The Mechanisms Of Sulforaphane On Apoptosis And Function Of Myeloid-derived Suppressor Cells

Background:Malignant tumor is a serious disease which can seriously threaten human health.,the morbidity and mortality of malignant tumors are also increasing globally.In recent years,traditional tumor treatment modalities have not made breakthroughs.In recent years,immunotherapy,including immune checkpoint blockade(ICBs)and chimeric antigen receptor T-cell therapy(CAR-T),has made breakthroughs in improving the prognosis and prolonging the survival of patients with hematologic tumors.However,it has little effect in the clinical treatment in solid tumors due to the presence of tumor microenvironment in solid tumors,limits the benefit of immunotherapy in patients with solid tumors,such as lung cancer,liver cancer and so on.Myeloid-derived suppressor cells(MDSCs)are one of the main components that constitute the tumor microenvironment and lead to the poor effect of solid tumor immunotherapy.Targeting MDSCs is an important breakthrough to improve the tumor microenvironment and improve the efficacy of immunotherapy in patients with solid tumors.Sulforaphane is a common phytochemical which was usually found in cruciferous vegetables such as broccoli and cabbage.It has been shown to exert anti-tumor effects in a variety of tumors,previous studies have shown that SFN promotes the differentiation of MDSCs into dendritic cells by inhibiting the expression of macrophage migration inhibitory factors,reducing the proportion of MDSCs and immunosuppressive function,but the related mechanism of SFN on MDSCs is still unclear.Therefor,cell experiments,animal experiments and clinical trials are used to explore the effect of SFN on MDSCs and the molecular mechanism of SFN regulating the proportion and function of MDSCs at multiple levels in this study,providing ideas and evidence for the clinical application of SFN in cancer patients.Objective1.The subcutaneous tumor-bearing mouse models of LLC and B16 were established and treated with SFN or DMSO,to investigate the effect of sulforaphane on tumor microenvironment in tumor-bearing mice.2.To clarify the way that SFN reduces MDSCs,combined with RNA-seq sequencing results,to explore IL12RB2-mediated SFN-induced apoptosis of MDSCs and mechanisms;and to explore the effect of SFN on the immunosuppressive function and glucose metabolism of MDSCs;3.Case-control study is aimed to investigate the distribution of MDSCs and T cells in tumor patients,and clinical trial explore the effect of SFN intervention on the MDSCs and T cell function in non-tumor patients.Methods1.The influence of Sulforaphane on tumor microenvironment in tumor-bearing mice(1)The subcutaneous tumor-bearing mouse models of LLC and B16 were established and treated with SFN(50mg/kg/d,injected intraperitoneally for 12 days)or DMSO after 9 days after tumor bearing;Flow cytometry was used to detect immune subpopulations in the spleen and tumor tissues in tumor-bearing mice.2.Study on the mechanism of SFN promoting MDSC apoptosis(1)MDSCs in tumor tissues were sorted by magnetic beads and then performed RNA seq sequencing to find the key molecules and related mechanisms of sulforaphane on MDSCs.(2)GM-CSF combined with G-CSF recombinant protein were adopted to induce mouse bone marrow cells to MDSCs for 72h,Flow cytometry and Western blot were used to detecte the proportion and level of apoptosis of MDSCs,PCR and other experimental techniques were uesd to evaluate expression of FasL,MMP3,IL12RB2.(3)IL12RB2 neutralizing antibody were used to verify the role of IL12RB2 and in vitro and in vivo in sulforaphane-induced MDSCs apoptosis.3.Effects of sulforaphane on the immunosuppressive function of MDSCs(1)PCR,Western blot and Flow cytometry were used to measure the expression and activity of MDSCs immunosuppression-related molecules such as PD-L1,Arg-1,iNOS2.Flow cytometry was used to detect the the effect of MDSCs on T cell proliferation and killing ability in Control and SFN groups(2)Seahorse energy assay was used to measure the effect of SFN on MDSCs glycolysis and oxidative phosphorylation.(5)GEPIA was used to analyze the correlation between MDSCs immunosuppression and metabolism in lung cancer patients.4.Clinical trial study on the effect of sulforaphane on MDSCs in non-tumor patientsFlow cytometry was used to detect the proportion of MDSCs and T cells in the peripheral blood of healthy and tumor patients in case-control study.Clinical trials was used to evaluate the effect of SFN on MDSCs in non-tumor patients,included subjects were supplemented with SFN(200 mg of SFN per day for 30 days),the changes of MDSCs as well as T cells in peripheral blood were detected by flow cytometry.Results1.The influence of Sulforaphane on tumor microenvironment in tumor-bearing mice(1)SFN delayed the tumor growth in LLC and B16 mouse models,the inhibition of SFN on LLC(NOD/SCID)was weaker than LLC(C57BL/6J).(2)SFN reduced the proportion of MDSC in spleen and tumor tissues and promoted CD8+T cells infiltration in tumor-bearing mouse models,P<0.05.(3)The proportion of MDSCs in tumor tissues was positively correlated with tumor weight(r=0.852,P<0.001),and negatively correlated with the proportion of infiltrated CD8+T cells(r=-0.560,P=0.024).2.Study on the mechanism of SFN promoting MDSC apoptosis(1)The proportion of MDSCs in the bone marrow of tumor-bearing mice had no difference in two groups(P>0.05),but SFN significantly reduced the proportion of MDSCs in blood and spleen.What’s more,proportion of MDSCs apoptosis and expression of cleaved-caspase3 on MDSCs significnatly increased.Similar results were obtained in in vitro level,P<0.05.(2)The results of RNA-seq indicated IL12RB2 was a key molecule for SFN-induced apoptosis of MDSCs,SFN up-regulated the expression of IL12RB2 on MDSCs,and regulated the MMP3(down-regulated)and FasL(up-regulated),which were downstream molecules of IL12RB2,and induce MDSC apoptosis by activating Cleaved-caspase3.(3)SFN intervention increased the level of IL-2,IFN-y and Ki-67 on CD8+T cells,enhance the killing function and proliferation ability of CD8+T cells.Neutralizion IL12RB2 could partially reverse SFN-induced apoptosis of MDSCs and promote MDSCs accumulation in tumor-bearing mice,reversed the effect of SFN on CD8+T in tumor-bearing mice,P<0.05.3.Effects of sulforaphane on the immunosuppressive function of MDSCs(1)SFN reduced the activity and expression of immunosuppressive molecules such as Arg-1 and iNOS2 on MDSCs and impaired the suppression on T cell proliferative and killing function by MDSCs.(2)SFN reduced the levels of oxidative phosphorylation and glycolysis on MDSCs.(3)The immunosuppressive function of MDSCs was positively correlated with the level of oxidative phosphorylation and glycosis.4.Clinical trial study on the effect of sulforaphane on MDSCs in non-tumor patients(1)The proportion of T cells in peripheral blood was decreased and the proportion of MDSCs was increased in tumor patients compared with healthy donors.The proportion of MDSCs was was negatively correlated with the proportion of CD8+T cells in peripheral blood,r=0.38,P<0.05.(2)The expression levels of FasL and IL12RB2 on MDSCs were lower in tumor patients compared with healthy donors,the expression of IL12RB2 and FasL on MDSCs was negatively associated with proportion of MDSCs.(3)SFN intervention decreased the proportion of MDSCs and increased the expression of IL12RB2 and FasL on MDSCs in non-tumor patients..(4)SFN intervention incresased the GITR+in CD8+T cells,promoted the activation of CD8+T cells without affect the proportion of CD8+T in non-tumor patients.Conclusions1.SFN could retard tumor progression by decreasing the proportion of MDSCs in tumor microenvironment and promoting infiltration of CD8+T cells.2.SFN could reduce the accumulation of MDSCs by inducing the apoptosis of MDSCs without affect the generation and migration.IL12RB2 is key molecular in SFN induced apoptosis.Neutralizion IL12RB2 could partially reverse SFN-induced apoptosis of MDSCs.3.SFN reduced the immunosuppressive function of MDSCs by remodeling the glucose metabolism of MDSCs.4.Intervention with SFN can reduce the proportion of MDSCs and promoted the activated of CD8+T cells in peripheral blood in non-tumor patients.