Metformin may improve the outcome of patients with colorectal cancer and type 2 diabetes mellitus partly through effects on neutrophil extracellular traps

Abstract

Background: Although metformin reduces the risk of cancer-related mortality in patents with type 2 diabetes, the mechanism of its anti-cancer effects has not been fully understood.

Method: Impact of metformin on survival was examined in patients who underwent curative colectomy for colorectal cancer (CRC). The effects of metformin in neutrophil extracellular traps (NETs) were examined with in-vitro experiments and multiplex immunohistochemistry of surgically resected CRC specimens.

Results: Prior intake of metformin prolonged relapse-free (P = 0.036) and overall survival (P = 0.041) in 289 patients with T2DM to the comparable levels to those of 1576 non-diabetic patients. Metformin reduced the production of NETs stimulated with lipopolysaccharide or HT-29 colon cancer cells to 60% of control. Neutrophils markedly suppressed the chemotactic migration of activated T cells in an NET-dependent manner, which was reversed by metformin treatment up to approximately half of the migration without neutrophils. Immunohistochemical analysis revealed a significant association between metformin intake and a reduction in the numbers of tumor-associated neutrophils (TANs) and NETs. Simultaneously, metformin intake was found to increase the presence of CD3(+) and CD8(+) tumor-infiltrating T cells (TILs), particularly at the tumor-invasion front, especially in areas with fewer TANs and NETs.

Conclusion: Metformin suppresses the diabetes-associated enhancement of NET formation, which can augment the infiltration of TILs in CRC tissues. The anti-tumor effect of metformin in patients with T2DM may be, at least partly, attributable to the inhibition of NETs.

Fig. 1. Metformin intake improves prognostic outcomes in colorectal cancer (CRC) patients with type 2 diabetes mellitus (T2DM) who underwent curative surgery.

a Relapse-free survival (RFS) and (b) overall survival (OS) after surgery for colorectal cancer in patients with (n = 1576) or without (n = 289) T2DM were evaluated according to the Kaplan–Meier method and P values were calculated by using the log-rank test. c RFS and (d) OS of the patients with T2DM who were taking (n = 62) or not taking (n = 227) metformin at colectomy. Gray lines show the survival curves of the patients without T2DM.

Fig. 2. Metformin alleviates impaired lymphocyte migration impairment by suppressing neutrophil extracellular trap (NET) formation.

a Neutrophils were purified from the peripheral blood of healthy volunteers and cultured in RPMI supplemented with LPS with or without metformin (10 to 100 μM). After 3 h, SYTOX Green was added, and the plate was viewed under a fluorescence stereomicroscope. Scale bars, 100 μm. b Neutrophils that showed characteristic strand staining for SYTOX Green were counted as NETs, and their ratios relative to total neutrophil counts were calculated in 3 randomly selected fields. Data are shown as the mean ± 1 SD of triplicates from one of two experiments. *P < 0.05, **P < 0.01. c Peripheral blood mononuclear cells, which consisted primarily of activated T cells, were cultured for 5 to 7 d in the presence of 10 ng/ml recombinant interleukin 2 on anti-CD3 mAb-coated plates. Cells were stained with calcein-AM, and 1 × 10⁵ stained cells were seeded into an upper chamber (3-μm pores); the cell-containing upper chambers were placed on the lower chambers, which contained or did not contain HT-29 cells. After 2 h, the proportion of T cells that migrated to the lower chamber was quantified by using flow cytometry as described in Materials and Methods. In some wells, neutrophils were preincubated with DNAse I (100 U/ml) or metformin (100 μM) for 30 min and added to the culture inserts containing activated T cells. Data are shown as mean ± 1 SD of triplicates from one of 3 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (d) Activated T cells were stained with PKH26, and the migration assay was performed in the presence of neutrophils with (lower panel) or without (upper panel) 100 mM metformin. After 2 h, the membranes of the culture inserts were stained with SYTOX Green. Photos taken under the wavelength for FITC (SYTOX Green) and rhodamine (PKH26) were superimposed. Scale bar, 50 μm.

Fig. 3. Metformin attenuates the density of tumor-associated neutrophil extracellular traps (NETs) while enhances the density of tumor-infiltrating lymphocytes (TILs) in the colorectal cancer (CRC) tissue.

a Representative image of multiplex immunostaining of tumor-associated neutrophils (TANs) and neutrophil extracellular traps (NETs) at the invasive front of colorectal cancer. Images stained for hematoxylin (blue), CD66b (green), and Cit-H3 (red) were merged. Arrowheads show CD66b(+) Cit-H3(+) NETs. The densities of TANs (b) and NETs (c), and the NET:TAN ratio (d), were calculated as described in the Materials and Methods and compared between 40 propensity-score–matched tumors from each group. e Representative image of multiplex immunostaining of tumor-infiltrating T cells (TILs). Images stained for hematoxylin (blue), CD3 (green), and CD8 (red) were merged. Arrows point to CD3(+)CD8(–) TILs, and arrowheads indicate CD3(+)CD8(+) TILs. The densities of CD3(+) TILs (f) and CD3(+)CD8(+) TILs (g), and the CD8(+):CD3(+) TIL ratio (h) were calculated. P values were calculated by using the Mann–Whitney U test. *P < 0.05, ***P < 0.001.

Fig. 4. An inverse correlation between neutrophil extracellular traps (NETs) infiltration and CD8-(+) T lymphocytes infiltration in colorectal cancer (CRC) tissues.

a Images of multiplex immunostaining of TANs, NETs, and TILs in different regions of the colorectal tumor specimen from a representative patient. Five images stained for hematoxylin (blue), CD66b (green), Cit-H3 (red), CD3 (cyan), and CD8 (magenta) were merged. Correlation between the density of CD3(+)CD8(+) TILs and that of TANs (b) or NETs (c) among all 80 tumor samples evaluated. Pearson’s simple linear regression analysis was used to calculate r and P values.