Mechanistic Study Of ATO-based Whole Cell Vaccine In Stimulating Anti-tumor Immune Responses

Cancer is acknowledged as a widespread and global health problem seriously threatening human health.Numerous patients are suffering from this arduous disease which has become a leading cause of death in recent years.Nowadays,surgery,chemotherapy,radiotherapy,and immunotherapy are still the main therapeutic strategies for cancer treatment.Tumor vaccine as the innovative immunotherapy,is one of the most attractive and potential therapeutic strategies,which can educate the immune system to recognize tumor cells through a series of immune reactions,and ultimately leads to immune elimination of cancer cells while avoids the impairment of normal cells.However,the low immunogenicity of tumor cells and immunosuppression of tumor microenvironment are the two major obstacles that predominantly attenuate the clinical therapeutic effect of tumor vaccine.Arsenic trioxide(ATO)is the main active ingredient of arsenics,which shows a broad-spectrum cytotoxicity to tumor cells.However,the clinical application of this drug is greatly restricted to its low target selectivity and strong cytotoxicity.The longterm successful cancer management predominantly depends on the activation of specific immune response,and tumor cells pretreated with cytotoxic drugs could act as the vaccine initiating anti-cancer immune responses.Thus,a variety of conventional chemotherapy agents were selected and compared and ATO were found to significantly induce the exposure of phosphatidylserine(PS)and calreticulin(CALR),the release of adenosine triphosphate(ATP)and high mobility group box protein(HMGB1),and increase expression of IFN-α/β.Besides,tumor cells treated with ATO showed stronger immunogenicity,promoted the proliferation of CD8+T cells and IFN-y secretion.The results of in vivo experiments indicated that the prophylactic vaccine prepared by ATO could significantly protect vaccinated mice from subsequent rechallenge in an immune-dependent manner.And we also found that ATO induced the production of a large amount of reactive oxygen species(ROS)in tumor cells,resulting in the increase of LC3-Ⅱ,cleaved-CASP3,phosphorylation of RIP3 and MLKL,and the degradation of GPX4.All these biological processes can be inhibited by N-AcetylL-cysteine(NAC)administration.Data mentioned above suggested that ATO-mediated ROS generation can activate various cell death modalities comprising autophagy,apoptosis,necroptosis and ferroptosis.Different knockout cell lines including Becn1-/-,Gpx4low,Acsl4-/-,Gsdme-/-,Gsdmd/-,Bax-/-,Bak-/-,Rip3-/-,Mlkl-/-were constructed to further investigate the function of different cell death modalities involved in ATO-mediated anti-tumor immunity.We analyzed the impacts of different cell death modalities on tumor immunogenicity and the involved regulatory mechanisms.The results indicated that ACSL4,RIP3,MLKL deficient tumor cells showed abolished the vaccine protection effect compared with WT cells.Furthermore,the release of various danger signal molecules was significantly reduced in ATO-treated Acsl4m-/-,Rip3-/-,Mlkl-/-tumor cells.It was because the membrane permeability was not significantly increased in these cells after ATO treatment compared with WT cells.In animal models,we found that intratumoral injection of ATO did not delay tumor growth,whereas subcutaneous injection of ATO-pretreated tumor cells significantly inhibited tumor growth.The great therapeutic efficacy also relied on host immunity and the intact expression of ACSL4,RIP3 and MLKL in tumor cells.Increased CD8+T,CD4+T,NK,DCs and macrophages were found in therapeutic vaccine setting of WT cells through analyzing the tumor microenvironment by flow Cytometry.In order to reverse the ineffectiveness of ATO-based Rip3-/-tumor cell vaccinelwe developed a personalized vaccine regimen combining several specific molecules.Importantly,the combination of PD-1 blockade therapy and ATO-mediated cell vaccine exhibited enhanced tumor suppressive effect and some mice achieved tumor-free survival(about 33%).In summary,we systematically studied the contributions of different cell death modalities on the immunogenicity of ATO-treated tumor cells and described the protective effects of ATO-mediated whole tumor vaccine by in vivo experiments.Moreover,in the early stage of cancer,combination treatment of PD-1 blockade with ATO-induced tumor vaccine significantly promotes immune cell infiltration and suppress tumor progress.This study provides new insights into therapeutic strategies and combination regimens for ATO clinical application.