| In the face of cancer,which is a serious threat to human life and health,emerging immunotherapy show broad prospects.In particular,the refinement of the tumor immune landscape has prompted T cell-based immunotherapy to enter the attention of researchers and show strong therapeutic potential.The immune response of T cells is pivotal in the tumorimmune cycle.After the major steps of antigen recognition,activation and differentiation,T cells initiate specific killing mechanisms in the tumor environment.Therefore,immunotherapy,based on the basic purpose of activating the immune system to kill tumors,has developed various therapeutic strategies such as tumor vaccines,immune checkpoint inhibitors,etc.Unfortunately,however,tumors have complex and diverse mechanisms of immunosuppression and immune escape.During antigen presentation,tumor cells have evolved multiple pathways to avoid antigen release and uptake,resulting in insufficient activation signals for T cells.Even after T cells are activated and infiltrate the tumor,highly expressed immune checkpoints release suppressive signals to the T cells,leaving them non-functional.Under the influence of unique conditions such as hypoxia,low glucose and chronic antigens in the tumor microenvironment,T cells accumulate depolarized mitochondria due to massive mitochondrial damage and inability to clear,which force T cells to produce depletion reprogramming and eventually to the fate of terminally exhausted T cells with complete loss of effector properties.These constraints make T cells at tumor sites often only powerless and unkillable,which is one of the main factors contributing to the current suboptimal responsiveness of clinical immunotherapy.We designed an injectable hydrogel O-TL@(N+S)to address the key issues of insufficient immunogenicity,T-cell depletion and programmed cell death protein 1(PD-1)-induced immune escape.O-TL@(N+S)is a hydrogel constructed based on tumor lysate(TL),chemically modified for the first time using sodium oxidized alginate(OSA)as a cross-linking agent for TL,and then encapsulated with the nicotinamide adenine dinucleotide(NAD)precursor drug nicotinamide ribose(NR)and glycogen synthase kinase 3(GSK-3)inhibitor SB415286.Among them,O-TL can form hydrogel intratumorally under the action of calcium ions,achieving a robust antigen reservoir to induce strong immunogenicity.Among them,O-TL can form hydrogel intratumorally under the action of calcium ions,creating an antigenic pool and inducing strong immunogenicity in vivo;meanwhile,NR can induce mitochondrial autophagy in T cells to clear depolarized mitochondria and inhibit exhaustion;while SB415286 inhibits PD-1 expression to prevent immune escape through GSK-3 pathway.The combination of the three modulates immune cells in situ to enhance T cell-based tumor immunity and achieve efficient tumor suppressionThe main research of this paper is as follows.1.OSA and O-TL were successfully synthesized by chemical reaction,and finally OTL@(N+S)was obtained.The preparation has good gel-forming properties in the presence of calcium ions and also possesses needle-through properties for intratumoral injection to form hydrogels.And this was confirmed by in vivo subcutaneous injection,which also showed that the hydrogel is degradable in vivo.The hydrogel formed by O-TL@(N+S)exhibits a classical reticular structure.In a release medium simulating the intratumoral environment,O-TL@(N+S)could continuously release active substances at a stable rate and was a potential therapeutic platform for in situ delivery of drugs.Hemolysis and cytotoxicity assays demonstrated that the hydrogel had no significant side effects on normal cells,providing preliminary evidence of safety.2.The immunomodulatory ability of O-TL@(N+S)was investigated in vitro by extracting and activating mouse bone marrow derived cells(BMDCs)and CD8+T cells.Cell maturation assays revealed that O-TL@(N+S)hydrogel could effectively stimulate DCs maturation to participate in subsequent immune initiation.Cell recruitment assays showed that the hydrogel could induce DCs recruitment to the gel site and increase the number of local DCs.On the other hand,the flow cytometry results of CD 8+T cells showed that O-TL@(N+S)hydrogel could reduce the proportion of depolarized mitochondria and thus inhibit exhaustion reprogramming,while reducing the expression of PD-1 on the T cell surface and blocking the PD-1/PD-L1 pathway.It also greatly enhanced T cell proliferation by inhibiting exhaustion,blocking PD-1 signaling and other mechanisms.The results of ELISA experiments showed a remarkable increase in IFN-γ secretion by T cells after hydrogel treatment,further demonstrating that T cell function was enhanced.The apoptosis of B16F10 cells in the co-incubation system directly revealed that the T cells after O-TL@(N+S)treatment were able to kill tumor cells more potently.3.A mouse tumor-loaded model of B16F10 was constructed to assess the in vivo immunomodulatory ability and the resulting tumor suppressive effect of O-TL@(N+S).In vivo imaging experiments first confirmed the advantages of the hydrogel in terms of drug intratumoral retention.Bilateral tumor experiments demonstrated the ability of hydrogels to significantly inhibit the growth of primary tumors and abscopal tumors without side effects.The results of flow analysis,immunofluorescence images and ELISA assay illustrated that OTL@(N+S)hydrogel elicited a strong immune response in situ as well as systemic immunity.Lung metastasis experiments confirmed that O-TL@(N+S)hydrogel could exert distant effects by activating immune responses in situ,thereby inhibiting tumor lung metastasis and prolonging survival.Memory model results showed that O-TL@(N+S)hydrogel could induce long-term immune responses in mice.In summary,an injectable tumor lysates hydrogel O-TL@(N+S)was designed and gelformed in situ after intratumoral injection.By continuously releasing active substances to induce immunogenicity,inhibit depletion reprogramming and suppress PD-1 expression,it modulated immune cells to enhance anti-tumor immunotherapy and successfully inhibited tumor growth,metastasis and recurrence,producing a powerful and sustained immune effect.This project builds injectable hydrogels based on tumor lysates to regulate T cell killing efficiency to open up new avenues for tumor immunotherapy. |
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