indirect effects on immunity in the tumor microenvironment.
- Supplementary Figure 2 - Metformin in the drinking water enhances PD-1 therapy in B16 melanoma.
Volume 5, Issue 1
