Study On The Anti-tumor Effect And Mechanism Of IFN-γ Modified Bacterial Vesicles Reprogramming Tumor-associated Macrophage

Background:Immune cell infiltration in the tumor microenvironment(TME)and its functional status plays a crucial role in tumor development and affects cancer patients’ clinical outcomes.Immunotherapy is essential for clinical cancer treatment;adoptive cell transfer(ACT)and immune checkpoint inhibitors(ICIs)have achieved compelling clinical outcomes in recent years.Still,their efficacy varies across patients,with only a tiny proportion of cancer patients benefiting from a response to therapy.Key constraints include the immunosuppressive mechanisms of tumor development that affect the effective production of anti-tumor effector cells and the immunosuppressive features of TME that limit the infiltration of effector cells into tumor tissue and the maintenance of anti-tumor activity in TME.As the most plastic and highest proportion of immune cells in TME,M2 tumor-associated macrophages(TAMs)are important cells causing tumor immune escape,which can suppress the anti-tumor activity of other immune cells and promote tumor growth and metastasis.However,when M2 TAMs are reprogrammed as M1 TAMs,they can release cytotoxic tumor-killing molecules such as reactive oxygen species(ROS)and nitric oxide(NO);in addition,M1 TAMs can phagocytose tumor cells,present tumor antigens as antigen-presenting cells,and secrete pro-inflammatory cytokines,which effectively synergize with adaptive immune cells to exert anti-tumor effects.Objective:Based on the functionalized modification of bacterial vesicles,this thesis aims to explore the effective strategy of reprogramming pro-tumor M2 TAMs into anti-tumor M1 TAMs to remodel the tumor immune microenvironment and enhance the immune response of anti-tumor cells,providing a novel idea for tumor immunotherapy.Method:1)High-pressure homogenization technique was used to drive recombinant E.coli cells expressing ClyA-IFN-γ fusion protein to extrude at high speed from the gap space and self-assemble into nanosized bacterial biomimetic vesicles(BBV),which were named as IFN-γ BBV.IFN-γ BBV was analyzed by SDS-PAGE electrophoresis,electron microscopy,particle size analyzer,and flow cytometry to analyze the physicochemical characteristics of the recombinant vesicles.2)Bone marrow-derived macrophage(BMDM)was isolated from mice,polarized to M2 phenotype using IL-4,then stimulated with IFN-γ BBV and detected by ELISA,Western blot,RT-PCR,and immunofluorescence.Marker changes and the ability of IFN-γ BBV to reprogram M2 macrophages to M1 macrophages were assessed at the cellular level.3)RNA sequencing of IFN-γ BBV-treated BMDMs and Western blot analysis to explore possible polarization mechanisms.4)Mice were injected intravenously with different doses of IFN-γ BBV(10 μg,40μg,100 μg,and 150 μof vesicles protein,respectively),and the safety of IFN-γ BBV was preliminarily evaluated by serum biochemical index analysis and H&E staining of primary tissues and organs.5)To evaluate the anti-tumor effects of IFN-γ BBV delivered via tail vein in TC-1 cervical cancer and CT26 colon cancer tumor models,respectively,and to examine the immune cell response characteristics in THE by flow analysis of M1 TAMs,M2 TAMs,MDSC,CTL and other immune cells in TME.6)In a 4T1 breast cancer model,tumor growth was investigated by the removal of macrophages in vivo using Clodronate Liposomes followed by intervention with IFN-γBBV delivered via the tail vein,in addition to stimulation of tumor-infiltrating lymphocytes with the 4T1 antigen CTL epitopes Zfp142 and Wdr33 and Th epitope M25 peptide,respectively,and anti-tumor effector T-cell responses were analyzed by flow cytometry and ELISPOT.7)Using the 4T1 model,single or combined interventions were performed to examine the effects of combined strategies of IFN-γ BBV and ICIs to inhibit tumor growth and metastasis.Results:1)SDS-PAGE showed a 39.7%loading of IFN-γ on IFN-γ BBV;TEM observed IFN-γBBV as 20-150 nm nano-vesicles;flow cytometry further confirmed IFN-γ exposure on the surface of recombinant bacterial vesicles.The results suggested that the nanostructured IFN-γ BBV surface presented a high density of IFN-γ.2)In vitro cellular assays showed that IFN-γ BBV stimulation significantly inhibited the levels of M2 markers Arg-1 and CD206 while significantly upregulating the expression of iNOS,CD86,and inflammatory cytokines IL-12,IL-1β,IL-6,and TNF-α,released NO,and directly killed tumor cells.The results showed that IFN-γ BBV reprogrammed M2 BMDMs to the M1 phenotype.3)RNA sequencing data showed activation of IFN-γ and TLR signaling pathways,upregulation of iNOS expression,and enhanced expression of pro-inflammatory chemokines and cytokines;Western blot detected the expression of IRF5,p-NF-κB,NFκB,p-STAT1,and STAT1 after IFN-γ BBV treatment.The results suggest that IFN-γBBV promotes macrophage reprogramming mainly through IFN-γ/JAK/STAT1 and TLR 4 pathways.4)Analysis of biochemical parameters in serum and H&E staining of different organs showed that 10 μg and 40 μg of IFN-γ BBV had no lethal toxic effects on mice and did not cause significant damage to tissues and organs,which tentatively suggested the safety of IFN-γ BBV.5)Cy7-labeled IFN-γ and IFN-γ BBV were injected into mice via the tail vein.The in vitro fluorescence signal analysis of significant organs showed that IFN-γ BBV injected mice had strong fluorescence signals,demonstrating the efficient tumor targeting and aggregating ability of IFN-γ BBV.6)IFN-γ BBV intervention significantly inhibited the growth of established cervical cancer TC-1 and colon cancer CT26 tumors.The results of flow analysis showed that IFN-γ BBV intervention significantly increased the proportion of Ml-type CD86+,iNOS+,and IL-12+TAMs and increased NO and ROS levels in tumor tissues while inhibiting M2-type CD206+TAMs,as well as immunosuppressed MDSCs and Treg levels;in addition,antigen-specific CTL cell responses were significantly enhanced.Further,immunofluorescence and TUNEL analysis of tumor tissues revealed enhanced CD8+and IFN-γ+anti-tumor effector cell infiltration and tumor cell regulation.The results suggest that IFN-γ BBV intervention successfully reprogrammed M2 TAMs to M1 phenotype,promoted tumor antigen-specific T cell immunity,released anti-tumor effector molecules,effectively remodeled the tumor microenvironment,and significantly inhibited tumor growth.7)In the 4T1 model,pretreatment with Clodronate Liposomes significantly inhibited the effect of IFN-γ BBV intervention on tumor growth.In addition,tumor CTL and Th epitope-specific effector T cell responses were significantly reduced in tumor tissues,indicating that the anti-tumor effect mediated by IFN-γ BBV intervention was dependent on macrophages.8)Tumor growth curve data showed that anti-PD-1 mAb alone was inefficient in treating"cold"tumors 4T1,while the combination of IFN-γ BBV and anti-PD-1 mAb significantly inhibited the growth of in situ breast tumors and lung metastasis compared to the single intervention strategy.Conclusion:This thesis shows that genetically recombinantly modified BBV can efficiently present IFN-γ,while engineered IFN-γ BBV can target enrichment in tumor tissue,promote reprogramming of macrophages from M2 to M1 phenotype through TLR4 and IFNγ/JAK/STAT1 signaling pathways,release effector molecules such as pro-inflammatory cytokines and NO,promote tumor antigen-specific CTL response and inhibit immunosuppressive cells MDSCs and Treg response,thus remodeling the tumor microenvironment and the anti-tumor immunity which significantly inhibit tumor growth and metastasis.Macrophages play a crucial role in IFN-γ BBV-mediated anti-tumor effects,and the combination of IFN-γ BBV and anti-PD-1 mAb produced stronger therapeutic effects than single interventions.In conclusion,this thesis establishes a reprogramming strategy based on IFN-γ BBV targeting tumor tissue macrophages for modulation,providing a new means and idea for tumor immunotherapy.