| ObjectiveHepatocellular carcinoma(HCC)is the most common type of liver cancer.Its unique and complex immune microenvironment plays a crucial role in the development of HCC and has been a hot topic in the field of HCC research.Previous studies have identified the role of various immune cells in tumor progression and developed therapeutic approaches using immune cells,but the clinical efficacy of these therapies is still limited or only has therapeutic effect on certain types of tumors.Under the influence of various factors in the liver cancer microenvironment,macrophages play different functions in the development of HCC by altering their polarization and influencing the activation or inhibition of other immune cells.Studies have shown that the suppressive immune microenvironment in HCC is not conducive to the anti-tumor function of CD8+T cells.Therefore,it is important to explore the mechanisms of CD8+T cell inactivation for the treatment of tumors.Insulin-like growth factor 2 mRNA binding protein 3(IGF2BP3)is an RNA-binding protein that has been shown to be lowly expressed in normal adult tissues.IGF2BP3 promotes tumor progression by promoting the malignant phenotype of tumor cells.It is up-regulated during the occurrence and development of tumors.Current studies in HCC have focused on the effects of IGF2BP3 on the tumor cells themselves and its relevance to malignant disease progression.However,the role of IGF2BP3 in the immune microenvironment of HCC has rarely been reported.In this study,we investigated the expression of IGF2BP3 and its effects on macrophages and CD8+T cells in the tumor microenvironment,and explored the cellular and molecular mechanisms involved.Our study also explored the feasibility of knocking out Igf2bp3 to activate anti-tumor immune cells combined with other immunotherapies at animal level to enhance the therapeutic effect,bringing new ideas and theoretical support for the treatment of HCC in clinical settings.MethodsFirstly,using public databases,a bioinformatics approach was used to analyze the expression of IGF2BP3 in different stages of liver cancer and the relationship with patient prognosis and immune cell infiltration in HCC.Single cell sequencing data were used to analyze the main cell types expressing IGF2BP3 in HCC.Hepa1-6 mouse HCC cell lines stably overexpressing and knocking out Igf2bp3 were constructed using lentivirus and Crispr-Cas9 techniques respectively and validated using qPCR,Western blot,Flow cytometry.The effects of IGF2BP3 on tumor cell proliferation,migration and apoptosis were examined by CCK-8,wound healing assay,Transwell and Flow cytometry,respectively.Secondly,a mouse subcutaneous transplantation tumor model was constructed by inoculating a mouse HCC cell line,Hepa1-6.The proportion and phenotype of various immune cells in tumors were analyzed by flow cytometry,and the subcutaneous xenograft tumor model after removing macrophages was further compared to verify the important role of macrophages in the promotion of tumors.Thirdly,to investigate the effect of IGF2BP3 on cytokine secretion by tumor cells,the culture supernatants of tumor cell were collected and the effect of IGF2BP3 expression on cytokine secretion by tumor cells was detected using cytokine arrays,ELISA,Western blot,immunohistochemical staining.Furthermore,the cytokines produced by tumor cells that mediate the regulation of macrophages and CD8+T cells by IGF2BP3 were verified using Transwell and Flow cytometry using neutralizing antibodies to CCL5 and TGF-β1,respectively.Fourthly,to investigate the regulatory mechanism of IGF2BP3 on CCL5 and TGF-β1,IGF2BP3-bound RNA fragments were enriched using RIP assays and the binding of IGF2BP3 to the target fragments was analyzed using PCR and qPCR methods.The binding was further verified using the TriFc system.Finally,the effect of IGF2BP3 on the half-life of CCL5 and TGF-β1 mRNA was examined using actinomycin D to inhibit RNA synthesis.Fifth,to investigate the feasibility of knocking out Igf2bp3 expression,activating immune cells and combining with other immunotherapies to enhance anti-tumor efficacy,the effect of knocking out Igf2bp3 combined with anti-CD47 on tumor development was compared with knocking out Igf2bp3 alone or using anti-CD47 alone in a mouse subcutaneous transplantation tumor model.Results1.Bioinformatics analysis revealed that high expression of IGF2BP3 in HCC was significantly correlated with high infiltration of macrophages.And high IGF2BP3 expression and high macrophage infiltration were negatively correlated with the survival time of patients.2.IGF2BP3 in tumor cells can promote macrophage migration and polarization towards M2.Using a mouse HCC subcutaneous transplantation tumor model,it was found that knockout of tumor cells Igf2bp3 resulted in a significant reduction in macrophage infiltration and an increase in the proportion of CD8+T cell infiltration in tumor tissue.3.Cellular mechanism studies revealed that IGF2BP3 in HCC cells upregulated the expression and secretion of CCL5 and TGF-β1.On the one hand,the secretion of CCL5 could recruit macrophages to infiltrate into the tumor microenvironment;on the other hand,IGF2BP3 induced macrophage M2-type polarization and inhibited CD8+T cell activation by promoting the expression of TGF-β1.4.Molecular mechanism studies revealed that IGF2BP3 could play a regulatory role by directly binding to the mRNA of CCL5 and TGF-β1.5.In a mouse subcutaneous transplantation tumor model of HCC,a therapeutic strategy of knockout Igf2bp3 in Hepa1-6 cells in combination with CD47 antibody significantly inhibited tumor growth and was more effective than either of the therapeutic strategies alone.ConclusionWe found that IGF2BP3 was mainly expressed in HCC cells.IGF2BP3 could promote macrophage infiltration in the tumor microenvironment by upregulating the expression and secretion of CCL5 and induce M2 polarization of macrophages by upregulating the expression and secretion of TGF-β1,while inhibiting the activation of CD8+T cells.Knocking out of Igf2bp3 in a mouse model of HCC activates anti-tumor immune cells in combination with CD47 antibodies has good therapeutic effects. |
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