1. Phenformin Inhibits Myeloid-Derived Suppressor Cells and Enhances the Anti-Tumor Activity of PD-1 Blockade in Melanoma 
Sun Hye Kim, Man Li, Sebastian Trousil, Yaqing Zhang, Marina Pasca di Magliano, Kenneth D. Swanson, Bin Zheng 
Journal of Investigative Dermatology 
Volume 137, Issue 8, Pages (August 2017)
DOI: /j.jid
Copyright © 2017 The Authors Terms and Conditions

2. Figure 1
Phenformin reduces the numbers of G-MDSCs in the spleens of tumor-bearing mice. (a) Mice bearing allograft tumors of BP01 mouse melanoma cells were treated with phenformin (Phen), metformin (Met), or vehicle control (Ctrl) for 10 days. Representative flow cytometry plots are shown. Expression of Ly6C and Ly6G in the gated CD11b+ cells from spleens of BP01 tumor-bearing mice were analyzed, and percentages of G-MDSCs (CD11b+Ly6GhiLy6Cint) and M-MDSCs (CD11b+Ly6GhiLy6Clo) in spleens were measured by flow cytometry. Numbers on the plots indicate percentages of cells among all live cells in the samples. One representative from each treatment group is shown. (b, c) Effects of metformin and phenformin on percentages of (b) G-MDSCs and (c) M-MDSCs in spleens of tumor-bearing mice. The results were obtained from two independent experiments. Individual data and mean are shown; n = 9–10 per group for MDSC subsets. ∗P < 0.05 (Student t test). Ctrl, vehicle control; G-MDSC, granulocytic myeloid-derived suppressor cell; M-MDSC, monocytic myeloid-derived suppressor cell; Met, metformin; ns, not significant; Phen, phenformin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
Phenformin selectively inhibits G-MDSC ex vivo. (a, b) Bone marrow from naïve FVB wild-type mice were co-cultured with BP01 melanoma cells for 3 days to induce differentiation of BM-MDSCs. Cells were then treated with vehicle control, phenformin, metformin, or AICAR for 24 hours. Percentages of (a) G-MDSCs and (b) M-MDSCs were assessed by flow cytometry as described in methods. (c) BM-MDSCs were treated with vehicle control, phenformin, and Compound C alone or in combination for 24 hours and the percentages of G-MDSCs were evaluated by flow cytometry. (d) Bone marrow from naïve FVB mice were cultured with BP01 conditioned medium for 3 days to induce differentiation of BM-MDSCs. Cells were then were positively selected with Ly6G antibody by magnetic-activated cell sorting. ROS levels were measured in Ly6G+ MDSCs and Ly6G– MDSCs by labeling cells with the oxidation-sensitive dye CellRox Red. Representative data from three biological replicates are shown. Line represents mean ± standard error of the mean. ∗P < 0.05 (Student t test). n = 6. (e, f) BM-MDSCs were treated with vehicle control, phenformin, or N-acetyl-l-cysteine (NAC) for 24 h. (e) ROS were measured in G-MDSCs (CD11b+Ly6GhiLy6Cint) by labeling cells with the oxidation-sensitive dye dichlorodihydrofluorescein diacetate. (f) Percentages of G-MDSCs upon various treatments were measured by flow cytometry. Representative data from three biological replicates expressed as mean ± standard error of the mean are shown. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Student t test). AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; BM, bone marrow; CC, Compound C; Ctrl, vehicle control; G-MDSC, granulocytic myeloid-derived suppressor cell; M-MDSC, monocytic myeloid-derived suppressor cell; Met, metformin; ns, not significant; NAC, N-acetyl-l-cysteine; Phen, phenformin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
Phenformin enhances the anti-tumor activity of anti-PD-1 antibody in the BRAF/PTEN melanoma mouse model. (a) Treatment scheme of BrafCA/+/Ptenlox/lox mice. Mice were treated with anti-PD-1 (αPD-1) or control isotype IgG (Ctrl) antibody by intraperitoneal injection every other day until day 13. Phenformin was administered ad libitum via drinking water throughout the duration of experiment. (b) Tumor growth curves of each treatment group in BrafCA/+/Ptenlox/lox mice. Results are represented as the mean tumor size (mm3) ± standard error of the mean; n = 7 for each group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < (Student t test). (c) Spaghetti plot showing individual tumor growth curves of all mice. Ctrl, control; i.p., intraperitoneal; Phen, phenformin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4
Phenformin decreases the number of G-MDSCs in the BRAF/PTEN melanoma mouse model. (a–d) Tumors and spleens were harvested 7 days after treatment and analyzed for G-MDSCs and M-MDSCs in single-cell suspension of BRAF/PTEN mouse tumors and spleens from different treatment groups as shown in Figure 3. Nine to 12 mice per group were analyzed from three independent experiments. Individual data and mean are shown. ∗P < 0.05; ∗∗P < 0.01; ns, not significant (nonparametric Mann-Whitney test). Ctrl, control; G-MDSC, granulocytic myeloid-derived suppressor cell; M-MDSC, monocytic myeloid-derived suppressor cell; Phen, phenformin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
Effects of the combination of phenformin and anti-PD-1 on CD11b and Gr1 double positive cells and arginase 1-expressing cells in the BRAF/PTEN mouse model. (a–b) Tumor samples from various treatment groups shown in Figure 3 were stained for CD11b (red) and Gr1 (green) by (a) immunofluorescence and (b) arginase 1 by immunohistochemistry. Ctrl, control; Phen, phenformin. Scale bar = 50 μm.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

7. Figure 6
Phenformin modulates the immune suppressive function of MDSCs in the BRAF/PTEN melanoma mouse model. (a–d) Tumors were harvested on day 7 after treatment as shown in Figure 3 and mRNA levels of arginase 1, S100A8, S100A9, and iNOS were quantified by quantitative PCR (n = 9–10). (e) Tumor-infiltrating T cells (CD45+CD3+CD8+) were analyzed by flow cytometry. Tumors of BRAF/PTEN mice were harvested 7 days after various treatments. Twelve to 16 mice per group from three independent experiments were analyzed. Individual data and mean are shown; ∗P < 0.05 (nonparametric Mann-Whitney test). (f) CFSE-labeled CD8+ T cells activated with anti-CD3/CD28 and co-cultured at the different ratios (1:1/1:2/1:4) with splenic MDSCs sorted from control, phenformin, or metformin treated BP01 tumor-bearing mice for 10 days. T-cell proliferation was monitored 72 hours after co-culture. Results are expressed as mean ± standard error of the mean from three independent experiments. ∗P < ns, not significant (Student t test). Arg-1, arginase 1; Ctrl, control; iNOS, inducible nitric oxide synthase; MDSC, myeloid-derived suppressor cell; Phen, phenformin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions