Mechanism Of Tumor-associated Macrophages Enhancing Epithelial-mesenchymal Transition And Invasion Of Prostate Cancer Cells In Microenvironment

BackgroudProstate cancer is the most common cancer affecting men in Western countries.In 2016,there will be over 180,000 new cases of prostate cancer in the USA alone,accounting for over one in five new cancer diagnoses.Metastatic spread of cancer is accountable for more than 90 percent of the nearly 8 million deaths caused by cancer worldwide each year.Metastasis is a complex,multistep process by which cancer cells spread from the primary tumor to surrounding tissues and distant organs.During tumor progression,circulating monocytes and macrophages are actively recruited into tumors where they alter the tumor microenvironment to accelerate tumor progression.Macrophages shift their functional phenotypes in response to various microenvironmental signals generated from tumor and stromal cells.In general,macrophages have been classified into two subsets:the classical M1 and the alternative M2 macrophages.The Ml macrophages serve as a critical cellular component involved in the inflammatory response and antitumor immunity.Conversely,the M2 macrophages exert anti-inflammatory and pro-tumorigenic activities.During late-stage tumor progression,TAMs generally switch to an M2-like phenotype.Such TAMs have been shown to provide a favorable microenvironment for tumor growth,tumor survival,and angiogenesis;produce multiple pro-inflammatory cytokines,such as IL-6,that are involved in the induction of genes important to tumor cell cycle progression and apoptosis suppression.Inducible nitric oxide synthase(iNOS)is a specific marker for type M1 macrophages,whereas arginase-1(Arg-1)is a TAM(M2)-specific marker.Macrophages are the most abundant inflammatory cells in the tumor environment.The interaction between tumor cells and cells in their microenvironment is known to be crucial for malignant progression.In our study,we speculated that tumor-secreted related factors could polarize macrophages from an M1 to an M2 phenotype(TAMs),which is likely to promote the invasion and progress of the tumor.ObjectiveTo investigate the mechanisms of interaction between tumor-associated macrophages and prostate cancer cells in microenvironment on invasion and progression of prostate cancer.Methods1.Human prostate cancer cells PC-3 and C4-2 cells,mice macrophages RAW264.7 cells were cultured in our experiment.PC-3 cells and RAW264.7 cells wereco-cultured for 24h,PBS were used as control group;C4-2 cells and RAW264.7 cells were co-cultured for 24h,PBS were used as control group.2.Western blot were used to detect the protein levels of iNOS and Arg-1 in RAW264.7 cells on the effect of PC-3 and C4-2 cells.3.Western blot were used to detect the protein levels of AMPK、p-AMPK、mTor、p-mTor in RAW264.7 cells the on effect of PC-3 and C4-2 cells.4.Immunofluorescent was used to measure the protein levels of iNOS、Arg-1 in RAW264.7 cells on the effect of PC-3 and C4-2 cells.5.Transwell migration experiments and cell wound-healing assay were used to measure the invasion and metastasis of PC-3 cells and C4-2 cells under the effect of RAW264.7 cells.6.Western blot were used to detect the protein levels of E-cadherin、N-Cadherin、Vimentin and Icam-1 in PC-3 cells and C4-2 cells on the effect of RAW264.7 cells.Results1.Macrophage morphology has changed significantly from rounded small cells to polygonal large and long spindle cells after co-cultured with human prostate cancer cells PC-3 or C4-2 cells for 24h.2.Western blot results showed that the protein expression of iNOS was declining and expression of Arg-lwas increased in RAW264.7 cells on the effect of PC-3 or C4-2 cells for 24h,compared with the PBS control group.3.Western blot results showed that protein expression of p-AMPK increased,while protein expression of p-mTor decreased on the effect of PC-3 and C4-2 cells for 24h,compared with the PBS control group.4.Cell immunofluorescence showed that the protein expression of iNOS was declining and expression of Arg-lwas increased in RAW264.7 cells on the effect of PC-3 and C4-2 cells for 24h,compared with the PBS control group.5.Transwell migration experiments showed that the number and migration ability of prostate cancer cells increased,compared with the PBS control group;Cell wound-healing assay showed that the speed of PC-3 or C4-2 cells migration significantly faster than that in the control group.6.Western blot results showed that protein expression of N-Cadherin、Vimentin、Icam-1 significantly increased,while protein expression of E-cadherin significantly decreased in PC-3 or C4-2 cells on the effect of RAW264.7 cells for 24h,compared with the PBS control group.Conclusion1.In the tumor microenvironment,prostate cancer cells can change the morphology of macrophages,and promote macrophages from M1 to M2 polarization by promoting phosphorylation of AMPK and inhibiting the expression of mTor.2.In the tumor microenvironment,polarized M2 macrophages(TAM)can accelerate the process of epithelial mesenchymal transition,increase the ability of invasion and metastasis of prostate cancer cells.3.The interaction between TAMs and tumor cells in their microenvironment is known to be crucial for malignant progression.