The Study Of Colorectal Cancer Postoperative Vaccine Therapy Based On Injectable Gel Material

Colorectal cancer(CRC)is the third most common cancer in the world,and its morbidity and mortality are increasing all over the world.In addition to ageing and eating habits in high-income countries,adverse risk factors such as obesity,lack of physical activity and smoking also increase the risk of colorectal cancer.The progress of pathophysiology increases the choice of treatment for local and advanced diseases and promotes the progress of individualized therapy.The standard treatments for colorectal cancer are surgery,chemotherapy and radiotherapy,which could be used in combination with patients.However,these treatments have many side effects because of their cytotoxicity to normal cell growth and division.In addition,many patients still have a relapse even after a series of treatments.Therefore,it is very important to find more effective alternative treatments to treat patients with colon cancer.Immunotherapy is one of the new options for cancer treatment.The strategy is to use the patient's own immune system to fight with cancer cells.The results of some cancer immunotherapy are amazing in some cases,but it is not in other cases depending on the state of the patient's own immune system.Patients who respond well to immunotherapy for cancer have better prognosis and quality of life.Cancer treatment is rapidly entering the age of immunotherapy,Cancer vaccine is considered to be the most promising cancer immunotherapy strategy.However,the progress of anticancer vaccine research is still limited because of the difficulties in identifying and obtaining tumor specific antigens.Considering that surgery is the first choice for tumor treatment in most cases,can we directly extract tumor antigens from resected tumors and design individualized tumor vaccines?Postoperative vaccine local immunotherapy based on injectable gel materials has become a promising strategy and approach for immunotherapy after tumor resection,including injectable hydrogels and hydrogels formed in situ.In recent years,studies have shown that this treatment has the characteristics of easy loading,degradability and controlled sustained release,showing great therapeutic potential.This kind of local immunotherapy can produce the effect of systemic anti-tumor immunity,and even achieve cure.Based on this idea,we designed a dynamic covalent hydrogel vaccine(DCHVax)based on injectable gel materials for personalized treatment after tumor surgery.The tumor resected protein was used as antigen,Cp G/polyethyleneimine complex as adjuvant,and multi-arm polyethylene glycol(8arm-PEG-NH2)/oxidized dextran(ODEX)dynamically crosslinked hydrogel as matrix.DCHVax was injected subcutaneously to recruit dendritic cells in situ and induce strong tumor-specific immune response.Our results show that it could effectively inhibit the growth of residual tumor in mouse tumor model.This simple and personalized method of cancer vaccine development is expected to develop clinically related postoperative cancer treatment strategies.Objective:A dynamic covalent hydrogel vaccine(DCHVax)based on injectable gel material was designed for the immunotherapy of advanced colorectal cancer after operation,to solve the problem of recurrence and metastasis of colorectal cancer after operation.Method:(1)Preparation of blank injectable dynamic covalent hydrogels:The crosslinked 8arm-PEG-NH₂ with ODEX and the crosslinked hydrogels were formed by the schiffbasis reaction of aldehyde group and amino group,the strength and adhesion of the injectable dynamic covalent hydrogels with different mass ratio were tested,and the best mass ratio was selected according to the test results;(2)Characterization of injectable dynamic covalent hydrogels current situation:the degradation of the hydrogels in vitro and in Vivo was tested under the optimal mass ratio;(3)Preparation of dynamic covalent hydrogel vaccine(DCHVax)containing model antigen,model Antigen OVA and Cp G/PEI were loaded into injectable dynamic covalent Hydrogel,and the sustained-release effect was tested;(4)Extraction of antigen protein from tumor:When the tumor volume reached about 200mm³,MC38 or CT26 tumor cells were excised from the body of mice to prepare the antigen,and the content of protein antigen was determined by BCA method;(5)Preparation of dynamic covalent hydrogel vaccine(DCHVax):Loading adjuvant(Cp G/PEI)and antigen protein extracted from tumor into injectable dynamic covalent hydrogel,and testing its sustained-release effect;(6)Verification of the efficacy of dynamic covalent hydrogel vaccine(DCHVax)in killing residual tumors:The CRC CT26?MC38cell models of BALB/c mice and C57BL/6 mice were constructed by subcutaneous incomplete resection.DCHVax was injected into the axillary lymph nodes of the mice,and the growth of residual tumors was monitored and recorded.The changes of immune microenvironment of residual tumor on day 3 and 10 after injection were analyzed;(7)Verification of the memory effect of DCHVax after DCHVax therapy:to monitor and record the tumor growth in mice treated with DCHVax;The changes of systemic immune including blood and spleen microenvironment were analyzed.Result:(1)When the mass ratio of 8-arm PEG-NH₂ to ODEX was 1:1,the injectable dynamic covalent hydrogel had higher strength and better tissue adhesion,and the degradation time of injectable dynamic covalent hydrogel was 18 days in vitro.While it was more than 18 days in vivo;(2)The model antigen B16-OVA was successfully loaded and verified,the CT26/MC38 antigen protein was successfully extracted,and Cp G/PEI was loaded into the injectable dynamic covalent hydrogel to prepare the hydrogel tumor vaccine DCHVax,the release time of antigen protein and Cp G/PEI was reached 18 days.(3)DCHVax was injected into the armpit lymph nodes of mice in the model of subcutaneously incomplete resection of tumor,which inhibited the growth of tumor significantly.The results of residual tumor immune microenvironment analysis showed that the innate immunity was activated rapidly,the infiltration of natural killer cell cells(Nature killer cells,NK cells)increased,the activation of dendritic cell cells(DCs)was enhanced,and the adaptive immunity was activated subsequently,the number of T cell infiltration in the tumor increased significantly(Day 6);(4)DCHVax-OVA prepared with OVA Model Antigen was used to treat B16-OVA mice with different subcutaneously incomplete resection of tumor,after DCHVax-OVA treatment,the tumor growth was monitored and recorded.The results of systemic immune analysis showed that the number of memory T cells in blood and spleen increased significantly;(5)DCHVax-MC38,which was prepared from MC38 model antigen extracted from tumor tissue,was used to treat different groups of MC38model mice,after DCHVax-MC38 treatment,the tumor growth was monitored and recorded.The results of systemic immunity analysis showed that the number of memory T cells in blood and spleen increased significantly;(6)In the subcutaneously incompletely resected tumor model,intraoperative injection of DCHVax-CT26 into the axillary lymph nodes of mice also significantly inhibited the tumor growth of CT26.Conclusion:In this study,we propose a DCHVax personalized cancer vaccine platform for postoperative cancer treatment.Our results confirmed that DCHVax could effectively inhibit tumor recurrence and prolong the survival time of mice.This platform is simple,efficient,cost-effective and universally applicable in comparison with the currently reported personalized cancer vaccine approaches.Therefore,we believe that the DCHVax platform is of great significance for the postoperative treatment of cancer,especially for the clinical application of advanced cancer.