Chimeric antigen receptor (CAR)-T cells directed to solid tumors have been less effective, due in part to the low or lost expression of specific tumor antigens. Herein, we developed a different strategy to enhance CAR-T cell persistence and efficacy by producing a multispecific CAR-T or vaccine based on immunogenic cell death (ICD). We demonstrated that ionizing radiation activates STAT1-IRF1-CD39 axis to upregulate CD39 expression to form an immunosuppressive tumor microenvironment (TME) to enhance radioresistance. CD39 blockade accumulates extracellular ATP, which activates NLRP3 inflammasome in dendritic cells via P2X7 receptor, thereby promoting radiation-induced ICD. Multispecific CAR-T cells in vitro prepared by elevated ICD suppress the growth of xenografts in nude mice. Radiation and CD39 inhibition-induced ICD of glioma stem cells as a vaccine enhance CAR-T expansion in peripheral blood, multifunctionality in the TME, and antitumor effect in a glioma model. The multispecificity of CAR-T cells, targeting CAR and tumor antigens, vastly enhances the function of conventional CAR-T cells, stimulates a native immune response, and overcomes obstacles of specific antigen loss or low expression of target cells in antitumor therapy.

Ectonucleotidase CD39 hydrolyzing extracellular ATP (eATP) functions as a key modulator of immune response in the tumor microenvironment, yet the role of CD39 in contributing tumor stem cells in a more immunosuppressive microenvironment remains elusive. Here we report that the upregulation of CD39 is crucial for the decrease of extracellular ATP concentration around glioma stem cells (GSCs) to maintain an immunosuppressive microenvironment. Adriamycin (ADM) is able to promote the release of ATP, which recruits dendritic cells (DCs) to phagocytose GSCs. CD39 inhibition further increased extracellular ATP concentrations following ADM treatment and DCs phagocytosis. In addition, GSCs upregulated CD39 expression by SOX2-binding CD39 promotor. In mouse tumor models, the combination of ADM and CD39 blockade increased immune cell infiltration and reduced tumor size. These findings suggest that GSCs upregulate CD39 expression by their biological characteristics to maintain an immunosuppressive microenvironment, and CD39 inhibition supplies a favorable tumor microenvironment (TME) for immunotherapeutic intervention and enhances the immune response induced by chemotherapy.

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Aim Immunotherapy for glioblastoma multiforme (GBM) is limited because of a strongly immunosuppressive tumor microenvironment (TME). Remodeling the immune TME is an effective strategy to eliminate GBM immunotherapy resistance. Glioma stem cells (GSCs) are inherently resistant to chemotherapy and radiotherapy and involved in immune evasion mechanism. This study aimed to investigate the effects of histone methyltransferases 2 (EHMT2 or G9a) on immunosuppressive TME and whether this effect was related to changes on cell stemness. Methods Tumor‐infiltrating immune cells were analyzed by flow cytometry and immunohistochemistry in orthotopic implanted glioma mice model. The gene expressions were measured by RT‐qPCR, western blot, immunofluorescence, and flow cytometry. Cell viability was detected by CCK‐8, and cell apoptosis and cytotoxicity were detected by flow cytometry. The interaction of G9a and F‐box and WD repeat domain containing 7 (Fbxw7) promotor was verified by dual‐luciferase reporter assay and chromatin immunoprecipitation. Results Downregulation of G9a retarded tumor growth and extended survival in an immunocompetent glioma mouse model, promoted the filtration of IFN‐γ + CD4+ and CD8+ T lymphocytes, and suppressed the filtration of PD‐1+ CD4+ and CD8+ T lymphocytes, myeloid‐derived suppressor cells (MDSCs) and M2‐like macrophages in TME. G9a inhibition decreased PD‐L1 and increased MHC‐I expressions by inactivating Notch pathway companying stemness decrease in GSCs. Mechanistically, G9a bound to Fbxw7, a Notch suppressor, to inhibit gene transcription through H3K9me2 of Fbxw7 promotor. Conclusion G9a promotes stemness characteristics through binding Fbxw7 promotor to inhibit Fbxw7 transcription in GSCs, forming an immunosuppressive TME, which provides novel treatment strategies for targeting GSCs in antitumor immunotherapy.

Anillin (ANLN) is a mitosis-related protein that promotes contractile ring formation and cytokinesis, but its cell cycle-dependent degradation mechanisms in cancer cells remain unclear. Here, we show that high expression of ANLN promotes cytokinesis and proliferation in esophageal squamous cell carcinoma (ESCC) cells and is associated with poor prognosis in ESCC patients. Furthermore, the findings of the study showed that the deubiquitinating enzyme USP10 interacts with ANLN and positively regulates ANLN protein levels. USP10 removes the K11- and K63-linked ubiquitin chains of ANLN through its deubiquitinase activity and prevents ANLN ubiquitin-mediated degradation. Importantly, USP10 promotes contractile ring assembly at the cytokinetic furrow as well as cytokinesis by stabilizing ANLN. Interestingly, USP10 and the E3 ubiquitin ligase APC/C co-activator Cdh1 formed a functional complex with ANLN in a non-competitive manner to balance ANLN protein levels. In addition, the macrolide compound FW-04-806 (F806), a natural compound with potential for treating ESCC, inhibited the mitosis of ESCC cells by targeting USP10 and promoting ANLN degradation. F806 selectively targeted USP10 and inhibited its catalytic activity but did not affect the binding of Cdh1 to ANLN and alters the balance of the USP10-Cdh1-ANLN complex. Additionally, USP10 expression was positively correlated with ANLN level and poor prognosis of ESCC patients. Overall, targeting the USP10-ANLN axis can effectively inhibit ESCC cell-cycle progression.