An Injectable Hydrogel Reshaping Adenosinergic Axis For Cancer Therapy

Tumor is one of the main threats to human health in the world today.In the current clinical treatment,radiotherapy,chemotherapy,surgery and other traditional treatment for advanced tumors and metastatic tumors are limited.With the development of technology,tumor immunotherapy has gradually become a powerful treatment method.Immunotherapy is a therapeutic strategy to specifically eliminate tumor cells by activating or strengthening innate immunity and adaptive immunity.It has remarkable therapeutic effects on renal cell carcinoma,non-small cell lung cancer,melanoma,nasopharyngeal carcinoma and other tumors,and is the most likely therapeutic strategy to eradicate tumors at present.Cellular immunity is the main way of the body’s anti-tumor immunity,which plays an important role in monitoring and defending tumorigenesis.However,the existence of multiple immune escape and immunosuppression mechanisms in solid tumors together constitute the tumor immunosuppressive microenvironment,resulting in a low clinical response rate to immunotherapy.Abnormal cell metabolism in the tumor microenvironment(TME)is an important factor that triggers immunosuppression.Targeting metabolic pathways in TME to reshape tumor immune metabolic microenvironment provides a new idea for improving clinical response rate of immunotherapy.Among them,adenosine triphosphate(ATP),as one of the most abundant metabolites in cells,is an important biological messenger molecule in the TME.Notably,the ATP metabolic pathway plays both anti-tumor and pro-tumor bidirectional roles during tumor immune response.Therefore,it is of great significance to reshape ATP metabolism pathway and eliminate its negative effect in tumor metabolism environment.In TME,the process of cell injury is accompanied by the release of a large amount of ATP,which plays a crucial role in initiating tumor-specific immune response by enhancing its immunogenicity.However,there is a negative feedback mechanism in the TME called adenosinergic axis that converts ATP to adenosine through the ectonucleotidases CD73 and CD39.The transformed adenosine regulates the proliferation and cytotoxicity of effector T cells(Teff)and promotes its differentiation into regulatory T cells(Treg)by binding to A2A adenosine receptor(A2AR)on the surface of T cells.Therefore,ATP-mobilized immunogenic TME is reversed into immunosuppressive TME,which is detrimental to anti-tumor immune response.In conclusion,adenosinergic axis causes ATP to be less effective in promoting anti-tumor immunity,and the regulation of the adenosinergic axis is critical.Previous strategies against adenosinergic axis mainly focus on two aspects:one is to reduce the conversion of ATP to adenosine by inhibiting CD73 activity;the other is to block the interaction between adenosine and A2AR located on T cells.Both strategies are based on the idea of blocking adenosinergic axis,but do not stop adenosine accumulation in TME.Therefore,exploring effective strategies for eliminating adenosine is important and challenging.Inosine is an alternative energy source of Teff in TME,which can restore the proliferation and cytotoxicity of Teff under glucose restriction,is an important immunoenhancer.Notably,adenosine deaminase(ADA)can catalyze the conversion of adenosine to inosine.Therefore,ADA is capable of reprograming the ATP metabolic pathway by converting the end-product of adenosinergic axis from an immunosuppressant to an immunoenhancer.Different from directly blocking adenosinergic axis,this strategy can not only achieve the elimination of adenosine inhibitory signal,but also generate an immunomodulator in situ to promote immune response.Extracellular ATP is mainly released by injured tumor cells.For chemotherapy,the autophagy-dependent release of ATP is key to its immunogenicity.The synergistic effect of chemotherapy and autophagy inducers can realize cascade increase of released ATP,while releasing tumor-associated antigens to initiate the anti-tumor immune response.Therefore,the combination of ADA,chemotherapy and autophagy inducers can not only ensure sufficient ATP to activate immune response,but also reshape the adenosinergic axis,reversing the negative feedback into promotion of anti-tumor immunity.Inspired by the above,this study designed an injectable hydrogel strategy.Due to the chelation of sodium alginate(SA)with calcium ions(Ca2+)in TME,the aqueous solutions of SA,ADA,autophagy inducer benzene-1 and 3-tricarboxylic acid(BTC)and chemotherapy drug doxorubicin(DOX)can gelatinize in tumor forming injectable hydrogel(S@ABD)to reshape adenosinergic axis.Under the synergistic action of DOX and BTC,the tumoricidal effect accompanied by a large amount of ATP release effectively improves tumor immunogenicity and initializes adaptive immune response.Meanwhile,ADA catalyzes the conversion of adenosine,the immune negative feedback product of ATP,into inosine,the energy substance of Teff,reversing the immunosuppressive mechanism of adenosinergic axis.The synergistic cooperation of the three drugs completely reshapes the metabolic balance of the adenosinergic axis and realizes the cascade amplification of anti-tumor immune response based on ATP.The specific research contents are as follows:The injectable hydrogel system(S@ABD)was constructed by simple physical mixing,which can quickly complete the sol-gel transition in response to Ca2+in vivo and in vitro.Scanning electron microscope(SEM)results show that it has a porous layered network structure and has the potential to store and release drugs in situ.At the same time,S@ABD has good injection performance,which can be used for in situ injection and conversion into hydrogel in vivo as a platform for in situ tumor therapy.To ensure the biosafety of SA and S@ABD hydrogel,the biocompatibility was comprehensively studied in vivo and in vitro.The results showed that the toxicity of SA hydrogel to epithelial cells was negligible in vitro,and no foreign body reaction was induced after injection.Routine blood test results showed that there were no toxicity of the blood system and systemic inflammation of SA hydrogel in vivo.Meanwhile,S@ABD hydrogel showed no obvious hemolysis in vitro,possessed good biocompatibility.B16F10 melanoma cells were selected as the in vitro cell model to verify the biological effect of S@ABD.The results showed that S@ABD had significant cytotoxicity on B16F10 tumor cells and could cause a large amount of damage-related molecular model(DAMPs)release including ATP,calreticulin(CRT)and high mobility group protein 1(HMGB1)while killing tumor cells.Therefore,tumor cells treated with S@ABD can significantly activate dendritic cells(DCs).T cell proliferation experiments show that ADA can effectively convert adenosine into inosine,relieve the inhibition of T cells and promote T cell proliferation.C57BL/6 mice were subcutaneously injected with B16F10 cells to construct tumor evaluation model in vivo.Intratumoral drug retention experiments demonstrated that intratumoral injection of S@ABD could form a drug reservoir in situ,achieve the sustained drug release.Three in vivo models of bilateral tumor,lung metastasis and immune memory were established to verify the anti-tumor and immunological effects of S@ABD hydrogel.In the bilateral tumor model,S@ABD hydrogel showed significant inhibition against both primary and abscopal tumors.Flow cytometric analysis showed that S@ABD treatment could significantly increase the maturity of DCs in lymph nodes and the proportion of cytotoxic T lymphocytes(CTLs)in both tumors and spleens.The results of cytokine detection showed that the contents of major inflammatory cytokines in tumor tissue and serum increased significantly after treatment.Therefore,S@ABD significantly boosted the anti-tumor immune response.At the same time,S@ABD hydrogel could effectively inhibit lung metastasis of tumor and form long-term immune memory,which plays a key role on preventing tumor recurrence for a long term.To sum up,an injectable hydrogel loaded with ADA,DOX and BTC was rationally designed to form a drug repository in situ after intratumoral injection,which can effectively reshape the adenosinergic axis through the synergistic release of the three drugs,continuously activate the anti-tumor immune response in vivo,inhibit tumor growth and metastasis and form long-term immune memory,which provides a new idea for tumor immunotherapy based on the regulation of tumor adenosine metabolism microenvironment.