Application And Mechanism Of Autophagy And TLR9 Synergetic Regulation Strategy Based On DNA Nanocage In Pancreatic Cancer Immunotherapy

Pancreatic cancer is a highly malignant tumor of the digestive system,with a five-year survival rate of less than 6%,known as the "king of cancer".Only 20%of patients can undergo surgery at the time of diagnosis.Patients who can not be treated by surgery mainly rely on chemotherapy,but there are few drug choices and serious side effects.Tumor immunotherapy provides hope for the treatment of many kinds of cancer.FDA has approved immunocheckpoint inhibitors for programmed death receptor-1(PD-1)and cytotoxic T lymphocyte associated antigen-4(CTLA-4),monoclonal antibodies and CAR-T cell therapy,and successfully used in the treatment of melanoma,gastric cancer,cervical cancer and diffuse large B-cell lymphoma and other cancers.However,many immunotherapies do not benefit well in pancreatic cancer.The biggest obstacle to immunotherapy for pancreatic cancer is the tumor microenvironment of pancreatic cancer.The main characteristics of TME in pancreatic cancer are insufficient infiltration of T cells and NK cells,massive infiltration of immunosuppressive cells,and inhibition of T cells and NK cells by immunosuppressive factors.Finally,TME,which is characterized by disability and even lack of T-cell-mediated adaptive immune response,is formed in pancreatic cancer,which is a typical "cold" tumor in the sense of immunology.As a classic immune stimulator,Toll-like receptor 9 agonists can activate TLR9 on the endosomal membrane of dendritic cells,and then activate two main TLR9 signal transduction pathways,IRF7 and NF-κ B.Thus,it mediates the rapid production of IFN-α,activates NK cells,blocks immunosuppression,promotes helper T cell 1 and CD8+T cells homing to tumor,stimulates DCs to up-regulate the expression of costimulatory molecules CD80,CD86 and CD40 on cell surface,and promotes the production and activation of tumor-specific CD8+T cells.Studies have shown that TLR9 agonists CpG oligonucleotides(CpG ODN),lefitolimod can not only inhibit the occurrence and development of breast cancer,melanoma,head and neck squamous cell carcinoma,small cell lung cancer and other cancers,but also inhibit the progression of distal tumors.TLR9 agonist has the immunological characteristics of activating body immunity and reversing tumor microenvironment,so it can be an effective strategy for the treatment of typical "cold" tumor-pancreatic cancer in theory.However,the study found that compared with the control group,the survival time of mice in the TLR9 agonist treatment group had no significant benefit and could not meet the treatment needs.Based on the concept of cancer-immune cycle,this paper further explores the reasons.To achieve effective killing of tumor by T cells,it is necessary to form a closed-loop anti-tumor immune response in the tumor microenvironment,including T cell initiation and activation,T cell chemotaxis,T cell infiltration,T cell recognition of tumor cells,T cells killing tumor cells,release of tumor antigen and presentation of tumor antigen.Studies have shown that pancreatic cancer cells reduce the expression of major histocompatibility complex I molecules on the surface of their own cells through autophagy.T cells can only recognize tumor antigens presented by MHC class I molecules.The low expression of MHC class I molecules blocks T cells’ recognition of pancreatic cancer cells.Therefore,there are two links in the tumor microenvironment of pancreatic cancer:insufficient T cell infiltration and T cell inability to recognize tumor cells.The application of TLR9 agonist can only restore the infiltration of T cells into the tumor,so it can not prevent pancreatic cancer cells from destroying the cancer-immune cycle.Chloroquine phosphate(CQP),as an autophagy inhibitor,can inhibit the binding of autophagosomes to lysosomes,block the process of autophagy,and restore the expression of MHC class I molecules in pancreatic cancer cells.The synergistic regulation of autophagy and TLR9 by the combination of CQP and CpGODN in pancreatic cancer may have a good effect.To sum up,TLR9 agonists alone can not reshape the complete cancer-immune cycle in pancreatic cancer TME.This paper further proposes a synergistic regulation strategy of TLR9 and autophagy to restore cancer-immune circulation in pancreatic cancer.In this paper,we designed and prepared DNA nano-cages(Fe@DN)which can target pancreatic cancer and cascade drug release.Fe@DN can restore DNAzyme8-17 activity in response to high concentration of H2O2 at tumor site,and release ferritin from DNA cage.DNA cage with CpGODN activity can activate DCsTLR9.Ferritin loaded CQP can target tumor cells to release drugs and inhibit tumor cell autophagy.In this paper,we explored the efficacy and mechanism of Fe@DN synergistic regulation of autophagy and TLR9 in pancreatic cancer in vivo and in vitro.In vitro experiments confirmed that Fe@DN could activate DCs,increase the expression of costimulatory molecules on the surface of DCs and the secretion of type I interferon,and up-regulate the expression of MHC class I molecules on the surface of pancreatic cancer cells.The synergistic effect of the two aspects finally improved the killing efficiency of lymphocytes of tumor-bearing mice to pancreatic cancer cells.Then in situ tumor-bearing mice were used to evaluate the curative effect.Compared with CpGODN or CQP alone,Fe@DN could significantly prolong the survival time of mice,significantly improve the infiltration of DCs and CD8+T cells in tumor microenvironment,up-regulate the expression of MHC class I molecules in pancreatic cancer cells,increase the secretion of pro-inflammatory factors such as TNF-α,IL-12 and IFN-γ in tumor-bearing mice,and realize the transformation of tumor microenvironment from "cold" to "hot".At the same time,Fe@DN has good safety in vivo.Fe@DN can realize the synergistic regulation strategy of autophagy and TLR9,which shows a good therapeutic effect on pancreatic cancer and provides a new idea and method for immunotherapy of pancreatic cancer.MethodsPreparation and characterization of Fe@DN:1.Fe@DN were constructed by pharmaceutical methods.2.The morphology,particle size,potential and entrapment efficiency of Fe@DN were determined by transmission electron microscope(TEM),Malvern particle size analyzer,trace protein concentration detection kit and ultraviolet spectrophotometry.3.The stability and safety of Fe@DN in vitro were evaluated by Malvern particle size analyzer and hemolysis test.4.Evaluation of drug release behavior of Fe@DN in vitro by trace protein concentration detection kit and UV spectrophotometer.Functional verification and mechanism of Fe@DN in vitro:1.Mice bone marrow-derived DCs(BMDCs)was extracted and induced,and its purity was detected by flow cytometry.2.Mice BMDCs was treated with Fe@DN in vitro.The expression of CD80,CD86,CD40 and other costimulatory molecules on DCs surface and the secretion of type I interferon were detected by flow cytometry and ELISA.3.PANC-02 cells were treated with Fe@DN in vitro,and the expression level of MHC class I molecules in PANC-02 cells was detected by Western-Blot technique.4.PANC-02 cells were treated with Fe@DN in vitro,and PANC-02 cell proliferation was detected by CCK-8 method.5.PANC-02 cells were treated with Fe@DN in vitro,and apoptosis of PANC-02 cells was detected by Annexin V-7AAD method.6.Spleen lymphocytes of tumor-bearing mice were co-incubated with PANC-02 cells and Fe@DN in vitro,and the killing effect of spleen lymphocytes on PANC-02 cells was detected by CFSE-7AAD method.In vivo pharmacodynamic evaluation and mechanism of Fe@DN:1.The tumor-bearing model of PANC-02 pancreatic cancer in situ was established,and Fe@DN was injected into the caudal vein.The in vivo distribution and tumor progression of Fe@DN were observed by in vivo imaging;the survival time of mice was counted;the infiltration of T cells and DCs in tumor was observed by immunofluorescence technique;the expression of MHC class Ⅰ molecules on the surface of pancreatic cancer cells was observed by immunohistochemical technique;the concentrations of TNF-α,IL-12 and IFN-γ in serum of mice were detected by ELISA;the safety of Fe@DN was observed by H&E staining.ResultsPreparation and characterization of Fe@DN:1.Fe@DN were successfully constructed.2.Fe@DN has clear morphology,good dispersion,average particle size of 81.75±2.05 nm,Zeta potential of-24.59±3.07 mV and suitable entrapment efficiency.3.Fe@DN remained stable in vitro and the hemolysis rate was less than 5%.4.Fe@DN have ideal drug release behavior and release efficiency in vitro.Functional verification and mechanism of Fe@DN in vitro:1.Mouse BMDCs with purity more than 90%was obtained.2.After BMDCs was treated with Fe@DN in vitro,the expression of CD80,CD86,CD40 and other costimulatory molecules on DCs surface and the secretion of type Ⅰ interferon were significantly increased.3.Fe@DN up-regulate the expression of MHC class Ⅰ molecules in PANC-02 cells.4.Fe@DN effectively inhibited PANC-02 cells’ proliferation.5.Fe@DN promoted apoptosis of PANC-02 cells.6.Fe@DN significantly improved the killing ability of spleen lymphocytes of tumor-bearing mice to PANC-02 cells.In vivo pharmacodynamic evaluation and mechanism of Fe@DN:1.Fe@DN had tumor targeting,prolonged the survival time of mice,controlled tumor progression,increased the infiltration of CD8+T cells and DCs in tumor,restored the expression of MHC class Ⅰ molecules on the surface of mice pancreatic cancer cells,increased the secretion of TNF-α,IL-12 and IFN-γ in mice,and had no damage to heart,liver,spleen,lung and kidney.Conclusions1.Fe@DN not only had the dual response and release ability of tumor high concentration H2O2 and lysosomal acidic environment,but also had good tumor targeting performance,and achieved accurate targeted delivery of CpG ODN and CQP grades.2.Fe@DN can effectively activate DCs,inhibit autophagy and up-regulate the level of MHC class I molecules in pancreatic cancer cells in vitro,effectively produce a synergistic effect,and promote the killing of lymphocytes of tumor-bearing mice to PANC-02 cells.3.The synergistic regulation strategy of autophagy and TLR9 realized by Fe@DN can restored the complete cancer-immunity cycle in tumor microenvironment of tumor-bearing mice,significantly controlled tumor progression,prolonged the survival time of mice,and showed a good therapeutic effect on pancreatic cancer.