Mesenchymal stem cell-derived exosomes enriched with miR-218 reduce the epithelial-mesenchymal transition and angiogenesis in triple-negative breast cancer cells.

BACKGROUND: The epithelial-mesenchymal transition (EMT) and angiogenesis are morphogenetic processes implicated in tumor invasion and metastasis. It is found that the aberrant expression of microRNAs (miRNAs) contributes to these processes. Exosomes are considered potential natural vehicles for miRNA delivery in cancer therapy. miR-218 is one of the tumor suppressor miRNAs and its downregulation is associated with EMT and angiogenesis. We aimed to use adipose mesenchymal stem cells-derived exosomes (ADMSC-exosomes) for miR-218 delivery to breast cancer cells and evaluate miR-218 tumor-suppressing properties in vitro. METHODS: Exosomes were isolated from conditioned media of ADMSCs. miR-218 was loaded to exosomes using electroporation. mRNA expression of target genes (Runx2 and Rictor) in MDA-MB-231 breast cancer cells was evaluated by qPCR. To explore the effects of miR-218 containing exosomes on breast cancer cells, viability, apoptosis, and Boyden chamber assays were performed. The angiogenic capacity of MDA-MB-231 cells after treatment with miR-218 containing exosomes was assessed by in vitro tube formation assay. RESULTS: miR-218 mimic was efficiently loaded to ADMSC-exosomes and delivered to MDA-MB-231 cells. Exposure to miR-218 containing exosomes significantly decreased miR-218 target genes (Runx2 and Rictor) in MDA-MB-231 cells. They increased the expression of epithelial marker (CDH1) and reduced mesenchymal marker (CDH2). miR-218 restoration using miR-218 containing exosomes reduced viability, motility, invasion, and angiogenic capacity of breast cancer cells. CONCLUSIONS: These findings suggest that ADMSC-exosomes can efficiently restore miR-218 levels in breast cancer cells and miR-218 can prevent breast cancer progression with simultaneous targeting of angiogenesis and EMT.

A systems biology approach and in vitro experiment indicated Rapamycin targets key cancer and cell cycle-related genes and miRNAs in triple-negative breast cancer cells.

An anticancer drug known as Rapamycin acts by inhibiting the mammalian target of the Rapamycin pathway. This agent has recently been investigated for its potential therapeutic benefits in sensitizing drug-resistant breast cancer (BC) treatment. The molecular mechanism underlying these effects, however, is still a mystery. Using a systems biology method and in vitro experiment, this study sought to discover essential genes and microRNAs (miRNAs) targeted by Rapamycin in triple-negative BC (TNBC) cells to aid prospective new medications with less adverse effects in BC treatment. We developed the transcription factor-miRNA-gene and protein-protein interaction networks using the freely accessible microarray data sets. FANMOD and MCODE were utilized to identify critical regulatory motifs, clusters, and seeds. Then, functional enrichment analyses were conducted. Using topological analysis and motif detection, the most important genes and miRNAs were discovered. We used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the effect of Rapamycin on the expression of the selected genes and miRNAs to verify our findings. We performed flow cytometry to investigate Rapamycin's impact on cell cycle and apoptosis. Furthermore, wound healing and migration assays were done. Three downregulated (PTGS2, EGFR, VEGFA) and three upregulated (c-MYC, MAPK1, PIK3R1) genes were chosen as candidates for additional experimental verification. There were also three upregulated miRNAs (miR-92a, miR-16, miR-20a) and three downregulated miRNAs (miR-146a, miR-145, miR-27a) among the six selected miRNAs. The qRT-PCR findings in MDA-MB-231 cells indicated that c-MYC, MAPK1, PIK3R1, miR-92a, miR-16, and miR-20a expression levels were considerably elevated following Rapamycin treatment, whereas PTGS2, EGFR, VEGFA, miR-146a, and miR-145 expression levels were dramatically lowered (p < 0.05). These genes are engaged in cancer pathways, transcriptional dysregulation in cancer, and cell cycle, according to the top pathway enrichment findings. Migration and wound healing abilities of the cells declined after Rapamycin treatment, and the number of apoptotic cells increased. We demonstrated that Rapamycin suppresses cell migration and metastasis in the TNBC cell line. In addition, our data indicated that Rapamycin induces apoptosis in this cell line. The discovered vital genes and miRNAs affected by Rapamycin are anticipated to have crucial roles in the pathogenesis of TNBC and its therapeutic resistance.

Overexpression of pigment epithelium-derived factor in breast cancer cell-derived exosomes induces M1 polarization in macrophages.

M2 macrophages, the major component of tumor microenvironment, are recognized as important player in tumor progression. M2 macrophages mediate this effect by promoting tumor angiogenesis, tumor metastasis, and suppression of tumor immunity. Reprogramming of M2 macrophages can serve as a promising strategy in cancer immunotherapy. In this study, we constructed pigment epithelium-derived factor (PEDF) expressing vector and transfected MDA-MB-231 cells with this construct. Then, exosomes were isolated from transfected cells and M2 macrophages were incubated with isolated exosomes from transfected cell. The effect of isolated exosomes on macrophage polarization was examined by real-time PCR and ELISA. The results demonstrated reprogramming of M2 macrophages after incubation with isolated exosomes from PEDF transfected cells. M2-to-M1 repolarization of macrophages was confirmed by upregulation of CD80, IRF5, MCP1, and IL-1ß and repression of CD206, Arg, TGM2, and TGF-ß. Therefore, these findings revealed that introducing PEDF into exosomes by genetic manipulation of parent cells may be a potential approach for reprogramming of M2 macrophages in cancer.

Evaluating the influence of Human Umbilical Cord Mesenchymal Stem Cells-derived exosomes loaded with miR-3182 on metastatic performance of Triple Negative Breast Cancer cells.

AIMS: Deregulation of microRNA (miRNA) function has been linked to numerous human cancers, such as Triple Negative Breast Cancer (TNBC). Exosomes, a subgroup of extracellular vehicles (EVs), can efficiently deliver many different cargo types to the target cell and have an extensive role in delivering therapeutic cargo for treatment. The present study intended to interrogate the effects of exosomal delivery of miR-3182 on TNBC cellular processes. MAIN METHODS: Human Umbilical Cord Mesenchymal Stem Cells (HUCMSCs) were cultured and exosomes were isolated and characterized using TEM, SEM, DLS, and Western blot. Exosomes were transfected with miR-3182 and added to the treatment groups. The expression level of miR-3182 and their target genes including mTOR and S6KB1 were evaluated using RT-qPCR. The effects of miR-3182 loaded HUCMSC-exosomes treatment on the cellular aspect of MDA-MB-231 cells including their viability, migration potency, cell cycle status and apoptosis were investigated. KEY FINDINGS: According to the results, exosomal miR-3182 significantly abolished cell proliferation and migration (P < 0.05). miR-3182 loaded exosomes also induced apoptosis in TNBC cells by down-regulating mTOR and S6KB1 genes (P < 0.05). SIGNIFICANCE: In nutshell, miR-3182-loaded HUCMSC-exosomes can suppress TNBC invasion, suggesting that exosomes containing miR-3182 could be a reliable therapeutic paradigm in TNBC therapy.

Transfer of miRNA in tumor-derived exosomes suppresses breast tumor cell invasion and migration by inducing M1 polarization in macrophages.

AIMS: Macrophage repolarization from M1 to M2 phenotype is one of the hallmarks of malignancy. M2 macrophages are the most represented population in the tumor microenvironment and play an active role in tumor progression. In recent years, microRNAs (miRNAs) have been identified as a regulator of macrophage polarization. MAIN METHODS: In this study, miR-130 was delivered to M2 macrophages using tumor-derived exosomes. Then, we evaluated the macrophage polarization status by assessment of specific markers and cytokines for M1 and M2 phenotype. The phagocytosis ability of macrophages was also investigated. Additionally, we performed migration and invasion assays to detect the effect of macrophage reprogramming on breast cancer cells migration and invasion. KEY FINDINGS: The findings of the current study indicated that exosomes efficiently delivered miR-130 into macrophages. Delivery of miR-130 into macrophages resulted in upregulation of M1 specific markers and cytokines, including CD86, Irf5, Nos2, TNF-α, and IL-1ß and downregulation of M2 specific markers and cytokines, including CD206, Ym1, Arg, TGF-ß, and IL-10. The phagocytosis ability of macrophages also enhanced after treatment with miRNA-loaded exosomes. Furthermore, migration and invasion assays demonstrated reduced ability of 4T1 breast cancer cells for migration and invasion after macrophages reprogramming. SIGNIFICANCE: These observations suggest that repolarization of M2 macrophages to M1 phenotype using miRNA-containing exosomes can be a therapeutic strategy against tumor invasion and metastasis in breast cancer.

Delivery of miR-381-3p Mimic by Mesenchymal Stem Cell-Derived Exosomes Inhibits Triple Negative Breast Cancer Aggressiveness; an In Vitro Study.

Recent investigations have emphasized the role of aberrant expression of microRNAs (miRNAs) in progression of almost all types of cancers. Exosomes, membrane-enclosed natural nanovesicles, transport cellular contents, including proteins, mRNAs, and miRNAs, between cells. Unique features of exosomes make them an appropriate carrier for drug delivery. miRNA-381 is one of the downregulated miRNAs in several cancers including triple-negative breast cancer (TNBC) and restoration of its expression in TNBC cells can restrict their migratory ability through targeting several signaling pathways. In current study, we exploited the exosomes isolated from adipose-derived mesenchymal stem cells (ADMSC-exosomes) to deliver miR-381 mimic to MDA-MB-231 cells to elucidate their effects on TNBC cells. The effects of miR-381 loaded ADMSC-exosomes on proliferation, apoptosis, migration, and invasion of MDA-MB-231 cells were analyzed. Our results indicated that ADMSC-exosomes were successfully isolated and internalized by MDA-MB-231 cells. miR-381 mimic was efficiently delivered to MDA-MB-231 cells by ADMSC-exosomes. miR-381 loaded ADMSC-exosomes significantly downregulated the expression of epithelial to mesenchymal transition (EMT) related genes and proteins. Notably, miR-381 loaded ADMSC-exosomes inhibited proliferation, migration, and invasion capacity of MDA-MB-231 and promoted their apoptosis in vitro. Taken together, we showed that ADMSC-exosomes could be used as efficient nanocarriers for RNA-based therapies. Graphical abstract.

Effect of mesenchymal stem cells-derived exosomes on tumor microenvironment: Tumor progression versus tumor suppression.

Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into different cell types. Owing to their immunosuppressive and anti-inflammatory properties, they are widely used in regenerative medicine, but they have a dual effect on cancer progression and exert both growth-stimulatory or -inhibitory effects on different cancer types. It has been proposed that these controversial effects of MSC in tumor microenvironment (TME) are mediated by their polarization to proinflammatory or anti-inflammatory phenotype. In addition, they can polarize the immune system cells that in turn influence tumor progression. One of the mechanisms involved in the TME communications is extracellular vesicles (EVs). MSCs, as one of cell populations in TME, produce a large amount of EVs that can influence tumor development. Similar to MSC, MSC-EVs can exert both anti- or protumorigenic effects. In the current study, we will investigate the current knowledge related to MSC role in cancer progression with a focus on the MSC-EV content in limiting tumor growth, angiogenesis, and metastasis. We suppose MSC-EVs can be used as safe vehicles for delivering antitumor agents to TME.