Experimental Study On The Therapeutic Activity Of Nanodiamond-polyglycerol-doxorubicin Composites Against Triple-negative Breast Cancer

Background: Triple-negative breast cancer(TNBC)has the poorest prognosis of all breast cancer subtypes and is one of the most fatal diseases for women.Because conventional endocrine therapy and molecular targeted therapy are ineffective,classic chemotherapy such as anthracycline doxorubicin(DOX)is still an important treatment for TNBC.In addition to its direct toxicity to tumor cells,the efficacy of DOX is also related to its immune effect.However,DOX also has significant problems of severe systemic toxicity and tumor chemoresistance.Immunotherapy is an emerging tumor treatment strategy,but tumor-induced systemic and local tumor immunosuppression poses a great hindrance to efficacious anti-TNBC immunotherapy.To solve the above problems,our study attempted to use a doxorubicin-polyglycerol-nanodiamond conjugates(Nano-DOX)with immunostimulation activity synthesized by us in the early stage to achieve "immunochemotherapy" for the TNBC model.The use of nanoparticles to deliver chemotherapeutic drugs to the tumor is considered to have many advantages and broad application prospects,but what are the differences between the nanoscale chemotherapeutic drugs and the prototype drugs in terms of cellular effects,efficacy,toxicity and drug resistance? The issue has yet to be explored in depth.Objective: Starting from tumor cells and their immune microenvironment,a comparative study was conducted on Nano-DOX and DOX in terms of anti-tumor efficacy,toxicity,immune regulation and tumor drug resistance,with a view to provide new strategies and ideas for the treatment of TNBC.Methods: Using mouse TNBC cells(4T1)and TNBC tumor-bearing mice as models,in vitro cell experiments and animal experiments were carried out,with DOX as the control:(1)To investigate the anti-TNBC efficacy and toxicity of Nano-DOX.(2)To investigate the effect of Nano-DOX on TNBC-induced systemic immunosuppressive and tumor local immunosuppressive microenvironment.(3)The endogenous mechanism(ATP binding cassette(ABC)transporters,autocrine cytokine IL-6)and environmental mechanism(paracrine IL-6,CXCL1,GM-CSF and myeloid-derived suppressor cells(MDSCs))of tumor cell resistance to DOX were used as entry points to investigate whether Nano-DOX induced TNBC drug resistance.Results:(1)DOX can induce apoptosis of TNBC cells,while Nano-DOX inhibits proliferation of TNBC cells but does not induce apoptosis of tumor cells.The therapeutic effect of Nano-DOX was weaker than that of DOX when tumor volume change was taken as index.However,DOX has significant systemic toxicity,and immune cells are particularly sensitive to DOX toxicity,while Nano-DOX has low systemic toxicity,and immune cells such as macrophages,dendritic cells,lymphocytes,and MDSCs have good tolerance to Nano-DOX.(2)Nano-DOX was shown to downregulate tumor-derived granulocyte-colony stimulating factor(G-CSF)and suppresses the induction and tissue filtration of MDSCs that are the principal effectors of TNBC-associated systemic immunosuppression.Nano-DOX also alleviated the phenotype of MDSCs induced by TNBC cells.At the same time,Nano-DOX can also stimulate TNBC cells to emit endogenous immune adjuvant---damage associated molecular patterns(DAMPs),which in turn stimulates macrophage-mediated innate anti-tumor immunity and dendritic cells-driven,lymphocytes-mediated adaptive anti-tumor immune response.(3)Up-regulation of ATP-binding cassette(ABC)transporters,especially P-glycoprotein(P-gp),multidrug resistance-related protein 1(MRP1)and breast cancer resistance protein(BCRP),is a key mechanism of TNBC cells resistance to DOX,while Nano-DOX does not induce drug resistance mediated by these transporters in TNBC.(4)DOX can stimulate TNBC cells to release IL-6,CXCL1 and GM-CSF by activating STAT3,NF-κB,MAPK signals.These cytokines exert autocrine and paracrine effects to promote TNBC resistance to DOX: 1)IL-6 can promote TNBC cells to resist DOX-induced apoptosis;2)GM-CSF can promote MDSCs to resist the toxicity of DOX,CXCL1 and GM-CSF can enhance the recruitment of tumor to MDSCs,while IL-6 can promote the release of IL-6 from MDSCs and act on TNBC cells,thereby enhancing their resistance to DOX.However,Nano-DOX does not induce drug resistance mediated by the above mechanism in TNBC.Conclusion: The effect,efficacy and toxicity of Nano-DOX on TNBC cells were significantly different from that of DOX.Nano-DOX has a significant immune regulatory effect and can reverse the TNBC-induced systemic immunosuppression and the local tumor immunosuppressive microenvironment.Nano-DOX can evade DOX resistance mediated by tumor cell endogenous mechanism and MDSCs environmental mechanism in TNBC.In conclusion,Nano-DOX provides a new strategy and means for TNBC immunochemotherapy.