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. 2023 May 5;11(5):e005719. doi: 10.1136/jitc-2022-005719

Figure 2.

Figure 2

Metformin improves tumor response to adoptive cell therapy (ACT) and CD8 T-cell infiltration in hypoxic TiRP tumor areas. (A) Tumor growth in TiRP mice that have received a single injection of cyclophosphamide (CTX, 100 mg/kg) or phosphate buffered saline (PBS) when the tumor size was around 400 mm3, followed (continuous lines) or not (dotted lines) by ACT of 10 million activated TCRP1A CD8 T cells 24 hours later and treated or not with metformin (0.5 mg/mL) in drinking water for 20 days after ACT or when any humane end point was reached (A, PBS no ACT: n=12; PBS+metformin no ACT: n=8; CTX no ACT: n=10; CTX+metformin no ACT: n=9; PBS and ACT: n=25; PBS+metformin and ACT: n=23; CTX and ACT: n=17; CTX+metformin and ACT: n=13). (B) Kaplan-Meier survival curve in TiRP mice treated as in (A) on ACT (B, PBS: n=18; PBS+metformin: n=9; CTX: n=17; CTX+metformin: n=12). (C) Fluorescence-activated cell sorting (FACS) analysis of CD8 T-cell tumor infiltration in TiRP mice treated as in (A) 4 days after ACT (C, PBS: n=21; PBS+metformin: n=23; CTX: n=20; CTX+metformin: n=19). (D) FACS analysis of TCRP1A CD8 T-cell tumor infiltration in TiRP mice treated as in (A), 4 days and 14 days after ACT (D, 4 days after ACT, PBS: n=20; PBS+metformin: n=22; CTX n=20; CTX+metformin: n=19; 14 days after ACT, PBS: n=23; PBS+metformin: n=28; CTX: n=25; CTX+metformin: n=18). (E, F) Immunofluorescence (IF) quantification and representative images of hypoxic CD8 T cells in TiRP tumors treated as in (A), 4 days after ACT (F, PBS: n=12; PBS+metformin: n=12; CTX: n=13; CTX+metformin: n=14). (G) FACS analysis of hypoxic TCRP1A CD8 T cells among alive cells in TiRP tumors treated as in A, 4 days after ACT (G, PBS: n=20; PBS+metformin: n=22; CTX: n=18; CTX+metformin: n=15). (H) IF analysis of the percentage of CD8 T cells (among total CD8 T cells) found at the indicated distance from the closest CD31pos cells in TiRP tumors treated as in (A), 4 days after ACT (H, PBS: n=12; PBS+metformin: n=14; CTX: n=13; CTX+metformin: n=10). (I), (J) FACS analysis of apoptosis in pimonidazole-negative or pimonidazole-positive TCRP1A CD8 T cells in TiRP tumors treated as in (A), 4 days after ACT (I) and 14 days after ACT (J) (I, PBS: n=17; PBS+metformin: n=21; CTX: n=17; CTX+metformin: n=14; J, PBS: n=23; PBS+metformin: n=27; CTX: n=26; CTX+metformin: n=17). All data are mean±SEM. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, calculated by two-way analysis of variance with Tukey’s multiple comparison correction in (A), log-rank (Mantel-Cox) test in (B), two-tailed unpaired t-test in (C–E, G–J).