| Background:CXC chemokine ligand 8(CXCL8)is highly expressed in colorectal cancer(CRC)and other tumors,and mediates the malignant progression of tumors by regulating tumor angiogenesis,cell migration,invasion and epithelial-mesenchymal transition.Recent studies have found that CXCL8 can also affect local anti-tumor immune responses by regulating the infiltration of M2-type macrophages in gastric and pancreatic cancer,thereby promoting malignant tumor progression.Studies have shown that many tumor-associated macrophages(TAMs),primarily M2 type,infiltrate in CRC microenvironment.However,the molecular mechanism of TAMs infiltration in CRC remains unclear.Therefore,it will be helpful to further explore the revealing the mechanism of CRC immune escape by uncovering the regulation of CXCL8 on CRC microenvironment.Objective:1、To analyzed the relationship between CXCL8 and the immune infiltration of M2 macrophages in the CRC microenvironment.2、To confirm the chemotactic effect of CXCL8 on M2 macrophages in vitro.3、To verify the chemotaxis of CXCL8 on M2 macrophages in TME in vivo.Methods:1 、 Online Bioinformatics tool “Clinical Bioscience Home” was used to analyzed the CXCL8 expression data in CRC and normal tissue samples from The Cancer Genome Atlas(TCGA)database,One-Sample GSEA Analysis(ss GSEA algorithm)was used to analyze the immune cell infiltration of CRC samples in TCGA database.2 、 Tissue microarray was performed by collecting 102 pairs of cancer and paired adjacent tissues from CRC patients from Shanxi Cancer Hospital.Immunohistochemical staining(IHC)was used to detect the expression differences of CXCL8 in cancer and adjacent tissues of CRC patients.The correlation between CXCL8 and CD163 in cancer tissues were also analyzed.3、Detect the basal expression level of CXCL8 in multiple CRC cells;construct human CRC cell lines(CXCL8/HCT116 and CXCL8/SW480)overexpressing CXCL8,and detect the overexpression efficiency of CXCL8 by WB and q RT-PCR;After HCT116、SW480 cells were stimulated by IL-1β,the expression and secretion of CXCL8 were detected by WB、q RT-PCR and ELISA.4 、 Acute monocytic leukemia cell line THP-1 was induced into M2macrophages(THP-1-M2)by PMA and IL-4 in vitro,q RT-PCR was used to detect the polarization efficiency.Chemotaxis of M2 macrophages was detected via Transwell assay after co-culture THP-1-M2 with CXCL8 overexpressed or IL-β stimulated CRC cell lines.5 、 Murine CXCL8-overexpressed cells were established by using mouse(C57BL/6J / BALB/c)colon cancer cell line MC38 and CT26,which were verified by WB and q RT-PCR.RAW264.7 cells were induced into M2 macrophages(RAW264.7-M2)by IL-4 in vitro.Polarization efficiency was detected by q RT-PCR.RAW264.7-M2 was co-cultured with murine CXCL8 overexpressed cells,followed with chemotaxis detection of M2 macrophages.6、BALB/c mouse subcutaneous xenograft model was established with CT26-CXCL8.Tumor growth was monitored.IHC was used to detect the infiltration of M2 macrophages in xenografted tumor tissue.Results:1 、 Bioinformatics analysis showed that compared with normal tissues,the expression of CXCL8 in cancer tissues of CRC patients was higher.The expression level of CXCL8 in CRC microenvironment was closely correlated with macrophages infiltration.2、IHC results of CRC samples showed that the expression level of CXCL8 was positively correlated with the infiltration of M2 macrophages.3、Upregulation of CXCL8 in human CRC cells could promote the chemotaxis of M2 macrophages.4、IL-1β can enhance the expression and secretion of CXCL8 in CRC cells,and further promote the chemotactic effect of CXCL8 on M2 macrophages.5、Upregulation of CXCL8 in murine CRC cells could promote the chemotaxis of M2 macrophages.6 、 Compared with control group,more infiltrated M2 macrophages were observed in CXCL8 overexpressed xenograft tumor tissues.Conclusion:CXCL8 was highly expressed in human CRC,and the elevated expression level of CXCL8 promotes the infiltration of M2 macrophages in the CRC microenvironment. |
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