Metformin prevents cancer metastasis by inhibiting M2-like polarization of tumor associated macrophages

Abstract

Accumulated evidence suggests that M2-like polarized tumor associated macrophages (TAMs) plays an important role in cancer progression and metastasis, establishing TAMs, especially M2-like TAMs as an appealing target for therapy intervention. Here we found that metformin significantly suppressed IL-13 induced M2-like polarization of macrophages, as illustrated by reduced expression of CD206, down-regulation of M2 marker mRNAs, and inhibition of M2-like macrophages promoted migration of cancer cells and endothelial cells. Metformin triggered AMPKα1 activation in macrophage and silencing of AMPKα1 partially abrogated the inhibitory effect of metformin in IL-13 induced M2-like polarization. Administration of AICAR, another activator of AMPK, also blocked the M2-like polarization of macrophages. Metformin greatly reduced the number of metastases of Lewis lung cancer without affecting tumor growth. In tumor tissues, the percentage of M2-like macrophage was decreased and the area of pericyte-coated vessels was increased. Further, the anti-metastatic effect of metformin was abolished when the animals were treated with macrophages eliminating agent clodronate liposome. These findings suggest that metformin is able to block the M2-like polarization of macrophages partially through AMPKα1, which plays an important role in metformin inhibited metastasis of Lewis lung cancer.

Keywords: AMPKα1; cancer metastasis; macrophage polarization; metformin.

Figure 1. Metformin inhibits M2-like polarization of macrophages induced by IL-13

A. RAW264.7 were treated with IL-13(10 ng/ml), metformin (1 mM), or the combination for 72 h. Both the expression of M2 marker CD206 and M1 marker CD86 were analyzed by FACS analysis. B. Bone marrow-derived macrophages (BMDMs) were treated with IL-13(10 ng/ml) and different concentrations of metformin as indicated for 72 h and the percentage of F4/80⁺ CD206⁺ macrophages were determined by FACS analysis. C. and D. Quantitative RT-PCR was carried out to assess mRNA expression of M2-marker genes (C) and M1-marker genes (D) when the macrophages were treated with IL-13(10 ng/ml) for 24 h, metformin (1 mM), or both of them. The histogram bars represent three independent experiments. *p,^#p < 0.05; **p,^##p < 0.01; as evaluated using Student's t test.

Figure 2. Metformin inhibits M2-like macrophages promoted migration of Lewis Cancer Cells (LLCs) in vitro

BMDM were treated with IL-13(10 ng/ml), metformin (1 mM), or the combination for 72 h and the culture medium were replaced by fresh medium without serum, 24 h later the supernatant medium was collected as macrophage-conditioned medium (CM). A. LLCs were cultured with CM for 24 h and cell survival was determined by SRB assay. B. LLCs were cultured with CM for 24 h and cell apoptosis was determined by FACS analysis. C. The effect of different CMs on LLCs migration was evaluated by trans-well assay in 24 h. D. BMDM were treated with IL-13(10 ng/ml), metformin(1 mM), or both of them for 24 h and the mRNA expression of metastasis-relative genes were determined by quantitative RT-PCR. The histogram bars represent three independent experiments. *p, ^#p < 0.05; as evaluated using Student's t test.

Figure 3. Metformin inhibits M2-like macrophages promoted angiogenesis in vitro

A. CMs were collected as above. Mouse pulmonary micro-vascular endothelial cells (MMVECs) were cultured with CMs for 72 h and cell survival was determined by SRB assay. B. For wood-healing assay, MMVECs were scratched with a pipette tip and then treated with CMs for 12 h. C. Trans-well assay was carried out to evaluate the impact of CMs on migration of MMVECs in 24 h. D. For tube formation assay, MMVECs were seeded in 96-well plates filled with matrigel and incubated with different CMs. After 6 h, cells were photographed under phase contrast microscopy (5X). E. Quantitative RT-PCR was carried out to measure the mRNA expression of angiogenesis-relative genes in macrophages treated with IL-13(10 ng/ml), metformin(1 mM), or both of them for 24 h. The histogram bars represent three independent experiments. *p, ^#p < 0.05; as evaluated using Student's t test

Figure 4. AMPKα1 is involved in metformin inhibited M2-like polarization of macrophages

A. AMPKα1 phosphorylation in either RAW264.7 or BMDMs was determined by Western-blotting when treated with merformin (1 mM) for indicated times. B. RAW264.7 was treated with IL-13 (10 ng/ml), AICAR (1 mM), or both of them for 72 h, the expression of CD206 or CD86 were determined by FACS analysis. C. AMPKα1 in RAW264.7 was knocked down by RNA interference. D. The percentage of CD206⁺ macrophages was determined by FACS analysis when RAW264.7 was treated with IL-13(10 ng/ml), metformin(1 mM), or both of them in the absence of AMPKα1. All experiments were repeated at least three times. *p,^#p < 0.05; **p,^##p < 0.01; as evaluated using Student's t test.

Figure 5. Merfromin inhibits metastasis of LLC in vivo by targeting macrophages

A–E. C57BL/6 mice were injected subcutaneously with LLCs in the flank and were treated with metformin (100 mg/kg), clodronate lipsome, or both of them. After 21days, animals were sacrificed and lungs were histologically analyzed for the occurrence of metastases. Total number as well as the number of small (diameter: < 50 μm), medium-sized (diameter: 50–200 μm), or large (diameter: > 200 μm) lung metastases determined (n = 8, A and B). The images show representative stained lung sections (C) Tumor volume and tumor weight were determined (D and E). F, G. C57BL/6 mice were injected intravenously with LLCs and treated with metformin for 21 days. Total number as well as the number of small, medium-sized, or large lung metastases determined (n = 8, F and G).

Figure 6. Metformin suppresses M2 polarization of TAMs and promotes tumor vessel maturation

A. The sections of tumor tissues from different group were double stained with macrophage marker F4/80 and M2-marker CD206. B. F4/80⁺ area (% of tumor area) and the F4/80⁺CD206⁺ area (% of F4/80⁺ tumor area) were qualified. C. The sections of tumor tissues from different group were double stained with CD31 and α-SMA. D. α-SMA⁺ CD31⁺ area (% of CD31⁺ tumor area) was qualified. ^#p < 0.05; **p < 0.01; ***p < 0.001; as evaluated using Student's t test.