The Effects Of Anti-cytokine Active Immunization In Combination With Tumor Immunogenic Cell Death In A Mouse Breast Cancer Model

Background Cytokines and related cells constitute a complex regulatory and effectors network during disease processes.Under normal physiological conditions,cytokine networks are in a state of equilibrium and stability;while under pathological conditions,certain cytokine deletions and functions inhibition or overexpression and abnormal accumulation will seriously damage the cytokine network homeostasis,affect the body's immune response,leading to serious diseases.At present,an important development direction of tumor immunotherapy research is to target the pathological cytokines and their signaling pathways in tumorigenesis and development,using monoclonal antibodies or soluble receptors against cytokines and their receptors,or cytokine signals.Pathway blockers perform regulatory interventions for tumor cell growth,tumor angiogenesis,and tumor immune microenvironment.Our previous research shows that the use of virus-like particles as a carrier to present cytokine epitopes can induce the production of cytokine-specific antibodies,effectively regulate the tumor immunosuppressive microenvironment,promote the production of anti-tumor effector cells,and aggregate into tumor tissues.Its anti-tumor effect suggests that active immunity against cytokines is an effective strategy for tumor immunotherapy.On the other hand,recent studies have found that after treatment of certain tumors with specific chemicals or radiation,tumor cells can be transformed from non-immunogenic cells into immunogenic cells while apoptotic,and thereby stimulate the body's resistance.The immunological killing effect of tumors,which is called tumor immunogenic cell death(ICD).ICD requires the participation of a series of signaling molecules and cytokines,including the expression and level of "eat me"signaling molecules on the cell membrane surface,and the synthesis and release of "find me" immune-important risk signaling factors.Induction of ICD can be a key strategy for tumor vaccine immunotherapy in response to tumor cells multiple antigens to stimulate a more comprehensive immune response and overcome tumor heterogeneity.It is a new strategy for tumor immunotherapy with unique application potential.Objective This study intends to combine the two strategies of anti-cytokine active immunization and tumor cell immunogenic death induction.On the one hand,the anti-cytokine active immunity is used to regulate the tumor immune microenvironment,which is beneficial to anti-tumor effect cells.On the other hand,it induces immunogenic death of tumor cells,stimulates the production of effective anti-tumor immune effect cells,and achieves significant tumor treatment effects through synergy between the two groups,thus providing a new strategy for clinical tumor immunotherapy intervention.Methods Based on the previous experimental basis,this study used gene recombination technology to obtain hepatitis B core antigen(HBcAg)virus-like particles(VLP)as a vector to present IL-33 full molecule and TGF-?1,FGF-2,IL-4,and IL-13 antigen peptide VLPs vaccine,respectively.Firstly,four kinds of commonly used chemotherapeutic drugs were injected intratumorally in tumor-bearing mice to investigate tumor growth,body weight and survival of mice,and to investigate the expression of key immunogenic molecules to obtain ICD in vivo.The type and concentration of chemotherapeutic drugs;then,in vitro,by tumor cell experiments,to investigate the tumor cell death caused by different concentrations,different time of action,different drugs,and to examine the expression of key immunogenic molecules,thereby obtaining ICD induced by tumor cells in vitro.Then,in the 4T1 cell transplanted mouse breast cancer model,the anti-cytokine immunological intervention was performed with a therapeutic immune strategy.After the tumor was established,the tumor size of the mouse was continuously monitored.When the tumor size of the mouse was 5-8 mm,the screen was selected.The chemotherapy drug is administered to the mice for intratumoral injection.The mice were sacrificed at the end of the experiment,and the specific IgG antibody levels in the serum were analyzed by ELISA;the tumor weight of the mice was weighed;the number of lung metastasis nodules in the mice was recorded;the immune effector cells in the mouse spleen cells were detected by flow cytometry.Levels of cytotoxic T lymphocytes(CTLs)and immunosuppressive cells such as regulatory T cells(Tregs)and bone marrow derived immunosuppressive cells(MDSCs).Results By HPLC and electron microscopy analysis,this study successfully purified and prepared a total of five virus-like particle vaccines that presented IL-33 full-molecules and presented TGF-?1,FGF-2,IL-4,and IL-13 antigen peptides.Immunization of mice with VLPs induces sustained,high levels of anti-cytokine-specific antibody responses.Through intratumoral injection of chemotherapy drugs in tumor-bearing mice,it was found that Mitoxantrone(MIT),Doxorubicin(DOXO),oxaliplatin(Oxa)and bortezomib(Bort)4 common chemotherapeutic drugs have 4T1 tumor killing activity in a dose-dependent manner,but high concentrations of chemotherapeutic drugs can cause significant weight loss and increased mortality in mice,while MIT and DOXO are 0.8 mg/kg/time,respectively.And the tumor-injected dose of 1.0 mg/kg/time is safe and has tumoricidal activity in mice.Meanwhile,by detecting the ICD key molecules ATP and HMGB1 in tumor tissues,it is found that ATP and HMGB1 of intratumoral injection of chemotherapeutic drugs.The expression level was higher than that of the control group,indicating that intratumoral injection of chemotherapeutic drugs can cause tumor tissue ICD.Further,through in vitro experiments of tumor cells,it was confirmed that MIT and DOXO could induce obvious apoptosis of tumor cells and expression of key molecules of CRT,ATP and HMGB1 after 12 hours of treatment at 10 ?M.Subsequently,ICD tumor cells treated with MIT and DOXO immunized mice in a prophylactic and therapeutic manner,which significantly inhibited the growth and metastasis of the inoculated tumor cells,further indicating the success of ICD cell induction and its ability to stimulate anti-tumor immunity in vivo.In vivo induced tumor tissue ICD and different anti-cytokine virus-like particle immune synergistic intervention studies,compared with the vehicle control group,the tumor size is inhibited to some extent;when the mouse tumor size is 5-8mm,further use MIT and Intratumoral injection of DOXO in mice,tumor growth of mice treated with MIT and DOXO alone was significantly inhibited,and combined with intratumoral injection of drugs and anti-cytokine immunization groups,combined intervention of mouse mammary gland tumor growth And lung metastasis was more significantly inhibited.In addition,the number of CD8a+in the spleen and tumor lymphocytes of the anti-TGF-?1 group increased significantly.Conclusion This study combines two strategies:active anti-cytokine active immunity and tumor cell immunogenic death induction.At the same time,considering the regulation of tumor immune microenvironment and anti-tumor immune effecter cell induction,synergy for tumor growth is obtained.Inhibition provides new strategies and means for clinical tumor immune intervention,and provides new ideas for the development of new tumor vaccines.