| ObjectiveMalignant tumors pose a serious threat to human health and are a crucial public health problem.Currently,the prognosis of most common malignant tumors is still poor.Identifying safe and effective anti-tumor medicine from natural compounds,such as dietary substances,is a growing hot topic in the field of cancer therapy.Sulforaphane(SFN)is an isothiocyanate,a phytochemical compound,with outstanding anti-tumor properties,mainly found in broccoli,cabbage and other cruciferous vegetables.Studies have shown that SFN is capable of inhibiting multiple phases of carcinogenesis through various molecular mechanisms.Epidemiological studies have also showed that the regular intake of cruciferous vegetables such as broccoli is beneficial in preventing cancer.The immune system plays a critical role in defending against cancers.However,it is unclear whether SFN inhibits tumor growth through regulating the immune system.Novel cutting-edge techniques such as chimeric antigen receptor(CAR)T cell therapy have made a break-through in clinical application,identifying CD8+T cells as the key effectors in the anti-tumor response.In this study,we investigated the in vitro effects of SFN on CD8+T cells,including phenotypes,functions and differentiation status.Furthermore,we investigated the influence of SFN on the anti-tumor activity of HER2-CAR T in vitro and in vivo using a murine model.In this study we explore a novel anti-tumor mechanism of SFN that may provide a new option for cancer immunotherapy.MethodsBlood samples were collected from healthy donors.Peripheral blood mononuclear cells were isolated and CD8~+T cells were purified by using immune magnetic beads.Anti-CD3/CD28 beads were used to activate CD8~+T cells in vitro,and different concentrations of SFN were added to the culture system for 3 days.The expression of surface costimulatory molecules were analyzed by flow cytometry and the secretion levels of cytokines were detected by intracellular staining.Annexin-V/PI staining was applied to detect the apoptosis of CD8~+T cells,and the expression of anti-apoptosis genes was measured by qRT-PCR.The differentiation status of CD8~+T cells was analyzed by flow cytometry.Western blot was used to detect the phosphorylation of the AMPK signaling pathway,and specific siRNA was used to knockdown mTOR expression.The HER2 expression on human lung cancer A549 cells and the phenotypes of HER2-CAR T cells were analyzed by flow cytometer.The effects of SFN on CD8~+T cell phenotypes and the cytotoxicity of HER2-CAR T cells against A549 cells were tested in vitro and in immunodeficient mice.ResultsSFN dramatically decreased the expression of surface inhibitory molecules such as PD-1(20.8±2.92%vs 11.8±2.32%,P=0.0045)and Tim-3(24.0±4.28%vs 14.1±1.38%,P=0.02),however it had no significant effect on surface activation molecules.Furthermore,SFN increased the expression levels of cytokines such as IFN-?(14.20±3.06%vs 30.2±8.88%,P<0.05)?IL-2(10.7±0.99%vs 26.0±6.91%,P<0.05)and TNF-?(14.1±4.18%vs 30.6±4.95%,P<0.03).In addition,SFN strongly inhibited activation-induced apoptosis of CD8~+T cells(11.3±2.64%vs5.7±0.78%,P<0.05),with high levels of anti-apoptosis genes Bcl-2 and Bcl-6.SFN promoted CD8~+T cell differentiation into memory precursor effector cells,with upregulated CD127(22.8±4.5%vs 55.5±8.0%,P=0.0064)and downregulated KLRG1(14.2±1.7%vs 3.0±0.6%,P=0.0178).Mechanistically,SFN inhibited the phosphorylation of MAPK signaling downstream molecules,mTOR and S6.SFN could upregulate the expression of CD107a on HER2-CART cells,showing better cytotoxicity against A549 cells in vitro.Finally,SFN-treated HER2-CAR T cells exhibited superior anti-tumor ability in mice,with decreased expression of inhibitory molecules PD-1 and Tim-3,as well as an increase in memory-related molecules and upregulated CD127 and downregulated KLRG1.Conclusion1.SFN can enhance the functions of CD8~+T cells.2.SFN can improve the anti-tumor ability of HER2-CAR T cells.3.SFN may provide a novel strategy to improve adoptive cell therapy. |
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