Basic Research On Anti-tumor Immunotherapy Based On Modulation Of Tumor Microenvironment

Malignant tumor seriously threatens the human health,and its prevention and treatment is a great challenge for tumor research.In recent years,tumor immunotherapy,which could inhibit and destroy tumor by improving the body’s anti-tumor immune response through reactivating and maintaining tumor-immune cycle,has become a hot field of medical research.Difference from traditional treatments,immunotherapy not only inhibit primary tumor,but also prevent tumor recurrence and metastasis.Although tumor immunotherapies have achieved major breakthrough,such as PD-1/PD-L1 immune checkpoints and CAR-T cell therapy,not every patient can benefit from them.With the development of tumor immunology,how to improve the immunogenicity of tumor cells and alleviate immunosuppressive tumor microenvironment（ITME）have become the key factors to enhance the efficiency of tumor immunotherapy.This study focuses on improving tumor immunogenicity and tumor immunosuppressive microenvironment to construct nano delivery system with enhanced antitumor efficacy,and explore the therapeutic effect in vitro and in vivo.The main contents and conclusions are as follows:1.Amplified cancer immunotherapy of microenvironment-regulated tumor-derived antigenic microparticle vaccineAs an important branch of tumor immunotherapy,tumor vaccine has always been concerned.Efficient cancer vaccines not only require the co-delivery of antigens and adjuvants to antigen presenting cells（APCs）to initiate immune system and enhance tumor immunogenicity,but also need to improve ITME to make it conducive to the immune response,so as to effectively enhance the effect of tumor immunotherapy.In this study,a novel tumor therapeutic vaccine by using tumor-derived antigenic microparticles（T-MPs）secreted by tumor cells as antigens was constructed,and its therapeutic potential in murine tumor prophylactic and therapeutic models were investigated.Firstly,the tumor cells were coincubated with small-sized nano-Fe₃O₄ and irradiated by ultraviolet.The Fe₃O₄ loaded T-MPs（Fe₃O₄/T-MPs）were obtained by gradient centrifugation.Then anti-tumor vaccine（Fe₃O₄/T-MPs-Cp G/Lipo）were obtained by tethered adjuvant Cp G-loaded liposomes（Cp G/Lipo）on the surface of Fe₃O₄/T-MPs through surface engineering strategy.The Fe₃O₄/T-MPs-Cp G/Lipo vaccine retains the antigen and adhesion molecules derived from its parent cells,which has the ability of immune activation and homologous targeting.Results from transwell test showed that the anti-tumor vaccine could recruit APCs and be phagocytized by them,thus promoting the maturation of APCs.The results of immune response in vivo showed that codelivery of Fe₃O₄/T-MPs and Cp G/Lipo to APCs could activate T cells by promoting the maturation and cytokine secretion of APCs,eliciting strong tumor antigen-specific host immune response.Meanwhile,vaccines in the TME could reverse infiltrated M2-mode tumor-associated macrophages（M2-TAMs）into a tumor-suppressive M1 phenotype by nano-Fe₃O₄,amazingly induce abundant infiltration of cytotoxic T lymphocytes,and transform a“cold”tumor into a“hot”tumor.A melanoma prophylactic study demonstrated that Fe₃O₄/T-MPs-Cp G/Lipo vaccine could specifically inhibit～85.7%of the progression of B16F10-bearing mice.Furthermore,the combination of Fe₃O₄/T-MPs-Cp G/Lipo vaccine deriverd from B16F10 cells and immune checkpoint PD-L1 blockade significantly improves the immune response rate of immune checkpoint blocker and extends the median survival time of mice from 18 d to 92 d.In BALB/C mice,the combination of CT26 cell-derived Fe₃O₄/T-MPs-Cp G/Lipo-CT26 vaccine and PD-L1antibody completely inhibited about 67%of colon cancer growth.Overall,this study suggests a general cell-engineering strategy tailored to a personalized vaccine from autologous cancer cell materials of each individual patient,which has a good application prospect in increasing tumor immunogenicity,improving immunosuppressive TME and enhancing tumor immunotherapy.2.Implantable bioresponsive nanoarray enhances postsurgical immunotherapy by synergistic pyroptosis and tumor microenvironment modulationAlthough tumor vaccine is the key direction of immunotherapy,the majority of development of tumor vaccine was stopped inⅢphase of clinical trials.At precent,surgical resection is still the primary therapeutic modality for most malignant solid tumors in clinical practice,but residual tumor cells that cannot be completely removed are likely to lead to serious postoperative tumor recurrence and metastasis.The severe ITME postsurgical not only directly promotes proliferation and invasion of cancer cells,but also inhibits the activities of various antitumor leukocytes.Based on these factors,this study constructed an implantable bioresponsive nanoarray scaffold that could not only eliminate residual tumor lesions but also regulate TME postsurgical,and investigated the anti-tumor efficacy of the nanoarray.Firstly,the polydopamine nanoparticles（PDA NPs）were synthesized by the polymerization of dopamine.At the same time,chemotherapeutics doxorubicin（DOX）and the epigenetic modulator JQ1 are coloaded into the nanoparticles and modified with hyaluronic acid（HA）to obtain tumor targeting nanoparticles（HP-DOX/JQ1NPs）.Then,the nanoarray was constructed by linking HP-DOX/JQ1 NPs up through a bio-responsive linker（RL）and further loading JQ1（DOX/JQ1-IBRN）.Under high level of H₂O₂ in postsurgical TME,the implanted DOX/JQ1-IBRN disaggregates and releases JQ1 while generating small-sized HP-DOX/JQ1 NPs for realizing ITME modulation and tumor targeting therapy.On one hand,HP-DOX/JQ1 NPs destroy residual tumor cells precisely and trigger gasdermin E（GSDME）-dependent pyroptosis.The pyroptosis of tumor cells could improve the tumor immunogenicity and activate the body’s anti-tumor immune response by increasing the infiltration of immune cells,promoting the maturation of APCs and enhancing the anti-tumor immune function of cytotoxic T lymphocytes.On the other hand,JQ1 selectively blocks PD-L1 mediated immune evasion and reduces regulatory T cells（Tregs）/-disruptive effect,which would alleviate immunosuppression and synergize the antitumor immunity based on pyroptosis.The results demonstrating that the implantation of DOX/JQ1-IBRN strongly prevents the residue tumors recurrence postsurgical and prolongs survival in multiple murine tumor models with negligible toxicity,and inhibited pulmonary metastatic nodules in lung metastasis model（15.2-fold lower than the surgery groups）.This cooperation in tumor pyroptosis and ITME modulation through the implantable nanoarray（a simple,valid,and safe scaffold）is expected to provide crucial insights for post-surgical treatment.