Valproic Acid Combined With Zoledronate Enhance γδ T Cell-mediated Cytotoxicity Against Osteosarcoma

Osteosarcoma is the most common primary bone malignant disease.It occurs predominantly in children and adolescents with a tendency of local invasion and early lung metastasis.The five-year survival of patients with osteosarcoma has improved to 60%-80% due to the therapeutic strategy of neoadjuvant chemotherapy combined with surgery,but has remained unchanged during the past 40 years.Especially for those with relapse or lung metastasis,current treatments are effecitveless and the prognosis is unsatifactory.Besides,the main agents in clinical first-line chemotherapy,including doxorubicin,methotrexate,cisplatin and ifosfamide,are generally limited due to drug resisitance,mild efficacy as well as their terrible toxicities and side effects.Therefore,novel therapy with effective efficacy and tolerated toxicity is especially urgent.Immunotherapy is considered as “the fourth therapy” in addition to surgery,chemotherapy and radiotherapy.Adoptive cell therapy(ACT)is one of the most promising immunotherapies,and has made significant achievements in the study of leukemia.Various immune cells including NK cells,macrophages and T cells have been studied for the application in ACT.T cells have been proved to play an important role in anti-tumor immune responses,making them to be the ideal candidates of ACT.However,lack of highly specific and strong expressed targets has greatly limited the application of ACT in most solid tumors,including osteosarcoma.γδ T cells,accounting for 1 to 10% of peripheral blood T cells,represent a potential candidate to kill tumor cells because of their direct recognition of tumor without the restriction of major histocompatibility complex(MHC)molecules.Activated γδ T cells will be able to directly kill target cells by engaging death receptors on the surface of tumor cells and producing cytotoxic granules and cytokines.Our previous study confirmed that ZOL could sensitize osteosarcoma and chondrosarcoma cells to the cytotoxicity of γδ T cells.Therefore,adoptive transfer of γδ T cells is expected to be a promising therapy against malignant solid tumors including osteosarcoma.However,results from in vitro studies have shown that high concentration of zoldronate was required to induce a strong immune reponse in γδ T cells,which exceeded the concentration that can be maintained for long time in in vivo experiments and clinical trials.Therefore,we need to find some effective adjuvants to enhance the effect of zoldronate,thereby reducing its required concentration to induce γδ T cells to exert significant immune efficacy.Valproic acid,a well-known FDA approved histone deacetylase inhibitor(HDAC-I),is commonly used as an antie-pileptic agent.Valproic acid was shown to inhibit tumor proliferation and exert immunostimulatory activities in vitro and in vivo.Moreover,valproic acid showed promising capability in augmenting the anticancer efficacies of other therapeutic regimens,including ionizing radiation,chemotherapy,and immunotherapy with little toxicity to nomal tissue.γδ T cells immunotherapy has been demonstrated to have more significant efficacy when in combination with chemotherapy or other strategies including HDAC-I(37).Recent study has revealed that valproic acid was related to the functional plasticity of γδ T cells.Taken together,we conjectured that valproic acid had the potential as an adjuvant to facilitate the antitumor activity of γδ T cells when combined with zoledronate.In this study,we found that valproic acid and zoledronate could synergistically enhance the antitumor effect of γδ T cells.Moreover,compared with single-agent stimulation,valproic acid combined with zoledronate induced γδ T cells to express higher levels of cytotoxicity-related markers,including CD107 a,Perforin,and IFN-γ.We further found that valproic acid and zoledronate could synergistically block the mevalonate pathway and cause the accumulation of intermediates,thereby activating γδ T cells.We also conducted adoptive transfer of γδ T cell experiments by establishing an immunodeficient mice model of osteosarcoma.Our results showed that valproic acid combined with zoledronate could significantly enhance the antitumor effect of γδ T cells,effectively inhibit the growth of osteosarcoma and promote the infiltration of T cells when compared with those treated with zoledronate only.Strongest blockade of mevalonate pathway was confirmed in combination group by IHC staining.We also obtained seven types of primary cells from osteosarcoma patients,including the cases with relapse,metastasis,and chemotherapy resistance.All the results showed that valproic acid combined with zoledronate could synergistically enhance the cytotoxicity of γδ T cells against osteosarcoma,making a contribution to the application of this therapy.Thus,our study confirmed the synergism of valproic acid and zoledronate in inducing γδ T cells cytotoxicity via blocking mevalonate pathway.