| Objective: The liver is a common solid tumor metastasis site,because it has organ-specific immunosuppressive properties,and the clinical effect on the treatment of liver metastasis is poor [1].Tumor associated macrophages(TAM)are common immune cells in the Tumor microenvironment(TME)and play an important role in the occurrence and development of tumors[2].Infiltration of TAM suggests poor tumor prognosis and can promote tumor function by promoting tumor angiogenesis,inhibiting immune response,promoting proliferation,and t tumor stem cell self-renewal.Increased infiltration of TAM in liver metastatic cancer has been previously reported [4],but the increased sources of TAM are inconclusive.Cluster of differentiation 36"CD36# is an important transporter of fatty acids,mainly responsible for the uptake and transport of long-chain fatty acids,and it has been reported that CD36 is highly expressed in a variety of tumors and can promote tumor metastasis[3].but the role of CD36 in TAM is unclear.Therefore,this study intends to explore in depth the role of CD36 in liver metastasis and its mechanism,and to find potential targets for the treatment of liver metastasis.Methods: Part Ⅰ: Construct a systemic CD36 knockout(CD36KO)mouse model against the background of C57BL/6J mice.Liver metastasis models were constructed by injecting tumor cells(LLC or B16F10)into WT and CD36 KO mice via the spleen.Liver tissue was obtained on the14 th day after tumor injection,mouse body weight,liver and tumor size were measured,HE staining to understand tumor area and differentiation,Ki67,CD31 immunohistochemical staining to understand tumor cell proliferation,angiogenesis.Database and flow cytometry to analyze the expression of CD36 in liver tissue cells.CL cleared macrophages in vivo to observe tumor formation in WT and CD36 KO mice.Myeloid-specific knockout CD36(CD36MKO)mice were constructed,and tumor liver metastases model was constructed on control and CD36 MKO mice.Tumor size,liver weight,body weight,and HE staining were measured on day 14 to determine the effect of macrophage CD36 on liver metastasis.Part Ⅱ: GEO database,immunohistochemical staining and flow cytometry to analyze immune cell infiltration in liver metastasis,and further flow cytometry to analyze the proportion of each subpopulation in liver metastatic carcinoma in TAM.CSFE-stained bone marrow cells were injected intravenously into WT normal and tumor-bearing mice,and liver tissue was obtained after 24 h to analyze the distribution of CSFE-stained bone marrow-derived monocytes in hepatic tissue.The database analyzed the correlation between CD36 expression and macrophage infiltration in liver metastatic carcinoma.Endothelial adhesion and Transwell migration experiments to understand the expression of CD36 in adherent and migratory of monocytes and macrophages.After extracting WT/CD36 KO mouse CD11b+ myeloid cells or BMDM cells using Tumor conditioned medium(TCM),endothelial cell adhesion and Transwell experiments were performed to analyze the adhesion and migration ability of monocyte macrophages.Construct CD36 overexpression stable cell lines of THP-1using lentiviral transfection.THP-1 NC/CD36 OE cell line and A549 supernatant treatment type adhesion and migration experiments were also used.Part Ⅲ: q PCR detects the expression of chemokines CCL2,CCL3,CCL5,CCL12 and MCSF in LLC liver metastases.q PCR detected LLC,BMDM,CD11b+ myeloid cell chemokines and receptor expression.Detection of chemokine receptor CCR2,CSF1 R m RNA expression in TAM.Endothelial adhesion and Transwell migration experiment were performed on THP-1WT or NC/CD36 OE cells using the pan-PI3 K inhibitor BKM120.WB detected BMDM WT/CD36 KO or THP-1NC/CD36 OE cells with PI3 K,p PI3 K,AKT,p AKT protein expression after tumor supernatant treatment.BODIPY and BODIPY FL C16 detect lipid deposition and fatty acid uptake in TAM.Lipidomics analyzes changes in lipid composition in TAM.Results: Part Ⅰ: In the preclinical model of liver metastasis,the formation of cancer of LLC and B16F10 liver metastases decreased after CD36 systemic knockout.Database and flow cytometry analysis suggest that CD36 is predominantly highly expressed on peripheral blood mononuclear cells(PBMCs)and liver macrophages compared with hepatocytes and other immune cells.Liver metastatic carcinoma was improved in both WT and CD36 KO mice after macrophage clearance,suggesting the contribution of macrophage CD36 to liver metastatic carcinoma.A liver metastasis model was further constructed on a mouse model of CD36 MKO,and the results suggested that myeloid CD36 knockout could reduce the occurrence of liver metastatic carcinoma.Part Ⅱ: Database,immunohistochemistry,and flow cytometry all indicate increased macrophage infiltration in hepatic metastatic carcinoma compared with normal liver tissue.Further analysis of in vitro bone marrow cell CSFE staining and flow cytometry suggests increased TAM mainly from peripheral blood mononuclear infiltrating macrophages(IM).Database analysis of CD36 expression was positively correlated with TAM infiltration in liver metastasis.Adhesion and migration experiments using WT CD11b+ bone marrow cells and bone marrow-derived macrophages(BMDM)suggested increased adhesion and migration capacity of CD36high-expression cell populations.Macrophage CD36 was confirmed using the CD36 MKO liver transfer model to reduce mononuclear macrophage infiltration.In vitro experiments have confirmed that CD36 KO can reduce the adhesion of monocytes and the migration ability of macrophages.Part Ⅲ: CCL2,CCL3,CCL12,MCSF expression is elevated in liver metastasis,while LLC cells mainly express CCL2,MCSF,BMDM and CD11b+ bone marrow cells with high expression of CCR2,CSF1 R.However,CD36 does not affect the expression of CCR2 and CSF1 R.Further adhesion screening PI3 K inhibitors inhibit the adhesion capacity of THP-1.WB prompts PI3K/AKT signal activation of BMDM and THP-1 after TCM processing.PI3 K inhibitors inhibit the increased adhesion and migration capacity caused by CD36 OE.After BMDM CD36 KO,the expression of phosphorylated PI3K(p-PI3K)and phosphorylated AKT(pAKT)decreased.In contrast,THP-1 CD36 OE cells increased expression of p-PI3 K and p-AKT.It has been previously reported that lipid accumulation in TAM can affect PI3 K.Further use of BODIPY staining and BODIPY FL C16 uptake suggests lipid accumulation and increased fatty acid uptake in TAM,and CD36 can promote lipid droplet deposition and fatty acid uptake in TAM.LC-MS suggests an increase in triglycerides(TG)and diglycerides(DG)in TAM,and CD36 promotes an increase in the total amount of TG in TCM and alters the composition of fatty acids in TG.Conclusion: CD36 can increase peripheral mononuclear macrophage infiltration and promote the occurrence of liver metastatic carcinoma,and CD36 can promote the adhesion and migration of mononuclear macrophages by reprogramming TAM fatty acid metabolism and regulating PI3K/AKT pathways. |
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