Oleanolic acid inhibits M2 macrophage polarization and potentiates anti-PD-1 therapy in hepatocellular carcinoma by targeting miR-130b-3p-PTEN-PI3K-Akt signaling and glycolysis

Background: Hypoxia promotes M2 polarization of macrophages and the formation of the immunosuppressive tumor microenvironment (TME) in hepatocellular carcinoma (HCC). Oleanolic acid (OA) has shown great potential in the treatment of HCC. However, the mechanisms of macrophage M2 polarization in hypoxic tumor TME and the regulating effect of OA is still unclear. Objective: To investigate the mechanisms of macrophage M2 polarization induced by hypoxic HCC cells-derived exosomes and examine the efficacy of OA in remedying the immunosuppressive TME and the anti-PD1 therapy potential. Methods: Hypoxic and normoxic HCC-derived exosomes (H-Exo and N-Exo) were collected by centrifugation. The microRNAs (miRNA) carried by the exosomes were sequenced and then screened to identify the functional miRNA. THP-1-induced macrophages were treated with exosomes or miRNAs to induce the M2 polarization of macrophages. Real-time RT-PCR and Western blotting were used to identify the direct target of miR-130b-3p and its downstream molecules. Hepa1-6 hepatoma-bearing mice were subjected to determine the efficacy of OA in regulating the TME and the anti-PD1 therapy potential. Results: H-Exo promotes macrophage M2 polarization, and thereby accelerates the migration and epithelialmesenchymal transition (EMT) of HCC cells. Exosomal miRNA sequencing and subsequent functional validation showed that miR-130b-3p was the mediator of H-Exo-induced macrophage M2 polarization. PTEN was identified as the target of miR-130b-3p, and downregulation of PTEN by miR-130b-3p led to the activation of PI3K/Akt signaling and macrophage M2 polarization. In addition, miR-130b-3p also enhanced the glycolysis. OA suppressed H-Exo and miR-130b-3p-induced macrophage M2 polarization, also inhibited miR-130b-3p-induced glycolysis. In vivo, OA treatment enhanced the efficacy of anti-PD1 antibody by decreasing the number of M2 macrophages and increasing the number of CD8+ T cells. Conclusion: Our findings uncover a new mechanism of hypoxic HCC cells-induced M2 polarization of macrophages through exosomal miR-130b-3p-PTEN-PI3K-Akt signaling. The combination therapy of OA with anti-PD1 antibody may lead to substantial improvements of the immunotherapy efficacy and expand the beneficiaries.