Degradable Biopolymer Immune Implants For Colorectal Cancer Postoperative Immunotherapy

Colorectal cancer(CRC)is one of the most common malignant tumors of the digestive tract.Surgical resection is the first-line therapy for colorectal cancer.However,for advanced CRC,the curative effect of surgical resection is limited due to subsequent relapse from residual tumor cells and metastatic tumor cells,leading to surgical failure and significant mortality.Systemic chemotherapy and radiotherapy are important options for the prevention of both local and metastatic tumor relapse following surgical resection.However,considering the post-surgery weak physical condition and incision healing of patients,systemic chemotherapy and radiotherapy are usually administered at least 2?3 weeks after surgery,and the optimal time point for eradicating residual cancer cells is often missed during this period.In addition,this period also creates early conditions for the tumor to relapse.Hence,finding appropriate postoperative management techniques to resolve tumor recurrence and metastasis in CRC remains an urgent clinical need.More recently,various cancer immunotherapies have been reported to inhibit tumor recurrence and may even provide the prospect of a cure.However,in the treatment of solid tumors,immunotherapy faces many challenges,such as low response to immunotherapy,drug resistance,on-target but off-tumor,and serious side effects,etc.For the past few years,some studies have proved that the above problems can be solved by local sustained release of immune drugs in/near the tumor site,and the safety is higher.For CRC,owing to the fact that surgeons have unique opportunities and direct access to tumors during tumor resection,in situ immunotherapy could be a promising cancer prevention strategy for postoperative management.Several recent studies have revealed the great potential of materials-based local immunotherapy for cancer,including implantable scaffolds,injectable hydrogels,and in situ formed hydrogels,among others.Theses immunotherapy results in superior anti-tumor effects and systemic anti-tumor immunity.The ease of cargo loading and controlled payload release have enabled materials-based local immunotherapy to be a promising means in cancer treatment,and should be emergent in CRC post-surgical management.To this end,we proposed a biopolymer implant fabricated with 4-arm poly(ethylene glycol)amine(4-arm PEG-NH2)and oxidized dextran(ODEX),and co-loaded with resiquimod(R848)and anti-OX40 antibody(aOX40)for CRC post-surgical treatment.Objective:Owing to the fact that surgeons have unique opportunities and direct access to the colorectal surgical site.Herein,we report a designed biopolymer immune implant for CRC post-surgical therapy.We hope the reported biopolymer immune implants may solve the problem of postoperative recurrence and metastasis of colorectal cancer.Method:(1)The biopolymer immune implants were prepared by cross-linking of 4-arm PEG-NH₂ and ODEX by Schiff base reaction between the primary amines and the aldehyde groups at different mass ratios;We performed strength and adhesion test for the formed hydrogels;Then we choose the best mass ratio according to the test results.(2)The degradation of the implants in vitro and in vivo was assessed at a best mass ratio of 1/1;(3)R848 was loaded inside the hydrogel by mixing it with the polymer during the mixing process.The aOX40 in solution was added to the lyophilized implant before use.The release of loaded cargos from the implants was investigated in vitro;(4)To test the therapeutic effects of biopolymer immune implants on post-surgery therapy,we established an incomplete tumour resection model.The biopolymer immune implants were placed in the tumour resection cavity and residual tumour growth was constantly monitored.We analyzed the immune microenvironment of the residual tumour niche at 3 and 10days after various treatments;(5)We performed tumor re-challenge tests to test the enduring immune memory effects.CT26 tumor cells were re-inoculated contralateral to the primary tumor site 30 days after original tumor resection and treatment,and fresh mice in the absence of any treatment were used as a control group.The tumour growth was constantly monitored.In addition,we also checked the immune cell status in spleens on day 10;(6)We further evaluated whether in situ treatment with immune impalnts was able to inhibit distant tumour growth.We established a bilateral tumor model by injecting CT26 cells to the two opposite flanks of the mice.Various treatments were applied to the primary tumour cavity when the tumor volumes reached 50?100 mm³,and tumor volumes on the controlateral site was monitored.We also analyzed the immune microenvironment of the distal tumor after various treatments.Result:(1)We increased storage moduli(G')at higher 4-arm PEG-NH2 ratios.The best adhesion appeared at a mass ratio of 1/1.As a result,we used a mass ratio of 1/1 in the following studies.In vitro degradation lasted for>9days,and in vivo degradation continued for>21 days.(2)R848 and aOX40 antibody were loaded inside the hydrogel.The release kinetics of R848 and Ig G from the implant is in accordance with the degradation kinetics of the implant.In buffers at p H 7.4 and p H 6.8,R848and Ig G-Cy5 were released from the implant in 9 days in a sustained manner,respectively;(3)In an incomplete tumour resection model on the BALB/c mice,BI(R848+aOX40)treatment obtained durable complete tumour eradication,and all the mice were completely cured by the end of the observation period.The number of NK cells in residual tumours was significantly increased in the R848-treated groups.In addition,p DCs and m DCs were also increased.Increased numbers of infiltrating T cells inside tumours were detected on day 10;(4)In tumor re-challenge tests,no tumour growth was observed in the mice previously treated with BI(R848+aOX40)during the 90 days observation period(maximum tumor volume reached 20 mm³ by day 5,and quickly regressed subsequently).In spleen,both CD4⁺and CD8⁺central memory T cells and effector memory T cells were clearly increased in the treated group;(5)In a bilateral tumor model,immune impalnts exhibited predominant inhibition of the growth of distal tumours.In addition,we observed increased CD4⁺and CD8⁺T cell numbers in distal tumours by flow cytometry.Conclusion:(1)we have demonstrated a simple post-surgical CRC immunotherapy strategy by placing previously formed therapeutic biopolymer immune implants(characterized with high tissue adhesion,sustained drug release)in the tumour resection cavity.The immune implants sequentially elicited innate and adaptive immunity,and immune memory effects,which not only resulted in complete residual tumour clearance,inhibit the growth of distal tumours and elicit immune memory effects to resist tumour re-challenge.(2)Our designed biopolymer immune implant was confirmed to be a logical choice for postoperative CRC treatment,and the experimental results suggested the potential clinical translation of this more specific,more effective,and less toxic method upon tumour resection.