Study On The Mechanism Of High Matrix Stiffness Enhancing M2 Polarization Of Macrophages

Objective: Matrix stiffness is an important mechanical parameter in tumor microenvironment,and its value is often higher in liver malignant tumors.Liver tissue stiffness greater than 12.0k Pa is an independent risk factor for hepatitis C patients with HCC,while liver tissue stiffness value of 8.5k Pa is the critical value for hepatitis B patients with HCC.At present,the relationship between matrix stiffness and cancer-related fibroblasts has been well elucidated.The activation of cancer-related fibroblasts will promote matrix deposition,and then aggravate the process of matrix stiffness increase.Under the condition of high matrix stiffness,cancer-related fibroblasts are more likely to be activated,thus forming a positive feedback cycle,leading to further matrix hardening and continuous activation of fibroblasts.In addition to cancer-related fibroblasts,macrophages are one of the most abundant immune cells in tumor microenvironment.Macrophages in tumor microenvironment(TME)are mainly divided into two polarization phenotypes: classical activated M1 and alternately activated M2.The proportion of M2 macrophages increased with the development of the tumor.M2 macrophages infiltrated a large number of M2 macrophages in HCC.It can accelerate tumor progression from many aspects,such as hindering anti-tumor immunity,stimulating tumor cell proliferation,promoting tumor angiogenesis and enhancing tumor metastasis.It was found that macrophages were highly sensitive to mechanical force,and the migration ability and polarization state of macrophages changed with the change of matrix stiffness.However,the relationship between matrix stiffness and macrophage polarization is still controversial.The purpose of this study is to reveal the relationship between high matrix stiffness and macrophage polarization,and the mechanism of the effect of high matrix stiffness on macrophage polarization,so as to provide a theoretical basis for the therapeutic strategy of targeting macrophages in TME.Methods:1.Materials: human hepatoma cell lines HCCLM3 and Huh7 and human monocytic leukemia cell line THP-1.2.Methods:(1)Direct cell co-culture: HCCLM3 and Huh7 cell lines were co-cultured with THP-1-derived PMA-induced macrophages in 4k Pa and 12 k Pa matrix stiffness petri dishes.(2)Indirect cell co-culture: the conditioned medium of hepatoma cells from 4k Pa and 12 k Pa matrix stiffness petri dishes was extracted and used to deal with macrophages in 4k Pa and 12 k Pa matrix stiffness petri dishes.(3)Cell culture: 4k Pa and 12 k Pa matrix stiffness petri dishes simulate different stiffness environments in vivo.(4)Western blot: used to detect macrophage polarization-related markers and M2polarization-related surface receptors of macrophages.(5)ELISA: used to detect some cytokines secreted by hepatoma cells or macrophages.(6)Real time PCR: used to detect the expression of CXCL12 in macrophages.(7)Flow cytometry: used to detect the relative number of M2 macrophages(CD86and CD206 positive cells)after direct co-culture.(8)Statistical analysis: the data were counted by the software SPSS22.0,and the experiments were repeated 3 times independently.The data is expressed in the form of mean ±standard deviation.Graph Pad Prism9.0 is used to draw charts.The statistical significance of two or more groups was compared by independent sample T test or single factor analysis of variance.When P < 0.05,we think the difference is statistically significant.3.Experimental scheme:3.1 Study on the relationship between high matrix stiffness and macrophage polarization.(1)To evaluate the M2 polarization of macrophages: the relative number of CD86(M1 marker)and CD163(M2 marker)positive cells was detected by flow cytometry.In western blot detection,CD206 and CD163 were used as M2 polarization evaluation indexes,and i NOS and CD86 as M1 polarization evaluation indexes.(2)Construction of direct co-culture model of hepatoma cells and macrophages:macrophages and tumor cells were co-inoculated in 4k Pa and 12 k Pa matrix stiffness petri dishes at 1:1 to achieve direct co-culture,which was used to evaluate the correlation between high matrix stiffness and macrophage polarization.(3)Construction of indirect culture model of hepatoma cells and macrophages: the conditioned medium of hepatoma cells from 4k Pa and 12 k Pa matrix stiffness petri dishes was extracted to deal with macrophages in 4k Pa and 12 k Pa matrix stiffness petri dishes to evaluate whether high matrix stiffness affects the regulation of hepatoma cell secretion on macrophage polarization.3.2 Study on the mechanism of the effect of high matrix stiffness on macrophage polarization.(1)The effect of high matrix stiffness on the sensitivity of macrophages to polarization signals: macrophages were directly inoculated into 4k Pa and 12 k Pa matrix stiffness petri dishes to evaluate the direct effect of high matrix stiffness on macrophage polarization.IFN γ / LPS or IL4/IL13 classical polarization-induced drugs were used to treat macrophages to explore whether high matrix stiffness affected the sensitivity of macrophages to polarization signals.The protein expression levels of CCR2,CSF1 R,IL10RA,CXCR4 and IL4 R were detected by western blot to explore whether high matrix stiffness affected the ability of macrophages to receive polarization signals.(2)To evaluate the M2 polarization-related cytokines secreted by macrophages:ELISA method was used to detect the secretion of CXCL12 in macrophage conditioned medium,and to explore the relationship between high matrix stiffness hepatoma cell conditioned medium and macrophage secretion of CXCL12.Real time PCR assay was used to evaluate the relative expression level of CXCL12 m RNA in macrophages treated with hepatoma cell conditioned medium,and to explore the relationship between high matrix stiffness hepatoma cell conditioned medium and macrophage expression of CXCL12.(3)M2 polarization related surface receptor inhibitor reversing M2 polarization of macrophages: in the experimental group treated with CSF1 R inhibitor PLX-3397,the secretion of CXCL12 by macrophages was measured by ELISA method to verify the correlation between CSF1/CSF1 R and CXCL12.The macrophages in 12 k Pa petri dish were treated with drugs(CXCR4 antagonist LY2510924 and CSF1 R inhibitor PLX-3397)and continuously added to 12 k Pa hepatoma cell conditioned medium.The expression of M2 polarization markers of macrophages was detected by western blot,and the effect of M2 polarization related surface receptor inhibition on M2 polarization of macrophages was evaluated.Results:1.High matrix stiffness enhanced M2 polarization of macrophages.1.1 High matrix stiffness enhances the M2 polarization of macrophages induced by hepatocellular carcinoma cells.1.2 High matrix stiffness enhanced the effect of hepatoma cell conditioned medium on M2 polarization of macrophages.2.High matrix stiffness enhances the sensitivity of macrophages to M2 polarization signals.2.1 High matrix stiffness promotes the expression of M2 polarization-related cell surface receptors in macrophages.2.2 High matrix stiffness enhances M2 polarization of macrophages by promoting the secretion of cytokines by hepatocellular carcinoma cells.2.3 High matrix stiffness enhances M2 polarization of macrophages through CSF1/CSF1R-CXCL12/CXCR4.Conclusion: High matrix stiffness can enhance the M2 polarization of macrophages by promoting the secretion of CSF1 by hepatoma cells and the expression of CXCR4 and CSF1 R in macrophages.