| Objective:Persuasive clinical and experimental evidence suggests that M2-like tumor-associated macrophages (TAM) provide support to tumor cells in malignant progression. At metastatic sites, M2-like macrophages promote tumor cell extravasation, survival, and subsequent growth. M2-like macrophages may represent vital new therapeutic targets. However, there are few compounds targeting M2-like macrophages in clinical use. Based on this, screening agents targeting M2-like macrophages is urgently needed.We found Imatinib inhibited M2 polarization of macrophages in vitro and then we assessed the influence of Imatinib on macrophage polarization as well as its effect on tumor cells at metastatic sites. Besides, we explored the mechanism of anti-M2 polarization function of Imatinib. Our study will provide new directions for the use of Imatinib in anti-metastatic therapeutic strategy. In addition, our study will offer the experimental proof for the application of agents targeting M2-like macrophage in anti-metastatic therapy.Methods:(1) The influence of Imatinib and the conditioned medium of macrophages on the proliferation of macrophages was detected by SRB cell viability assay; (2) Flow cytometric analysis was used to explore the expression of CD206, the M2 marker, and CD 86, the M1 marker, and F4/80, the typical marker of macrophages; (3) The relative mRNA level of M2 genes and M1 genes was determined by RT-PCR; (4)Transwell assay was performed to detect the invasion ability of LLC cultivated with conditioned medium or treated with IL-13 and/or Imatinib; (5) The anti-metastasis function of Imatinib was assessed in LLC subcutaneous model and LLC intravenous model; (6) The metastatic nodes were detected by H&E staining and the metastatic nodes in the surface of lungs were counted; (7) F4/80 positive and CD206 positive cells in primary tumors and at metastatic sites were detected by immunofluorescence or immunohistochemical staining; (8) The expression of specific proteins was investigated by western blot; (9) Immunofluorescence was performed to determine the location of STAT6.Results:(1) Imatinib suppressed macrophage M2 polarization, but exerted no effect on M1 polarizationThe expression of CD206, M2 marker, in RAW264.7 or BMDM induced by IL-13 or IL-4 was detected by flow cytometry. The results showed the percentage of CD206 positive cells was dramatically increased by IL-13 from 1.51±0.97% to 32.65±3.79% (p<0.01). When treated with IL-13 and 5μM Imatinib, the percentage was significantly decreased to 9.78±0.55% (p0.01). Similar results were found in IL-14-induced RAW264.7 cells. The relative mRNA level of M2 genes was investigated by Real-time PCR including Argl, Mgl2, Mrcl, CDH1 and CCL2. The result demonstrated Imatinib restrained transcription of M2 genes up-regulated by IL-13. For example, the relative mRNA level of Argl was up-regulated to 71.7± 13.1 by IL-13, which was significantly down-regulated to 4.6±2.2 by Imatinib (p<0.001).We also assessed the effect of Imatinib on M1 polarization induced by LPS and IFNy. Flow cytometric analysis was performed to detect the expression of CD86, the M1 marker. Results displayed that the percentage of CD86 positive cells went up from 2.29±0.09% to 43.56±4.4% (p<0.01), when the cells were treated with LPS and IFNy. However, there was no significant difference between RAW264.7 cells incubated with LPS and IFNγ and the ones treated with LPS, IFNγ and Imatinib (p>0.05). Real-time PCR was used to investigate relative mRNA level of M1 genes including iNOS, CCL5 and TNFa. The results illustrated that the relative mRNA level of these three genes was respectively upregulated to 6.1±1.6,1.7±0.6,2.0±0.3 by LPS and IFNy, which Imatinib exerted no effect on (p>0.05).(2) Imatinib blocked tumor metastasis by suppressing M2-like TAMTranswell assay was performed to detect the invasion ability of LLC cultivated with conditioned medium of BMDM treated with IL-13 and/or Imatinib. The results showed that the number of migrated LLC cells incubated with conditioned medium of BMDMs treated with IL-13 was remarkably increased from 118±14 to 211±27 (p<0.01). Meanwhile, those incubated with conditioned medium of BMDMs treated with IL-13and Imatinib significantly decline to 120±17 (p<0.01). Furthermore, Imatinib and IL-13 had no influence on the migration of LLC cells.Imatinib blocked tumor metastasis in either LLC subcutaneous model or LLC intravenous model. The proportion of M2-like TAMs in primary tumor was analyzed. Besides, the percentage of M2-like TAMs was detected at the 7th day and the last day of LLC intravenous model. Results demonstrated Imatinib inhibited the percentage of M2-like macrophage at the 7th day and the last day of this model. It suggested Imatinib reduce the percentage of M2-like TAMs at the metastatic site and suppressed subsequent growth of tumor cells.(3) The mechanism for anti-M2 polarization activity of ImatinibThe influence of Imatinib on STAT6 in the process of macrophage M2 polarization induced by IL-13 was determined by western blot. The result displayed Imatinib effectively inhibited the phosphorylation of STAT6. Immunofluorescence was performed to determine the location of STAT6. The result showed Imatinib blocked translocation of STAT6 into nucleus.Conclusions:(1) Imatinib suppressed macrophage M2 polarization, but exerted no effect on M1 polarization.(2) Imatinib blocked tumor metastasis by suppressing M2-like TAM.(3) Imatinib inhibited macrophage M2 polarization by repressing the phosphorylation of STAT6 and its translocation into nucleus. |
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