Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems.
ABSTRACT: Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy.
Project description:Multiple studies show that chronic lymphocytic leukemia (CLL) cells are heavily dependent on their microenvironment for survival. Communication between CLL cells and the microenvironment is mediated through direct cell contact, soluble factors, and extracellular vesicles. Exosomes are small particles enclosed with lipids, proteins, and small RNAs that can convey biological materials to surrounding cells. Our data herein demonstrate that CLL cells release significant amounts of exosomes in plasma that exhibit abundant CD37, CD9, and CD63 expression. Our work also pinpoints the regulation of B-cell receptor (BCR) signaling in the release of CLL exosomes: BCR activation by ?-immunoglobulin (Ig)M induces exosome secretion, whereas BCR inactivation via ibrutinib impedes ?-IgM-stimulated exosome release. Moreover, analysis of serial plasma samples collected from CLL patients on an ibrutinib clinical trial revealed that exosome plasma concentration was significantly decreased following ibrutinib therapy. Furthermore, microRNA (miR) profiling of plasma-derived exosomes identified a distinct exosome microRNA signature, including miR-29 family, miR-150, miR-155, and miR-223 that have been associated with CLL disease. Interestingly, expression of exosome miR-150 and miR-155 increases with BCR activation. In all, this study successfully characterized CLL exosomes, demonstrated the control of BCR signaling in the release of CLL exosomes, and uncovered a disease-relevant exosome microRNA profile.
Project description:Extracellular vesicles, including exosomes, have recently been implicated as novel mediators of immune cell communication in mammals. However, roles for endogenously produced exosomes in regulating immune cell functions in vivo are just beginning to be identified. In this article, we demonstrate that Rab27a and Rab27b double-knockout (Rab27DKO) mice that are deficient in exosome secretion have a chronic, low-grade inflammatory phenotype characterized by elevated inflammatory cytokines and myeloproliferation. Upon further investigation, we found that some of these phenotypes could be complemented by wild-type (WT) hematopoietic cells or administration of exosomes produced by GM-CSF-expanded bone marrow cells. In addition, chronically inflamed Rab27DKO mice had a blunted response to bacterial LPS, resembling endotoxin tolerance. This defect was rescued by bone marrow exosomes from WT, but not miR-155-/-, cells, suggesting that uptake of miR-155-containing exosomes is important for a proper LPS response. Further, we found that SHIP1 and IRAK-M, direct targets of miR-155 that are known negative regulators of the LPS response, were elevated in Rab27DKO mice and decreased after treatment with WT, but not miR-155-/-, exosomes. Together, our study finds that Rab27-dependent exosome production contributes to homeostasis within the hematopoietic system and appropriate responsiveness to inflammatory stimuli.
Project description:Exosomes, membranous nanovesicles, naturally carry bio-macromolecules and play pivotal roles in both physiological intercellular crosstalk and disease pathogenesis. Here, we showed that B cell-derived exosomes can function as vehicles to deliver exogenous miRNA-155 mimic or inhibitor into hepatocytes or macrophages, respectively. Stimulation of B cells significantly increased exosome production. Unlike in parental cells, baseline level of miRNA-155 was very low in exosomes derived from stimulated B cells. Exosomes loaded with a miRNA-155 mimic significantly increased miRNA-155 levels in primary mouse hepatocytes and the liver of miRNA-155 knockout mice. Treatment of RAW macrophages with miRNA-155 inhibitor loaded exosomes resulted in statistically significant reduction in LPS-induced TNF? production and partially prevented LPS-induced decrease in SOCS1 mRNA levels. Furthermore, exosome-mediated miRNA-155 inhibitor delivery resulted in functionally more efficient inhibition and less cellular toxicity compared to conventional transfection methods. Similar approaches could be useful in modification of target biomolecules in vitro and in vivo. From the clinical editor: In this study, exosome-based delivery of miRNA-155 mimicker or inhibitor was found to have significant biological response in hepatocytes and macrophages. Exosome-based approaches may be useful in the modification of other target biomolecules.
Project description:BACKGROUND:Exosomes are extracellular vesicles containing a variety of biological molecules including microRNAs (miRNAs). We have recently demonstrated that certain miRNA species are selectively and highly enriched in pancreatic cancer exosomes with miR-1246 being the most abundant. Exosome miRNAs have been shown to mediate intercellular communication in the tumor microenvironment and promote cancer progression. Therefore, understanding how exosomes selectively enrich specific miRNAs to initiate exosome miRNA signaling in cancer cells is critical to advancing cancer exosome biology. RESULTS:The aim of this study was to identify RNA binding proteins responsible for selective enrichment of exosome miRNAs in cancer cells. A biotin-labeled miR-1246 probe was used to capture RNA binding proteins (RBPs) from PANC-1 cells. Among the RBPs identified through proteomic analysis, SRSF1, EIF3B and TIA1 were highly associated with the miR-1246 probe. RNA immunoprecipitation (RIP) and electrophoretic mobility shift assay (EMSA) confirmed the binding of SRSF1 to miR-1246. Lentivirus shRNA knockdown of SRSF1 in pancreatic cancer cells selectively reduced exosome miRNA enrichment whereas GFP-SRSF1 overexpression enhanced the enrichment as analyzed by next generation small RNA sequencing and qRT-PCR. miRNA sequence motif analysis identified a common motif shared by 36/45 of SRSF1-associated exosome miRNAs. EMSA confirmed that shared motif decoys inhibit the binding of SRSF1 to the miR-1246 sequence. CONCLUSIONS:We conclude that SRSF1 mediates selective exosome miRNA enrichment in pancreatic cancer cells by binding to a commonly shared miRNA sequence motif. Video Abstract.
Project description:Breast cancer remains the most prevalent cause of cancer mortality in woman worldwide due to the metastatic process and therapy resistance. Resistance against cancer therapy is partially attributed to cancer stem cells (CSCs). These cells arise from epithelial cells undergoing epithelial-to-mesenchymal transition (EMT) and might be responsible for tumor recurrence. In this study, we reported the relevance of miR-155 upregulation in chemoresistant cells associated with EMT. Notably, we found miR-155 induction in exosomes isolated from CSCs and resistant cells, followed by resistant cells' exosome transfer to the recipient sensitive cells. Functionally, miR-155 mimic assay showed an enrichment in miR-155 from exosome concomitant with miR-155 exosome transfer to breast cancer cells. In parallel to these effects, we also observed EMT change in miR-155 transfected cells. The chemoresistance phenotype transfer to sensitive cells and the migration capability was analyzed by MTT and scratch assays and our results suggest that exosomes may intermediate resistance and migration capacity to sensitive cells partly through exosome transfer of miR-155. Taken together, our findings establish the significance of exosome-mediate miR-155 chemoresistance in breast cancer cells, with implications for targeting miR-155 signaling as a possible therapeutic strategy.
Project description:The cancer drug gemcitabine (GEM) is a key drug for treating pancreatic ductal adenocarcinoma (PDAC), but PDAC cells develop chemoresistance after long-term administration. Since the tolerance was immediately spread to every PDAC tissue in a patient, it is assumed that some certain efficient mechanisms underlay in the development of chemoresistance. Changes in the levels of particular microRNAs or alterations in intercellular communication play a dominant role in chemoresistance development, and recent data also suggest that exosomes play an important role in this process. In this study, we revealed that the loop conferred chemoresistance in PDAC cells. The loop was as follows; 1, The long-term exposure of GEM increased miR-155 expression in PDAC cells. 2, The increase of miR-155 induced two different functions; exosome secretion and chemoresistance ability via facilitating the anti-apoptotic activity. 3, Exosome deliver the miR-155 into the other PDAC cells and induce the following function. The target therapy to miR-155 or the exosome secretion effectively attenuated the chemoresistance, and these results were validated with both clinical samples and in vivo experiments. This mechanism represents a novel therapeutic target in GEM treatment to PDAC.
Project description:Rationale: Exosomes are small extracellular vesicles secreted by most cells that are found in blood and other bodily fluids, and which contain cytoplasmic material and membrane factors corresponding to their cell type of origin. Exosome membrane factors and contents have been reported to alter adjacent and distant cell behavior in multiple studies, but the impact of cancer-derived exosomes on chemoresistance is less clear. Methods: Exosomes isolated from three pancreatic cancer (PC) cell lines displaying variable gemcitabine (GEM) resistance (PANC-1, MIA PaCa-2, and BxPC-3) were tested for their capacity to transmit chemoresistance among these cell lines. Comparative proteomics was performed to identify key exosomal proteins that conferred chemoresistance. Cell survival was assessed in GEM responsive PC cell lines treated with recombinant Ephrin type-A receptor 2 (EphA2), a candidate chemoresistance transfer factor, or exosomes from a chemoresistant PC cell line treated with or without EphA2 shRNA. Results: Exosomes from chemoresistant PANC-1 cells increased the GEM resistance of MIA PaCa-2 and BxPC-3 cell cultures. Comparative proteomics determined that PANC-1 exosomes overexpressed Ephrin type-A receptor 2 (EphA2) versus exosomes of less chemoresistant PC cell lines MIA PaCa-2 and BxPC-3. EphA2-knockdown in PANC-1 cells inhibited their ability to transmit exosome-mediated chemoresistance to MIA PaCa-2 and BxPC-3, while treatment of MIA PaCa-2 and BxPC-3 cells with soluble EphA2 did not promote chemoresistance, indicating that membrane carried EphA2 was important for the EphA2 chemoresistance effect. Conclusion: Exosomal EphA2 expression could transmit chemoresistance and may potentially serve as a minimally-invasive predictive biomarker for PC treatment response. Further work should address whether additional exosomal factors regulate resistance to other cancer therapeutic agents for PC or other cancer types.
Project description:Circulating miRNAs can be found in extracellular vesicles (EV) and could be involved in intercellular communication. Here, we report the biodistribution of EV associated miR-155 using miR-155 KO mouse model. Administration of exosomes loaded with synthetic miR-155 mimic into miR-155 KO mice resulted in a rapid accumulation and clearance of miR-155 in the plasma with subsequent distribution in the liver, adipose tissue, lung, muscle and kidney (highest to lowest, respectively). miR-155 expression was detected in isolated hepatocytes and liver mononuclear cells of recipient KO mice suggesting its cellular uptake. In vitro, exosome-mediated restoration of miR-155 in Kupffer cells from miR-155 deficient mice augmented their LPS-induced MCP1 mRNA increase. The systemic delivery of wild type plasma to miR-155 KO mice also resulted in a rapid accumulation of miR-155 in the circulation and distribution to the liver and adipose tissue. In summary, our results demonstrate tissue biodistribution and biologic function of EV-associated miR-155.
Project description:Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.
Project description:Exosomes are nanovesicles (30-100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a few dozen to thousands of different proteins. First crude exosome preparations from four placentas (normal pregnancy) were obtained here using several standard centrifugations but then were additionally purified by gel filtration on Sepharose 4B. Individual preparations demonstrated different gel filtration profiles showing good or bad separation of exosome peaks from two peaks of impurity proteins and their complexes. According to electron microscopy, exosomes before gel filtration contain vesicles of different size, ring-shaped structures forming by ferritin and clusters of aggregated proteins and their complexes. After filtration through 220 nm filters and gel filtration exosomes display typically for exosome morphology and size (30-100 nm) and do not contain visible protein admixtures. Identification of exosome proteins was carried out by MS and MS/MS MALDI mass spectrometry of proteins' tryptic hydrolyzates after their SDS-PAGE and 2D electrophoresis. We have obtained unexpected results. Good, purified exosomes contained only 11-13 different proteins: CD9, CD81, CD-63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, serotransferin, and probably human serum albumin and immunoglobulins. We assume that a possible number of exosome proteins found previously using crude preparations may be very much overestimated. Our data may be important for study of biological functions of pure exosomes.