Boosting Anti-PD-1 Therapy With Metformin-Loaded Macrophage-Derived Microparticles

Programmed cell death 1(PD-1)checkpoint blockade therapy has demonstrated encouraging clinical outcomes in numerous cancer types.However,the lack of sufficient T cell infiltration,tumor immunosuppressive microenvironment,and inadequate tumor accumulation and penetration of anti-PD-1 antibody due to complex physical barriers in tumors led to the resistance of anti-PD-1 therapy.Thus,how to resolve the above problems to overcome the resistance of anti-PD-1 antibody remains a big challenge.M2-like tumor-associated macrophages(TAMs),one of the most abundant tumor infiltrating leukocytes,play a crucial part in facilitating tumor growth,metastasis,and immune escape.Repolarization of M2-like TAMs is a promising strategy to enhance T cell-mediated antitumor immunity and ameliorate the tumor immunosuppressive microenvironment,which might contribute to the enhanced anticancer activity of anti-PD-1 antibody.In this study,in view of the natural tumor-targeting capacity of macrophages and high expression of mannose receptor CD206/MRC1 in M2-like TAMs,mannose(Man)-modified macrophage-derived microparticles(Man-MPs)loading metformin(Met@Man-MPs)are developed.And the antitumor efficacy and mechanism of Met@Man-MPs combining with anti-PD-1 antibody are investigated in detail.The main research contents and results are as follows:1.The preparation,characterization and M2-like TAMs repolarization capacity of Met@Man-MPsMurine or human macrophages were incubated with DSPE-PEG-Man to obtain Man-engineered cells.Man-engineered macrophages were then treated with Met,followed by ultraviolet irradiation for Met-packaging Man-MPs(denoted as Met@Man-MPs)production.The diameter of Met@Man-MPs was about 440 nm,and zeta potential was about-11.6 m V.The drug loading capacity for Met@Man-MPs was about 0.69μg of Met perμg protein.Met@Man-MPs were relatively stable in physiological conditions.Met@Man-MPs efficiently targeted to M2-like TAMs,resulting in the repolarization into M1-like phenotype.The Met@Man-MPs-repolarized M2-like TAMs efficiently enhanced the cytotoxicity and phagocytosis of tumor cells as well as decreased the tumor formation and growth of the co-implanted H22 cells.2.Antitumor effects and regulation of tumor immune microenvironment induced by Met@Man-MPsMet@Man-MPs exhibited strong anticancer activity in subcutaneous H22 tumor-bearing mice and orthotopic 4T1 breast tumor-bearing mice.Moreover,Met@Man-MPs obviously inhibited the lung metastasis of 4T1 tumors and prolonged the survival time of tumor-bearing mice without significant side effects.In vivo studies showed that Met@Man-MPs significantly decreased the numbers of M2-like TAMs in tumors of H22 tumor-bearing mice.Meanwhile,Met@Man-MPs significantly increased the numbers of M1-like TAMs,CD8~+T cells and activated CD8~+CD69~+T cells,while decreased the numbers of myeloid-derived suppressor cells(MDSCs)and regulatory T cells(Tregs).Furthermore,Met@Man-MPs promoted the secretion of pro-inflammation cytokines,and reduced the level of anti-inflammatory,resulted in the improvement of tumor immune microenvironment.3.Mechanisms of Met@Man-MPs-mediated infiltration of CD8~+T cells and the enhanced accumulation and penetration of anti-PD-1 antibody in tumor tissuesMet@Man-MPs expressed MMP9(Matrix metalloproteinase 9)and MMP14,exhibiting tumor collagen degradation capacity.Meanwhile,Met@Man-MPs-reset M2-like macrophages efficiently recruited CD8~+T cells by upregulated TNF-α.The Met@Man-MPs-induced degradation of tumor collagen and recruitment of CD8~+T cells contributed to the infiltration of CD8~+T cells into tumor tissue and the increased number of cytotoxic CD8~+CD69~+T cells and CD8~+IFN-γ~+T cells.Meanwhile,the collagen degradation of Met@Man-MPs significantly enhanced tumor accumulation and penetration of anti-PD-1antibody.4.Antitumor effect of the combination of Met@Man-MPs and anti-PD-1 antibodyThe combination of Met@Man-MPs and anti-PD-1 antibody generated remarkable anticancer activity in subcutaneous H22,orthotoptc 4T1 tumor-bearing mice,and azoxymethane(AOM)/dextran sodium sulfate(DSS)-induced colitis-associated cancer.Meanwhile,combination of Met@Man-MPs and anti-PD-1 antibody also showed strongest synergistic anticancer effects in organotypic ex vivo slice culture model of fresh human HCC tumors.Furthermore,the combination treatment remodeled tumor immune microenvironment by decreasing the ratio of M2-like TAMs and increasing the ratio of M1-like TAMs,CD8~+T cells as well as the activated cytotoxic CD8~+T cells.Moreover,the combination treatment evoked long-term antitumor memory immunity against tumor recurrence.In summary,our results showed that Met@Man-MPs efficiently target M2-like TAMs to repolarize into M1-like phenotype,leading to the recruitment of CD8~+T cells into tumor tissues and the remodeled tumor immunosuppressive microenvironment.More importantly,the collagen-degrading capacity of Met@Man-MPs contributes to the infiltration of CD8~+T cells into tumor interiors and the enhanced tumor accumulation and penetration of anti-PD-1 antibody.With these unique features,Met@Man-MPs efficiently improved the anticancer activity of anti-PD1 antibody therapy,inducing long-term memory immunity.Our results support Met@Man-MPs as a potential drug to improve tumor resistance to anti-PD-1 therapy.