Effect Of Fatty Acid Transporter CD36 On Hepatic Metastasis And The Underlying Mechanisms

Objective:Study on the role of CD36 in experimental liver metastases to provide a new direction and theoretical basis for the improvement and treatment of liver metastasis.Methods:Part I:Mouse Lewis lung cancer cells were used to construct an experimental liver metastasis model by spleen injection,in which 8 weeks old C57BL/6 background male wild type mice（WT）were selected as the control group,and CD36 gene-knockout mice(CD36^-/-)were selected as the experimental group.The mice were dissected at 12d and 18d to collect liver tissue and compared for differences in tumor metastases by HE staining.Part II:Liver-specific CD36 knockout mice(CD36^△hep)were generated using the Cre/Loxp recombinase system.The genotype was identified by agarose gel electrophoresis after amplification of the rat tail DNA.The liver DNA was extracted to verify Cre recombinase activity,and the efficiency of liver-specific CD36 knockdown was verified by Q-PCR and Western blot.An experimental liver metastasis model was constructed of LLC cells to the control CD36^fl/fll/fl mice and CD36^△hepmice.The liver tissue was dissected at 12 days and the area of metastaticlesionswascountedbyHEstaining.PartⅢ:Immunohistochemistry and TUNEL staining were used to detect cell proliferation and apoptosis in tumor metastases,respectively.The expression of F4/80 and M1 and M2 macrophage-associated factors were detected by Q-PCR.WT mice and CD36^-/-mice were intraperitoneally injected with Clodronate liposomes to observe the effect of depleted macrophages on hepatic metastases.Results:Part I:Liver tissues of metastasis model constructed by LLC cells showed that the number of liver metastases in CD36^-/-mice was significantly reduced than that in WT mice at 12 days（P<0.05）.HE staining of liver paraffin sections showed a significant decrease in the area of tumor metastases in CD36^-/-mice compared with WT mice.The results of 18d liver tissue showed that the relative tumor area of CD36^-/-mice were also significantly decreased than that of WT mice,suggesting that systemic knockout of CD36 inhibits the development of hepatic metastases.Part II:Compared with the control group mouse CD36^fl/fl,CD36^△hepmouse liver Cre recombinase plays a role.PCR and Western blot results showed that the expression level of CD36 in the liver of CD36^△hephep mice was significantly decreased compared that of the control mice.The expression of CD36 protein in other tissues was not statistically changed.These results indicated the successful generation of liver-specific CD36knockout mouse.In the liver metastasis model constructed by LLC cells,the number and relative area of tumor metastases in CD36^△hepmice tended to be increased compared with CD36^fl/fll/fl mice,but the difference did not reach statistically significant,indicating that CD36 on liver parenchymal cells does not affect liver cell metastasis.PartⅢ:Immunohistochemistry and TUNEL staining showed that systemic CD36 deletion did not affect cell proliferation and apoptosis in tumor metastases.The expression of F4/80 in liver tumor tissues of CD36^-/-mice was significantly decreased by Q-PCR（P<0.05）.The expression of M1 macrophage-associated factors iNOS,TNF-αand IL-1βwas increased in liver tumor tissues of CD36^-/-mice,and the expression of M2 macrophage-associated factors CD206 and CD68 was decreased,indicating systemic CD36 deficiency may inhibit the development of experimental liver metastases by increasing the number of M1 macrophages and reducing the number of M2 macrophages in tumor metastases.CD36^-/-mice cannot inhibit the progression of liver metastases after consumption of macrophages.Conclusion:Systemic CD36 deletion significantly inhibited the progression of hepatic metastases,but CD36 deletion in hepatocytes did not affect the colonization of tumor cells,suggesting that CD36 deletion may be inhibited in liver non-parenchymal cells.Systemic CD36 deletion increases M1 type macrophage and decreases M2 type macrophages in tumor metastases,which may affect the polarization of macrophages.This situation causes a microenvironment that is not conducive to tumor colonization to ultimately inhibit the progression of liver metastases.