Macrophage GSK3 beta-deficiency inhibits the progression of hepatocellular carcinoma and enhances the sensitivity of anti-PD1 immunotherapy

BackgroundGlycogen synthase kinase 3 beta (GSK3 beta) was originally discovered to regulate glycogen synthesis and show a relationship to tumors. However, the biological functions of GSK3 beta in tumor-associated macrophages (TAMs) in cancers including hepatocellular carcinoma (HCC) remain unclear.MethodsThe enrichment of GSK3 beta in tumor tissues was assessed by Gene Expression Omnibus (GEO) database. The in vitro and in vivo assays assisted in evaluating how GSK3 beta in TAMs affected HCC in terms of proliferation, invasion and migration. Immunofluorescence was used to assess GSK3 beta expression in TAMs in the anti-PD1 therapy non-responsive HCC group and the responsive group. Western blot and coimmunoprecipitation were performed to demonstrate the interaction between GSK3 beta and PD-L1. We carried out in vivo experiments in a C57BL/6 mouse model of HCC established through subcutaneous injection.ResultsGEO single-cell RNA sequencing data suggested that GSK3 beta was highly enriched in TAMs of HCC. According to in vitro and in vivo experiments, reducing GSK3 beta in TAMs inhibits the cancer cell proliferation, invasion, and migration. The immunofluorescence and immunohistochemistry results confirmed that the GSK3 beta is significantly upregulated in TAMs of the anti-PD1 therapy non-responsive group in comparison with the responsive group. In vitro and in vivo experiments confirmed that reduced GSK3 beta in TAMs are capable of enhancing the sensitivity of anti-PD1 immunotherapy for HCC by decreasing PD-L1 ubiquitination. Mass spectrometry results suggested that high expression of CD14(+)GSK3 beta(+) in the peripheral blood mononuclear cell (PBMC) can predict non-responsive to anti-PD1 treatment. Moreover, escitalopram is confirmed to act as GSK3 beta inhibitor that can increase the sensitivity of anti-PD1 immunotherapy for HCC.ConclusionsThis study revealed that macrophage GSK3 beta deficiency can inhibit the development of HCC by inhibiting the M2 phenotype and enhance the sensitivity of anti-PD1 immunotherapy for HCC by decreasing PD-L1 ubiquitination. The expression of CD14(+)GSK3 beta(+) in PBMC can noninvasively predict anti-PD1 sensitivity in HCC patients, which provides novel strategies to predict anti-PD1 sensitivity, increase anti-PD1 therapeutic effect, and bring new hope for HCC patients.