The Study On The M2 Polarization Of Macrophages In The Hypoxic Hepatoma Microenvironment

Objective: Liver cancer ranks fourth among the new tumors in China,and the death caused by liver cancer ranks second among all tumor related deaths,posing a huge burden on the healthcare system in China.At present,the main methods of liver cancer can only benefit partial liver cancer patients,but there are still a large number of liver cancer patients who cannot receive effective treatment.Hypoxia is one of the most prominent characteristics of solid tumors such as liver cancer.With the development of tumor,due to the abnormal distribution and dysfunction of tumor blood vessels,liver cancer cells are often in the hypoxic microenvironment.Driven by hypoxia,liver cancer cells can not only reprogram their internal metabolic mode,but also secrete information substances to transmit to cells in the microenvironment to adjust the phenotypes of tumors for survival.Macrophages are the most infiltrating immune cells in liver cancer tissue and have multiple functions during the occurrence and development of liver cancer.Tumor associated macrophages(TAMs)can polarize into two different phenotypes when responding to different microenvironment conditions,namely classic activated type(M1type)and alternative activated type(M2 type).M1 type tumor macrophages exert cytotoxic functions,thus inhibiting tumor progression.M2 macrophages have anti-inflammatory activity,which often promotes tumor progression.Tumor hypoxia and M2 type macrophages are closely related to the poor prognosis of patients with liver cancer.At the same time,studies have confirmed that hypoxia microenvironment has a profound impact on macrophages,while changes in macrophage metabolism can further aggravate tumor hypoxia.However,whether hypoxic microenvironment can induce M2 type polarization of macrophages remains controversial.A large number of studies have confirmed that hypoxia microenvironment in liver cancer is closely related to macrophages.For example,hypoxia has a chemotaxis effect on macrophages,which makes them infiltrate more in liver cancer tissue;Target and reduce hypoxia in liver cancer can reduce tumor-promoting M2 macrophages and increase tumor-inhibiting M1 macrophages.However,whether hypoxia in liver cancer can directly induce M2 type polarization of macrophages,and whether macrophage polarization in hypoxic microenvironment depends on a specific condition remain unknown.These scientific questions still need further exploration.Therefore,to reveal the relationship between hypoxic liver cancer microenvironment and M2 type polarization of macrophages,this project explores whether the hypoxic microenvironment of liver cancer is related to M2 macrophage,then explores whether the hypoxic microenvironment of liver cancer is directly related to macrophage M2 type polarization or whether there is an indirect relationship between them,and finally preliminarily explores the specific mechanism of macrophage M2 type polarization in the hypoxic microenvironment of liver cancer.Methods:1.Materials: Human liver cancer cell lines Hep G2 and Huh7,human monocytic leukemia cell line THP-1,clinical liver cancer samples.2.Methods:(1)Cell coculture: Hep G2 and Huh7 cell lines were cocultured with THP-1 in the upper and lower chambers of transwell with a pore size of 400 nm,respectively.(2)Cell culture: Culture cells under 21% oxygen concentration to simulate a normal oxygen concentration environment,while culture cells under 1% oxygen concentration to simulate a low oxygen concentration environment.(3)Immunohistochemistry: to detect of HIF-1α to divide the liver cancer samples into hypoxic and non-hypoxic samples,and detect CD206 in clinical liver cancer samples to determine M2 macrophage infiltration.(4)Differential ultracentrifugation: to extract exosomes.(5)Transmission electron microscopy: to observe the particle size and morphology of exosomes.(6)Nanoparticle tracing analysis: to detect the particle size distribution of exosomes.(7)Western blot: to verify the purity of exosomes and to detect the activation of classical pathways in M2 macrophages.(8)Microscopic observation: to determine whether THP-1 cells undergo polarization.Unpolarized cells appear in a single suspended state,and polarized macrophages appear in adherent clusters.(9)Real time PCR: to detect the expression of M2 type markers(CD206,CD163,and Arg-1)in macrophages.(10)Flow cytometry: to detect the proportion of M2 type macrophages(CD11b and CD163 double positive cells).(11)Statistical analysis: The data was analyzed using software SPSS 22.0,and the experiments were repeated independently three times.The data is represented in the form of mean ± standard deviation.Statistical significance was determined through Student’s t-test and analysis of variance,and there was a statistical difference when P<0.05.3.Protocol:3.1.The correlation between the hypoxic microenvironment and the M2 polarization of macrophages in hepatocellular carcinomaCollect liver cancer samples.Determine whether the hypoxic microenvironment of liver cancer is related to M2 macrophages by dividing liver cancer samples into hypoxic and non-hypoxic samples,and evaluating the infiltration of M2 macrophages in hypoxic and non-hypoxic liver cancer via immunohistochemistry.3.2.the study on how the hypoxic liver cancer microenvironment effect M2 polarization of macrophages(1)Evaluating the M2 polarization of macrophages: The M2 polarization was comprehensively determined by morphological observation,PCR,and flow cytometry.(2)Determine whether hypoxia can directly induce macrophage M-type polarization:Evaluate the M2 type polarization of THP-1 cells cultured under hypoxia and normal oxygen concentrations.(3)Determine whether liver cancer cells can induce macrophage M2 type polarization in hypoxic environments: THP-1 and liver cancer cell coculture systems,as well as THP-1 cell culture systems,were cultured under hypoxic conditions to evaluate the M2 type polarization of THP-1 cells in both systems.(4)Extract and verify exosomes: Exosomes were extracted by differential ultracentrifugation.Evaluate the purity of exosomes via transmission electron microscopy,western blot,and nanoparticle tracing analysis.(5)Evaluate the uptake of exosomes by macrophage: use Di D dye to label exosomes and coculture exosomes and THP-1 cells.Ab Fluor 488-Phalloidi labele THP-1cell membrane and DAPI labeles THP-1 cell nucleus.Then,the uptake of exosomes was observed through fluorescence confocal microscopy.(6)Determine whether exosomes released by liver cancer cells in hypoxic environments can induce M2 type polarization of macrophages: THP-1 cells treated with equal amounts of PBS were used as negative controls,it was evaluated that the M2 type polarization of THP-1 cells induced by exosomes released by liver cancer cells at 1% or21% oxygen concentrations.To further verify whether the exosomes released by hepatoma cells in the hypoxic liver cancer microenvironment can induce M2 polarization of macrophages,the exosome inhibitor GW4869 was added to the coculture system of hepatoma cells and THP-1 cells,and the THP-1 cells cultured separately,then it was evaluate that the M2 polarization of THP-1 cells.3.3.Preliminary exploration on the mechanism for the M2 polarization of macrophages in hypoxic liver cancer microenvironmentUsing THP-1 cells treated with equal amounts of PBS as negative controls,Western blot was used to detect the total protein level and phosphorylation level of three M2 polarization classical pathways STAT3,STAT6,and NF-κB in THP-1 cells induced by exosomes released from liver cancer cells at 1% and 21% oxygen concentrations.Results:1.More M2 type macrophages infiltrate in the hypoxic liver cancer samples.2.M2 polarization of macrophages in hypoxic liver cancer microenvironment requires exosomes released by liver cancer cells: hypoxia cannot directly induce the M2 polarization of macrophages;hepatoma cells participate in the M2 polarization of macrophages in the hypoxic liver cancer microenvironment;the exosomes released by hepatoma cells in hypoxic microenvironment induce the M2 polarization of macrophages.3.M2 polarization of macrophages in hypoxic liver cancer microenvironment may be related to STAT6 signal pathways.Conclusion: The exosomes released by hepatoma cells in the hypoxic hepatoma microenvironment induced the M2 polarization of macrophages,which led to more M2 type macrophages infiltrating in the hypoxic hepatoma region.The M2 polarization of macrophages in hypoxic liver cancer microenvironment may be related to STAT6 signal pathways.