Synergistic Therapeutic Strategy Of Macrophage Immunotherapy And Tumor Ferroptosis In Cancer

The morbidity and mortality of tumors are increasing rapidly worldwide.In particular,the recurrence and metastasis of solid tumors are one of the leading causes of death.Tumor therapy includes both direct action on the tumor and indirect modulation of the tumor microenvironment,thereby sensitizing tumor cells to treatment,with overlap and interactions between direct and indirect modification strategies.Cancer immunotherapy is a promising approach to activate a patient’s immune system to fight cancer.Tumor-associated macrophages(TAMs)are critical in the tumor microenvironment.Mainly divided into 2 types,namely the M2 type that promotes tumor growth and a small amount of inflammatory anti-tumor M1 type macrophages.Remodeling of TAMs to an "immunostimulatory" M1 phenotype offers an opportunity to reverse the tumor microenvironment.In this article,we designed 2 treatment systems for primary,recurrent and metastatic tumors,and we hope to make new breakthroughs in tumor combination therapy or immunotherapy.In Chapter 1,we designed a novel multifunctional nanotherapeutic platform based on nanotechnology.Purpose: The system is based on superparamagnetic iron oxide nanoparticles core,and the surface is coated with biomimetic nano-hydrogels,and the immune adjuvant Cp G ODNs and DOX are effectively loaded,thereby realizing the synergistic therapeutic effect of immune regulation,efficient ferroptosis and chemotherapy of tumors,and revealed the synergistic therapeutic synergistic mechanism.Methods: Preparation of iron oxide nanoparticles and composite nanoparticles;particle size,morphology and drug loading rate were characterized by dynamic light scattering(DLS),transmission electron microscopy and UV absorption spectroscopy.The endocytosis and distribution of nanoparticles observed by laser confocal microscope under different conditions;CCK8 assay and flow cytometry were used to study the killing and combined therapeutic effects of nanoparticles on tumor cells;hydrogen peroxide detection,flow cytometry,Western blotting,Enzymelinked immunosorbent assay,etc.were used to explore the remodeling effect of macrophages;CCK8 assay,flow cytometry,western blotting,immunofluorescence,etc.,were used to study the ferroptosis-inducing effect of nanoparticles on tumor cells.Animal-level tumor-inhibitory effect study: tumor growth curve,immunofluorescence,H&E staining,etc.were used to verify in situ tumor inhibition and ferroptosis-inducing effect;flow cytometry was used to verify the effect of nanoparticles on macrophage remodeling in tumor microenvironment and systemic immune regulation.Recurrence and distant models: tumor growth curve,flow cytometry was used to verify the inhibitory and immune memory effects of nanoparticles on recurrent and distant tumors;blood metastasis model: small animal in vivo imaging,H&E staining,etc.were used to verify that nanoparticles inhibit tumor blood lung metastasis effect.Results: The composite nanoparticles continuously release the loaded effective molecules in response to the tumor microenvironment,which remodeled the M2 type of macrophages into the tumor-killing M1 type to release killing factors,to reverse the immunosuppressive microenvironment,and increase the sensitivity of chemotherapy and effectively kill tumor cells.In addition,ferromagnetic particles induce ferroptosis in tumor cells and replenish tumor antigens in the tumor microenvironment,thereby activating adaptive immune responses and long-lasting immune effects to achieve significant inhibit primary,recurrence,and metastasis of tumors.Conclusion: We systematically demonstrate the strong potential of the synergy of macrophage immunomodulation,tumor ferroptosis,and chemotherapy as a novel tumor treatment modality.In Chapter 2,we prepared a multifunctional,natural orthotopic tumor vaccine against postoperative tumor recurrence and metastasis.Purpose:We prepared fused cell membranes from M1 macrophages and tumor cells,and co-encapsulated them into calcium alginate hydrogels with immune adjuvant Cp G ODNs to develop a tumor vaccine FM+@N,and explored its immune regulation and tumor recurrence inhibitory effect.Methods:The classical PEG method was used to fuse mouse bone marrow-derived M1 macrophages and 4T1 breast tumor cells.Gradient centrifugation was used to obtain the fusion membrane,and the tumor vaccine FM+@N was prepared by physical cross-linking of calcium ions.Fluorescence imaging,flow cytometry,western blotting,etc.were used to verify fusion cells;CCK8 assay,flow cytometry,fluorescence imaging were used to study the killing effect of fusion membrane on tumor cells;flow cytometry,western blotting,enzyme-linked immunosorbent assay,etc.were used to expounded the macrophage remodeling effect;flow cytometry was used to verify the fusion membrane antigen presentation effect;imaging,scanning electron microscopy,swelling experiments,CCK8 tests,etc.were used to verify the successful preparation of the vaccine.Animal-level tumor suppressive effect study: tumor growth curve,in vivo imaging,immunofluorescence,H&E staining,etc.were used to explore the tumor suppressive effect of in situ recurrence;flow cytometry was used to verify the tumor vaccine on tumor microenvironment macrophage remodeling and systemic immune regulation effect.Distant models: Tumor growth curve and flow cytometry were used to explore tumor vaccine inhibition on distant tumors,macrophage remodeling,immune memory effects.Results: As an effective "immune molecule library",FM+@N vaccine continuously releases effective molecule of FM+,to remodel tumorassociated macrophages M2 type into M1 type macrophages,and realize the suppression of recurrent tumors.The dead tumor cells and the tumor cell membrane antigens(in the fused membrane)to achieve selfsupplementation of tumor antigens in the tumor microenvironment,activate an effective adaptive immune response,and improve the immunosuppression of the tumor microenvironment.In addition,the memory immune effect was activated to achieve a long-lasting immune response and inhibited the distant metastasis of tumors.Conclusion: The multifunctional,natural and effective hybrid membrane vaccine achieves the inhibition of primary recurrence and distant metastasis of tumors,opening a new field of macrophage-related vaccines.