Melanoma Microparticles Promote Lung Metastasis Via A Macrophage-dependent Pathway

Objective:Metastasis is a challenge during the progress of tumor.In particular,the frequency of lung metastases and its associated mortality rates are extremely high,but it is still not clear how tumor cells colonize and metastasize to the lungs.In our research,microparticles（MPs）from the tumor cells which found to influential in the process of lung metastasis.However,the mechanism is unknow.This article focuses on the mechanism of melanoma lung metastasis via MPs.Methods:（1）B16-MPs,4T1-MPs and LLC-MPs were extracted.The size distribution of these MPs was analyzed by NS300 system.（2）To discuss the alteration of melanoma lung metastasis after B16-MPs treatment,B16-F10 tumor-bearing mice were treated with PBS or B16-MPs,after 21 days,tumor metastasis in the lungs were analysed by HE staining.（3）To investigate the function of macrophage after B16-MPs treatment,clodronate liposomes（a widely used macrophage depleting agent）pretreated B16-F10 melanoma tumor-bearing mice were treated with B16-MPs and clodronate liposomes.After 21 days,tumor nodules in the lungs were counted.（4）PKH-26 labeled B16-MPs were injected into the mice,24h later,macrophages and B16-MPs were analysised by confocal;at the same time,macrophages in the lungs were analyzed by flow cytometry and fluorescence microscopy after injections of B16-F10 or splenocytes MPs;splenocytes MPs was a control to exclude the influence of microparticles from all immune cells in the tumor microenvironment.（5）In order to explore the originate of lung macrophages,B16-or S-MPs were injected into C57mice,24h later,CCL2 expression in lung macrophages was detected by PCR,then lung resident macrophages were isolated from mice and cultured in the presence of B16-or S-MPs.After 24-or 48-hour,the expression of CCL2 was analyzed by realtime PCR and ELISA;clodronate liposomes pretreated mice or CCR2^-/-mice injected with B16-MPs,after 48 hours the percentage of inflammatory monocytes in lung tissues was analyzed by flow cytometry.（6）To detect the macrophage polarization after MPs treatment,mice injected with S-and B16-MPs,macrophage phenotype relative cytokine and gene were assessed by flow cytometry and PCR.（7）To investigate the relationship between lung IL-6 levels and lung metastasis.IL-6 in lung was measured by ELISA after B16-MPs treatment;B16-F10 tumor-bearing mice was injected with IL-6 antibody and B16-MPs/IL-6 antibody,after 21 days,a subset of mice was sacrificed,and tumor nodules in lung were counted,others were for survival time.（8）Our lab has established an effective soft 3D fibrin gel culture system to generate Stem cell–like tumorigenic cells（3D cell）which are thought to be the key to repopulate a metastatic tumor in distant organs.Mice injected with PKH-26 labeled3D B16 cells followed by B16-F10 MPs treatment,24h later,B16-F10 cells within lung analyzed by confocal;MPs treated mice were challenged with 3D B16-F10 cells,tumor nodules of partly mice were counted after 21 days,others were for the long-term survival.（9）Fibrin within lung of mice were detected after treatment with S-and B16-MPs;Mice treated with B16-MPs or B16-MPs/plasmin was challenged with B16-F10 cells,after 21 days tumor nodules in the lungs were counted.（10）To investigative the reason of fibrin deposition,the concentration of VEGF-A in the lung of B16-MPs and B16-MPs/GW2580 treated mice was detect by ELISA;The expression of VEGF-A of bone marrow-derived macrophages and lung macrophages was measured by Elisa after treatment with B16-MPs 48h.Results:（1）The diameter of microparticles was 100-1000 nm,and the microparticles were not uniformly distributed,the intermediate particle size were the most.（2）After treatment of microparticles,lung metastasis is significantly enhanced.（3）Macrophage was crucial to lung metastasis of melanoma.（4）B16-MPs could get into the lungs and be taken up by macrophages,pulmonary macrophages increased significantly by B16-MPs treatment rather than S-MPs.（5）The number of inflammatory monocytes in lung increased definite after treatment with B16-MPs,and this effect was associated with CCL2 expression of macrophages,which chemotactic monocytes into the lungs.（6）Mice treated with B16-MPs promoting lung macrophages with partial functions of M1 and M2 phenotypes.（7）The concentration of IL-6 within the lungs increased with the treatment times,but after treatment with IL-6 antibody,lung metastasis which promoted by B16-MPs disappeared.（8）B16-F10 tumor-repopulating cells migrated to the lungs enhanced by B16-MPs（9）After mice treated with B16-MPs,fibrin deposition in the lungs increased,plasmin could inhibit the melanoma lung metastasis induce by B16-MPs.（10）The concentration of VEGF-A in lungs rised,and it decreases after macrophages were suppressed in vivo.Same result was acquired when macrophages were treated with B16-MPs in vitro.Conclusion:We show that B16-MPs readily enter the lung parenchyma where they are taken up by local macrophages and induce CCL2 production.CCL2 recruits inflammatory monocytes to the lungs where they mature into macrophages that not only produce IL-6 but also trigger fibrin deposition.IL6 and the deposited fibrin facilitate the survival and growth of tumor-repopulating cells in the lungs by providing chemical and mechanical signals,respectively,thus,setting the stage for lung metastasis.