Lung Cancer-promoting Mechanism Of Macrophage Polarization Microenvironment And Drug Therapy

The malignant progression of tumor is closely related to the microenvironment of macrophage polarization. During tumor initiation, polarized M2 macrophage create an inflammatory environment to promote tumor growth. Unfortunately, the relationship between macrophage polarization microenvironment and tumor progression is complex and remains ambiguous. Therefore, it is extremely important to clarify why macrophage polarization microenvironment promotes lung cancer development. In this study, we focus on the mechanism of macrophage polarization microenvironment in lung cancer-promoting efficacy and related drug therapy, object is to provide effective measures for prevention and treatment of lung cancer.Firstly, we investigated whether there is a direct effect of macrophage polarization on LLC cells in vitro. The mouse peritoneal macrophages(PEMs) were extracted and stimulated by 10 ng/ml lipopolysaccharide(LPS) to obtain the M1-type or stimulated by 10 ng/ml IL-4 to obtain the M2-type. Macrophage phagocytosis of LLC was observed by fluorescence staining. In vitro LLC cells, the effect of macrophage conditioned medium on the cell proliferation was detected by MTT assay and PCNA immunofluorescence assay; cell scratch assay and Transwell assay were used to test the effect of macrophage conditioned medium on cell migration and invasion. Meanwhile, the effect of macrophage polarization microenvironment on angiogenesis in vitro was also studied in human umbilical vein endothelial cells(HUVECs). The effect of macrophage conditioned medium on cell proliferation was examined by MTT assay and PCNA immunofluorescence assay, cell permeability was detected by evans blue infiltration; chick chorioallantoic membrane(CAM) model was used to study angiogenesis. Further, to explore the cancer-promoting mechanism of macrophage polarization microenvironment, cell apoptosis was evaluated by AO/EB fluorescence staining; the expression of autophagy related LC3-B and P62 protein was detected by immunofluorescence assay; DQ-Green BSA degradation experiment was used to observe the cell lysosomal activity; DAF-2 DA and DCF-DA were used to detect the intracellular NO and ROS. Finally, we establish a urethane-induced lung cancer model in mice, the phenotype of macrophages in the alveolar cells was detected by flow cytometry and Western Blot in mice with lung cancer; the contents of IFN-?, IL-12, TNF-?, TGF-?, IL-10, IL-4 in the BAL and 5-HIAA in the serum of mice were detected by the ELISA kit; LC3-B, P62 and CD31 protein expression in mouse lung was detected by immunohistochemistry. In addition, macrophage depletion agents, autophagy inhibitors and vascular protective agents were used to confirm the relationship between macrophage polarization and tumor angiogenesis in lung cancer.In this paper, the results showed that the phagocytic capacity was significantly stronger in M1 macrophages and was weaker in M2 macrophages than pre-polarized populations. The polarized M1 or M2 macrophage-conditioned medium had no significant effect on the proliferation, migration and invasion in LLC cells, unexpectedly, we found that it was M2 but not M1 macrophage-conditioned media promoted cell proliferation and permeability in vitro HUVECs, and increased angiogenesis in chick chorioallantoic membrane(CAM). Further mechanism exploration showed that the M2 macrophage-conditioned media significantly increased the expression of autophagosome and LC3-B protein but decreased the degradation of P62 protein and DQ-Green BSA in HUVECs under 1% serum-starved-induced autophagy condition, whereas chloroquine as a autophagy inhibitor less than the used concentration of affecting cell proliferation decreased cell permeability in HUVECs. In addition, we found that M2 macrophage conditioned media increased intracellular NO and ROS levels which was blocked by chloroquine in HUVECs, Finally, we observed in vivo experiments that urethane-induced lung carcinogenesis increased M2 macrophages phenotype, promoted the LC3-B expression and prevented P62 degradation in lung tissue. Simultaneously. urethane exposure promoted angiogenesis indicated by CD31 immunohistochemical staining, increased vascular abnormality indicated by serum 5-HIAA level, and enhanced pulmonary vascular permeability. Clodronate liposome-induced M2 macrophage depletion, chloroquine-induced autophagic prevention or salvianolic acid B-induced vascular protection significantly decreased the number of lung tumors, CD31 protein expression, serum 5-HIAA level and pulmonary vascular permeability, abnormal angiogenesis and prevented lung carcinogenesis.In summary, these findings indicate that the mechanism of M2 macrophage microenvironment in cancer-promoting efficacy is not to directly stimulate tumor proliferation but to induce autophagic vascular abnormalities. Inhibiting the formation of M2 macrophage, reducing autophagy or protecting blood vessels can be the promosing measures for lung cancer prevention and treatment.