Leptin Regulates Crosstalk Between Cancer Cells And Tumor Macrophages To Promote Invasion And Metastasis Of Breast Cancer

Objective:To investigate the contribution of leptin in regulating the crosstalkbetween breast cancer cell and tumor associated macrophages on theinvasion and metastasis of breast cancer and to explore its molecularmechanisms that modulate leptin effects.Methods:1.Human monocyte cells THP1treated with PMA and IL-4wasdifferentiated into M2macrophages. The cell morphology was observed byinverted microscope. And the phenotype (CD206, IL-10, IL-12and TGF-β)of M2macrophage was detected by flow cytometry.2.The expression of key cytokine secreted from M2macropages whichwere treated with leptin was detected by qRT-PCR and Western blotingmethods.3.The expression of Ob-R in THP1and M2macrophage was detectedby immunofluorescent staining, qRT-PCR and Western bloting methods.And the expression of Ob-R in M2macrophage treated with leptin wasdetected. 4.The effect of IL-8in migration and invasion of breast cancer MCF7and MDA-MB-231cells was detected by scratch test and Trasswell assay.And the migration and invasion of breast cancer cells in coculture systemof breast cancer cells-TAMs which was neutrilized IL-8by anti-IL-8antibody were detected.5.The signaling pathways(MAPK/ERK1/2and p38/MAPK) activatedby leptin that regulated the expression of IL-8in TAMs, which werepretreated with several common signaling pathway inhibitors, was detectedby qRT-PCR and Western bloting methods. And further to prove theactivation of key signaling pathways was dependent the leptin-Ob-R axis.6.The expressions of CD68, Ob-R and IL-8in human breast cancersamples were detected by immunohistochemical analysis.7.Establishment of breast cancer allograft model in situ female Babl/cnude mice (6to8-week-old) mammary fat pad of orthotopic injection ofMDA-MB-231cells. Intraperitoneal (i.p.) injection of Clophosome-Clodronate Liposomes (neutral) for macrophage depletion, and micereceived intratumor injection of leptin when the volume of tumor wasappropriate, then observed the growth of tumor, the survival time of miceand the metastasis of tumor. Immunohistochemical analysis of CD68, Ob-Rand IL-8in the tumor of mice.Results:1.Human monocyte cells THP1treated with PMA and IL-4wasdifferentiated into M2macrophages. The cell morphology was fromsuspension into adherence, and several cells produced pseudopodiums. TheM2macrophage surface markers CD206was positive in M2macrophages.And the cytokine profiles of M2macrophages were high TGF-β, IL10, andlow IL12, compared to THP-1. 2.Leptin up-regulate the expression of IL-8in TAMs. The migration andinvasion of breast cancer MCF7and MDA-MB-231cells were enhancedby IL-8. And the migration and invasion of breast cancer cells in coculturesystem of breast cancer cells and TAMs was decreased by neutralized theIL-8.3.The Ob-R was highly expressed in THP1and M2macrophage, andthe expression of Ob-R in M2macrophage was increased by leptin. Leptinbinding to Ob-R, activated the MAPK/ERK1/2and p38/MAPK signalingpathway and increased the expression of p-ERK1/2and p-p38toup-regulate the expression of IL-8in M2macrophage.4.The expression of CD68, Ob-R and IL-8was higher in humanmetastatic breast cancer samples than in benign breast tissue samples andprimary breast cancer samples.5. In the tumor-bearing nude mice, leptin could increase the growth oftumor and decrease the survival time. And the pulmonary metastasis ofbreast cancer might be associated with the expression of IL-8in TAMsregulated by leptin.Conclusion:1.Leptin binding to Ob-R, activated the MAPK/ERK1/2andp38/MAPK signaling pathway to increase the expression of IL-8in M2macrophages. Leptin up-regulated the expression of IL-8in M2macrophages to promote the migration and invasion of breast cancer cellsin coculture system of breast cancer cells-TAMs.2.The number of TAMs and the expression of Ob-R and IL-8werehigher in human metastatic breast cancer samples than in benign breasttissue samples and primary breast cancer samples. 3. In the tumor-bearing nude mice, leptin could increase the growth oftumor and decrease the survival time. And the pulmonary metastasis ofbreast cancer might be related with the expression of IL-8in TAMsregulated by leptin.