The Role Of TLR4 In Tumor-associated Macrophage Polarization Of Lung Cancer

Objective:To investigate the role of TLR4 and its molecular mechanism in the polartization of tumor associated macrophage in Lung cancer.Methods:1. The correlation of TLR4 and CD68 level in lung cancer patient tumors with relapse-free survival;The expression of TLR4 in tumor associated macrophage of human lung cancer was detected by Immunoflourence.2. Mouse Lewis Lung cancer cells were injected subcutaneously into the flanks of C57BL/6 and TLR4-/- mice.The tumor size of cell primary tumors was compared between C57BL/6 and TLR4-/- mice.3. Tumors were separated from the flanks of C57BL/6 and TLR4-/- mice,the expression of Ml markers(INOS?TNF-??IL-1??IL-6) and M2 markers (Argl? IL-10?MRC1)were assayed by quantitative PCR and Western Blot.4. RAW 264.7 cells were transefected with scramble control and TLR4 shRNA to establish the stable TLR4 knockdown cell line,and then were stimulated by LCM. The interference efficiency was analyzed by quantitative PCR and Western Blot;The expression of Glycosis genes (SLC2A1?HK1?PFKP?PKM2?Aldolase?LDH-5?) was assayed by quantitative PCR;ATP and Lactate concetration were detected by ATP and Lactate assay Kit.5. Flow cytometric analysis was performed to analyze the expression of the quantity and function of mitochondrial and mito-ROS.6. Tumor associated macrophages was separated from C57BL/6 and TLR4-/- mice.The expression of M1 markers(INOS?TNF-??IL-1??IL-6),M2 markers (Argl?IL-10? MRC1) and Glycosis genes (SLC2A1?HK1?PFKP?PKM2?Aldolase?LDH-5?) was assayed by quantitative PCR;ATP and Lactate concetration were detected by ATP and Lactate assay Kit.7. Stable TLR4 knockdown cell linewas stimulated by LCM. And Tumor associated macrophages were separated from C57BL/6 and TLR4-/- mice.The expression of p-AKT,p-mTOR,HIF-1?,Bcl-6 was assayed by Western Blot.8. The cell proliferation markers(Ki67,PCNA) in tumors of C57BL/6 and TLR4-/-mice was analyzed by Immunohistochemical.Lewis Lung cancer cells were separated from C57BL/6 and TLR4-/-mice,and were treated with Genfitinib by MTS and by TUNEL to detect apoptosis;The invasion and migration were analysed by Transwell;The sternness was analysed by Soft agar and Sphere formation assay.9. PGL3-HIF-1? promoter was constructed in report gene plasmid,and they were transfected into RAW264.7 cell together.Results:1. TLR4 is overexpression in tumor associated macrophage of human lung cancer tissues and has negative correlation of patient relapse-free survival.2. The size of tumor from TLR4 knockout mice was smaller than C57BL/6 mice.3. The expression of M1 makers(INOS?IL-6. TNF-? IL-1?) were significantly up-regulated in tumor of TLR4 knockout mice.However The expression of M2 makers(Arg1?IL-10?MRC1) were significantly found down-regulated in tumor of TLR4 knockout mice.4. TLR4 was stably knockdown in RAW264.7 cell line.Knockdown of TLR4 caused the increasd expression of Glycosis genes (SLC2A1?HK1?PFKP?PKM2?Aldolase? LDH-5?) and Lactate concertration.Neverthless TLR4 increased ATP concertration.5. The expression of the quantity and function of mitochondrial and mito-ROS were down-regulated in RAW264.7 TLR4 knockdown cell line.6. The expression of Ml makers(INOS?IL-6? TNF-a?IL-1?),Glycosis genes (SLC2A1? HK1? PFKP?cPKM2? Aldolase? LDH-5a)and Lactate concentration in tumor associated macrophage of TLR4 Knockdown mice were up-regulated. However TLR4 up-regulated M2 makers(Argl. IL-10? MRC1) and increased ATP concertration.7. The expression of p-AKT, p-mTOR, Bcl-6 was down-regulated in Stamble TLR4 knockdown cell line and Tumor associated macrophages separated from C57BL/6 and TLR4 knockout mice. However,The expression of HIF-la was up-regulated.8. The cell proliferation markers(Ki67,PCNA) were down-regulated in tumors of TLR4 kockout mice. TLR4 can help tumor associated macrophage decrease Lewis Lung cancer cells's apoptosis.Futhermore,The invasion and migration of Lewis Lung cancer cells were all increased influenced by TLR4.More importantly,TLR4 strongly enhance The sternness of Lewis Lung cancer cells.9. TLR4 specifically decreased pGL3-HIF1? promoter luciferase activity,which was with overexpression of Bcl-6 in LLC supernatant-stimulated macropahge.10. TLR4 specifically decreased HIF1? expression through the activation of AKT-mTOR-Bcl-6 pathway.Conclusions:1. TLR4 was overexpressed in tumor associated macrophage of Lung cancer.2. TLR4 regulated LCM macropahge and promote M2 polarization through enhacing mitochondrial metabolism and inhibiting glycosis in Lung cancer.3. TLR4 helped tumor associated macrophage improve the growth,cell invasion and migration,stemness of LLC in Lung cancer.4. TLR4 directly influenced AKT-mTOR pathway to target Bcl-6 and exerts a positive role on its expression,Bcl-6 inhibits HIF-la activation by modulating its promoter upon LCM stimulation.