GDF15 Inducing Tumor-Associated Macrophage Reprogramming Via CD48 Leading To Tumor Escape Of Hepatocelluar Carcinoma

BackgroundThe incidence rate and death rate of hepatocellular carcinoma(HCC)in China are the highest in the world.Traditional therapies are not effective in treating HCC,however,the emergence of immunotherapy has brought new hope to HCC patients.Immunotherapy has been shown to be effective in basic research and clinical practice for HCC.However,the efficacy of immunotherapy for HCC is still far from satisfactory compared with other approved tumors.The search for more effective immunotherapeutic targets for liver cancer and the development of corresponding therapeutic agents is crucial.As an important frontline immune organ of the body,the liver maintains a suppressed immune tolerance state.This immunosuppressed state promotes the formation of a suppressive immune microenvironment(Tumor microenvironment,TME)for HCC when carcinogenesis occurs,further exacerbating immune escape.Uncovering the formation mechanism of suppressive TME in hepatocellular carcinoma is crucial to address the problem of low responsiveness of immunotherapy in HCC.In recent years,with the indepth study of TME in hepatocellular carcinoma,it has been found that a large number of suppressive immune cells exist in TME,which can mediate immunosuppression in HCC through mechanisms such as promoting effector immune cell depletion and enhancing immune tolerance.Among these suppressive immune cells,tumor-associated macrophages were the most abundant,which accounts for more than half of the infiltrating immune cells in TME of HCC.TAM has a key role in the formation of suppressive TME in HCC.As an important immune effector cell,macrophages mainly performing phagocytosis and antigen presentation functions.Macrophages can polarize into different subtypes in different microenvironments,which are mainly divided into two types,M1 subtype and M2 subtype.Most of the infiltrated TAM in TME present M2 type,which can inhibit the antitumor response and promote the formation of immunosuppressive TME in HCC,and is closely related to poor healing of HCC.It was demonstrated that reversal of M2 subtype macrophages to M1 subtype in TME of HCC restored the antitumor effect.This suggests that reversal of M2 subtype macrophages in TME is a highly promising therapeutic strategy in the treatment of HCC.Therefore,there is an urgent need to find the initiating factors that induce TAM subtype transformation in HCC and to elucidate the molecular mechanisms of subtype transformation to provide new targets for HCC immunotherapy.Growth differentiation factor 15(GDF15)is a member of the transforming growth factor(TGF-β)superfamily.GDF15 was originally named Macrophage inhibitory cytokine1(MIC1)because it inhibits the secretion of tumor necrosis factor alpha(TNF-α)by macrophages.Subsequently,it was shown that GDF15 could attenuate the immune surveillance of pancreatic cancer by TAM,suggesting that GDF15 has a regulatory function on macrophages.However,in recent years,studies on GDF15 have mainly focused on appetite control and energy metabolism,while its regulatory role on macrophages and specific mechanisms have not been studied in depth.Our group discovered that GDF15 can be a potential target for immunotherapy of HCC,and identified the receptor CD48 on immune cells for the first time.Moreover,we found that TAM distribution was significantly changed in TME of GDF15 gene knockout liver cancer tissues in mice.Therefore,this study will investigate the role and mechanism of GDF15 on the subtype distribution of tumorassociated macrophages,and further expand the basis that GDF15 can be used as a therapeutic target for immunotherapy in HCC.AimsTo investigate the regulatory role of GDF15 molecules on TAM subtype distribution and explore its molecular mechanism.To clarify the application potential of GDF15 as a tumor immunotherapy target,and to provide new ideas for immunotherapy of liver cancer targeting TAM.Methods1.The correlation between GDF15 expression levels and TAM subtype distribution was confirmed by bioinformatics analysis of published HCC single-cell sequencing data;2.The correlation between GDF15 and different subtypes of TAM in HCC TME was confirmed by bioinformatics analysis of liver cancer samples from TCGA database;3.The effect of GDF15 stimulation on macrophage polarization was investigated by a series of in vitro experiments,and the effect of GDF15 on macrophage phagocytosis and induction of apoptosis in tumor cells by macrophage was investigated by co-culture experiments;4.The detrimental effect of GDF15 on HCC in vivo was investigated by establishing the orthotopic transplantation HCC mouse model by GDF15 gene knockout cells or Gdf15knockout(KO)mice,and the effect of GDF15 deletion on the distribution of infiltrating macrophage subtypes in the tumor immune microenvironment was analyzed by flowmass spectrometry(Cy TOF);5.The receptor of GDF15 on macrophages was identified by immunofluorescence colocalization and IP-western blotting and elucidation of specific molecular mechanisms mediating the biological effects of GDF15;6.The effect of GDF15 on the expression profile of M1 and M2 polarized systems was demonstrated by RNA-seq and the pathway mediating the biological role of GDF15 was explored by enrichment analysis.Results1.GDF15 positively associated with the elevation of TAM distribution of M2 subtype in TME and positively correlated with the expression of marker molecules of M2 macrophages and negatively correlated with the expression of M1 marker molecules;2.GDF15 promotes the polarization of macrophages to the M2 subtype and inhibits their polarization to the M1 subtype,thereby inhibiting the antitumor immune response in the TME and mediating immune escape from HCC;3.GDF15 mediates macrophage transformation to M2 subtype through macrophage surface receptor CD48.Conclusion1.We found that the expression of GDF15 was positively correlated with infiltration of M2 subtype macrophages in HCC TME by analyzing HCC single cell sequencing set and TGCA database HCC samples;2.GDF15 is a key molecule to induce macrophage transition to M2 subtype,which inhibits macrophage polarization to M1 subtype and induces it to M2 subtype.GDF15 can inhibit the ability of macrophages to phagocytose and induce apoptosis of tumor cells,which can exacerbate the immunosuppressive state of the tumor microenvironment and thus mediate immune escape of HCC;3.GDF15 binds CD48 and affects the downstream signaling pathway of macrophages,thereby mediating the transition of macrophages to the M2 subtype.