Extracellular Vesicles Mediated MiR-424 Delivery In Treatment Of Triple-negative Breast Cancer

Breast cancer has become the second leading cause of cancer death among women after lung cancer in the world.Triple-negative breast cancer(TNBC)is a subpopulation characterized by a lack of expressions of estrogen receptors(ER),progesterone receptors(PR)and the amplification of human epidermal growth factor 2(HER2)and comprises 15-20% of all breast cancers.This subtype typically tends to be more aggressive with poor prognosis,strong invasiveness,early metastasis,high rates of relapse and rapid disease progression.There are no targeted therapies of TNBC due to the lack of ER,PR and HER2 which are usually emerged as targets.Surgery and chemotherapy are still the most common clinical treatment options for patients with TNBC,and offer disappointing clinical benefits.However,recent studies have reported that TNBC is the most immunomodulatory subtype since it has increased expression of programmed death ligand-1(PD-L1)and the existence of tumor-infiltrating lymphocytes(TILs)in the tumor microenvironment is clearly associated with clinical outcome and improved survival.These findings suggest that TNBC may show significant responsiveness to immunotherapy through blocking the PD-1/PD-l1 axis.Mesenchymal stem/stromal cells(MSCs)derived from adipose tissue(AT-MSCs)are adult stem cells with multi-differentiation potential,and are characteristically recruited to the hypoxic tumor microenvironment.They contribute to the disease progression through immunoregulation and interaction with cancer cells.Exosomes originating from endosomes with sizes ranging from 30-150 nm are released by all cell types and are found in almost all body fluids,including blood,saliva and urine.The contents of exosomes packaged from the lipid bilayer membrane consists of various types of lipids,proteins,and nucleic acids including mRNAs and miRNAs.MiRNAs is a non-coding single-stranded RNA containing 18-25 nucleotide sequences.Through binding to the 3’ untranslated region,miRNAs prevent translation or promote the degradation of mRNA.Due to the small size,miRNAs uploaded in exosomes can be transferred and regulate the expression of target genes in recipient cells;hence,they are involved in intercellular communication.In addition to natural ability to home in to tumor sites,exosomes inherit low immunogenicity with low expression of MHC class I from their parental MSCs.The composition of exosomes is based on their AT-MSC source and may facilitate the transport of exosomes to recipient breast cancer cells and the release of their miRNA cargos.Therefore,exosomes derived from AT-MSCs appear to be a promising miRNA drug delivery system for TNBC.In this study,we compared the expression levels of miR-424 and PD-L1 in different breast cancers and verified the relatively high expression of PD-L1 and the decreased expression of miR-424 in TNBC tumor tissues and TNBC cell lines.Furthermore,low miR-424 and high PDL1 expression were significantly correlated in TNBC.We predicted that miR-424 targeted the3’ UTR of PD-L1 according to an in-silico analysis and validated that the overexpression of miR-424 reduced PD-L1 expression through binding to the 3’-UTR.We compared the efficacy of different methods to upload miR-424 in exosomes.According to the miRNA expression patterns of exosomes derived from different types of MSCs,all four miRNAs including miR-424 that specifically expressed in exosomes derived from AT-MSCs theoretically targeted at PD-L1.We found that lipofectamine transfection can increase the miR-424 expression quickly and effectively compared to lentivirus transfection and electroporation.Moreover,it did not hurt the cell viability and secretion of exosomes.Finally,we established in-vitro and in-vivo models to investigate the anti-cancer function of EVs-424.The co-culture system of tumor cells and peripheral blood mononuclear cells(PBMCs)was used as the in-vitro model.After exposure to EVs-424,the amounts of inflammatory IFN-y and TNF-a were increased,while the amounts of immunosuppressive IL-6 and IL-10 were decreased in the medium.In addition,the proliferation rates of tumor cells were inhibited and the apoptosis of tumor cells was enhanced after incubation with EVs-424.Moreover,the numbers of CD8+ T cells were not decreased.To validate the clinical relevance,we subcutaneously transplanted MDA-MB-231-D3H2 LN cells along with stimulated PBMCs into BALB/c mice,followed by the intratumoral administration of EVs-424.It was observed that EVs-424 significantly slowed down the cancer progression in the mouse models.Our findings provide a novel approach for immune checkpoint PD-1/PD-L1 blockage via EVmediated miR-424 delivery that enhanced the immune responses against tumor cells in TNBC.