Tumor Cell-Derived Cancer Vaccines For Combination Cancer Immunotherapy

Objective:With the booming development of tumor immunology,cancer vaccine and its combination therapy have achieved potent activation of the immune response,and the functional remodeling of the inhibitory immune microenvironment,thus effectively controlling tumor progression,recurrence,and metastasis.Recent studies have shown that tumor cell-derived vesicles or whole tumor cells could effectively retain intact epitopes of antigen and trigger a wide range of innate and adaptive immune responses,thus showing good prospects of clinical transformation.However,tumor cell-derived vesicles are faced with difficult problems of efficient loading of functional drugs and antigen retention under complex operations.Moreover,whole tumor cell vaccines have poor clinical benefits due to their low immunogenicity.Given the above dilemma in the development of cancer vaccines,this thesis proposed two strategies of cancer vaccines based on tumor cell-derived vesicles and whole tumor cells,respectively.We aim to explore new paradigms of combination immunotherapy,and further elucidate the in vivo mechanism of immune activation,thus providing a beneficial reference for the development of cancer vaccines.Methods:Through the specific pretreatment of tumor cells,two types of tumor cell-derived cancer vaccines have been developed.Firstly,tumor cell-derived gold nanovesicle(Au@NV)was developed based on the biosynthesis of chloroauric acid in tumor cells.Electron microscopy and flow cytometry were used to investigate the morphology and biological information retention of Au@NV.The biosynthesis mechanism of Au@NV was elucidated through the intervention by specific chemical reagents and physical stimulation.In addition,the frozen dying tumor cell vaccine(FDT)was constructed by integrating immunogenic cell death and liquid nitrogen cryogenic inactivation technology.Western blot,RNA sequencing,and in vitro immunology analysis were conducted to evaluate the immunogenicity and then the dominant component of the vaccine was screened.The in vivo safety was evaluated by high-dose vaccination and blood compatibility analysis.In vivo imaging system was used to investigate the distribution of the above cancer vaccines and explore their tumor targeting and lymphatic drainage ability.A variety of orthotopic and metastatic tumor models were constructed to investigate the anti-tumor therapeutic effect.Furthermore,the in vivo immune mechanism was explored by flow cytometry,immunohistochemistry,and immunofluorescence staining.Results:In the first chapter,the tumor cell-derived vesicle vaccine efficiently loaded with functional gold nanoparticles was developed,aiming at the dilemma of drug loading in vesicle vaccines.Results showed that a variety of redox-active substances in tumor cells could participate in the regulation of chloroauric acid biosynthesis.The biogenetic gold nanovesicles could retain the key biomolecules of tumor cells and exhibited good homologous targeting properties.In vitro radiobiology studies showed that Au@NV could effectively enhance the ROS generation and DNA damage induced by radiation,thus aggravating the apoptosis of tumor cells.In MC38 colorectal cancer model,Au@NV combined with radiotherapy exhibited a tumor inhibitory rate of 87.7%.Au@NV radiosensitization could promote DC maturation and T cell activation,and induce the abscopal effect,resulting in effective inhibition on the lung metastatic tumors in non-irradiated areas with an inhibitory rate of 53.5%.In addition,in the 4T1 triple-negative breast cancer model,this strategy still demonstrated versatility,which could effectively inhibit tumor progression and metastasis.Further combination of Au@NV radio-sensitization and immune checkpoint inhibitors could significantly prolong the overall survival time of tumor-bearing mice.In the second chapter,an efficient whole tumor cell vaccine was developed to solve the problem of low immunogenicity and avoid the in vivo safety risk.Tumor cells underwent immunogenic cell death exhibited enhanced immunogenicity.Cryogenic freezing could eliminate the proliferative activity of tumor cells.High-dose inoculation had not shown tumorigenic risk or treatment-related adverse reactions.After 6 months of cryogenic storage in liquid nitrogen,FDT still had good immune-activation capacity and exhibited effective tumor suppression on B16-OVA melanoma with an inhibitory rate of 71.2%.Compared with other derivatives,FDT had satisfactory efficiency in promoting antigen cross-presentation,DC maturation,and pro-inflammatory cytokines secretion,thus significantly increasing the activation of T lymphocytes.FDT could be efficiently drained to lymph nodes and internalized by antigen presenting cells,triggering both humoral and cellular immune responses.FDT induced an 11.13-fold and 4.42-fold increase in germinal center B cells and follicular helper T cells proliferation compared with PBS,respectively.Moreover,FDT could promote T cell infiltration in the tumor microenvironment,and regulate the polarization of tumor-associated macrophages towards pro-inflammatory phenotype,thus exerting a synergistic anti-tumor effect.FDT,combined with IL-12 and a PD-L1,could significantly delay the progression of B16-OVA malignant melanoma and achieved complete clearance of peritoneal carcinomatosis of MC38 colon cancer in mice.Conclusion:In summary,to fully utilize the advantages of tumor cells and their derivatives as cancer vaccines,this thesis has innovated and developed two types of tumor cell-derived cancer vaccines by focusing on the inherent properties of tumor cells or their special form of death.These two proposed vaccine systems were simple and efficient,and exhibited certain versatility.They have displayed good in vivo safety and therapeutic efficacy in a variety of cancers.Further combination with radiotherapy or other immunotherapies could effectively regulate the immune microenvironment in draining lymph nodes and tumor tissues,thus synergistically enhancing anti-tumor responses.Related research could provide diversified ideas and reliable foundations for the development and clinical translation of cancer vaccines.