PKH67-labeled Exosomes Research Articles

The Exosomal Long Noncoding RNA aHIF is Upregulated in Serum From Patients With Endometriosis and Promotes Angiogenesis in Endometriosis.

The transfer of long noncoding RNAs (lncRNAs) via exosomes to modulate recipient cells represents an important mechanism for disease progression. Antisense hypoxia-inducible factor (aHIF) is a well-known angiogenesis-related lncRNA. Here, we aimed to investigate the clinical implications of aHIF and exosomal aHIF in endometriosis and the involvement of exosome-shuttled aHIF in endometriosis angiogenesis. The distribution and expression of aHIF in ectopic, eutopic, and normal endometria was evaluated. Serum exosomal aHIF levels in patients with endometriosis were tested. The correlation between serum exosomal aHIF and aHIF expression in ectopic endometria was analyzed. Endometriotic cyst stromal cells (ECSCs)-derived exosomes were characterized. The internalization of exosomes by human umbilical vein endothelial cells (HUVECs) was observed. A series of in vitro assays were conducted to investigate the roles and mechanisms of exosomal aHIF in endometriosis angiogenesis. Clinically, aHIF was highly expressed in ectopic endometria and serum exosomes in patients with endometriosis. Serum exosomal aHIF was significantly correlated to aHIF expression in matched ectopic endometria. In vitro, PKH67-labeled exosomes derived from aHIF high expression ECSCs were effectively internalized by recipient HUVECs. Notably, exosome-shuttled aHIF was transferred from ECSCs to HUVECs, which in turn elicited proangiogenic behavior in HUVECs by activating vascular endothelial growth factor (VEGF)-A, VEGF-D, and basic fibroblast growth factor, thereby facilitating endometriosis angiogenesis. Our study illustrates a potential cell-cell communication between ECSCs and HUVECs in an ectopic environment, provides a novel mechanistic model explaining how ECSCs induce angiogenesis from the perspective of the "exosomal transfer of aHIF," and highlights the clinical value of circulating exosomal aHIF in endometriosis.

The release of tryptase from mast cells promote tumor cell metastasis via exosomes

BackgroundCancer cells release exosomes and can be taken up by mast cells (MCs), but the potential functional effects of MCs on tumor metastasis remain unknown.MethodExosomes were isolated from the lung adenocarcinoma cell line A549, and the uptake of PKH26-labeled exosomes by bone marrow MCs was examined via flow cytometry and fluorescence microscopy. Cytokines and tryptase in MC supernatant were analyzed using an ELISA kit, and the presence of tryptase was evaluated by Western blotting. Cell proliferation and migration were determined through CCK-8 and transwell assays. Proteins in the tryptase-JAK-STAT signaling pathway were detected by Western blotting.ResultsIn this study, we show that exosomes from A549 cells can be taken up by MCs. Moreover, A549 exosomes contain stem cell factor (SCF) to MCs and subsequently induce the activation of MCs through SCF-KIT signal transduction, which leads to MC degranulation and the release of tryptase. Tryptase accelerates the proliferation and migration of human umbilical vein endothelial cells (HUVECs) through the JAK-STAT signaling pathway.ConclusionsOur results reveal a mechanism for metastasis in which exosomes can transfer SCF to and activate MCs, which can affect the release of tryptase and the angiogenesis of HUVECs.

Exosomes derived from microRNA-30b-3p-overexpressing mesenchymal stem cells protect against lipopolysaccharide-induced acute lung injury by inhibiting SAA3

Mesenchymal stem cells (MSCs) have been widely documented for their potential role in the treatment of various clinical disorders, including acute lung injury (ALI). ALI represents a clinical syndrome associated with histopathological diffuse alveolar damage. Recent evidence has demonstrated that exosomes derived from MSCs may serve as a reservoir of anti-apoptotic microRNAs (miRs) conferring protection from certain diseases. Hence, the current study was performed with the aim of investigating whether MSCs-exosomal miR-30b-3p could confer protection against ALI. A bioinformatic analysis and a dual luciferase assay were initially performed to verify that SAA3 was highly-expressed in ALI which was confirmed to be a target gene of miR-30b-3p. Next, the lipopolysaccharide (LPS)-treated type II alveolar epithelial cells (AECs) (MLE-12) were transfected with mimics or inhibitors of miR-30b-3p, or sh-SAA3. It was revealed that LPS induced AEC apoptosis, which could be inhibited by overexpressing miR-30b-3p by down-regulating the expression of SAA3. After co-culture of PKH26-labeled exosomes with MLE-12 cells, we found that the number of PKH26-labeled exosomes endocytosed by MLE-12 cells gradually increased. Furthermore, the LPS-treated MLE-12 cells co-cultured with MSC-exosomes overexpressing miR-30b-3p exhibited increased miR-30b-3p, decreased SAA3 level, as well as increased cell proliferation, accompanied by diminished cell apoptosis in LPS-treated MLE-12 cells. Finally, the protective effect of MSCs-exosomal miR-30b-3p on the AECs in vivo was investigated in an ALI mouse model with tail vein injection of MSC-exosomes with elevated miR-30b-3p, showing that overexpression of miR-30b-3p in MSC-exosomes conferred protective effects against ALI. Taken together, these findings highlighted the potential of MSC-exosomes overexpressing miR-30b-3p in preventing ALI. The exosomes derived from MSCs hold potential as future therapeutic strategies in the treatment of ALI.

Circulating exosomes regulate T-cell-mediated inflammatory response in oral lichen planus.

Exosomes are newly recognized natural nanocarrier and intercellular messenger that emerge as important mediators of signal transmission. Exosomes have been reported to modulate the inflammatory response of a number of diseases. This study investigated the effects of circulating exosomes from oral lichen planus (OLP) on T cells. Plasma-derived exosomes were purified from both OLP patients and control groups. T cells were observed under a confocal laser scanning microscope after co-cultivation with PKH67 labeled exosomes for 12, 24, and 48hours. The effects of exosomes exposure on T cells were analyzed with several functional assays, investigating proliferation, apoptosis, and migration. Production of interleukin (IL)-2, -4, -10, and interferon (IFN)-γ was measured via enzyme-linked immunosorbent assay. PKH67-labeled exosomes were taken up by T cells in a time- and dose-dependent manner. Several biological functions of T cells were promoted. In particular, the circulating erosive OLP exosomes significantly enhanced T-cell proliferation and attenuated the apoptosis. The migration capacity of T cells increased remarkably in response to erosive OLP exosome treatment. In addition, the ratio of IFN-γ/IL-4 was significantly elevated in OLP patients. Our findings indicate that the circulating OLP exosomes are involved in the biological functions of T cells, potentially promoting the OLP progression by regulating the T-cell-mediated inflammatory response.

Cluster of Differentiation 46 Is the Major Receptor in Human Blood-Brain Barrier Endothelial Cells for Uptake of Exosomes Derived from Brain-Metastatic Melanoma Cells (SK-Mel-28).

Brain metastasis is a frequent complication of cancer and may be mediated, at least in part, by the internalization of cancer-cell-derived exosomes into brain capillary endothelial cells. Clarifying the mechanism(s) of this internalization is of interest because it could help us to develop ways to block brain metastasis, as well as affording a potential new route for drug delivery into the brain. Therefore, the purpose of the present study was to address this issue by identifying the receptors involved in the internalization of exosomes derived from a brain-metastatic cancer cell line (SK-Mel-28) into human blood-brain barrier endothelial cells (hCMEC/D3 cells). The combination of sulfo-SBED-based cross-linking and comprehensive proteomics yielded 20 proteins as exosome receptor candidates in hCMEC/D3 cells. The uptake of PKH67-labeled exosomes by hCMEC/D3 cells measured at 37 °C was significantly reduced by 95.6% at 4 °C and by 15.3% in the presence of 1 mM RGD peptide, an integrin ligand. Therefore, we focused on the identified RGD receptors, integrin α5 and integrin αV, and CD46, which is reported to act as an adenovirus receptor, together with integrin αV. A mixture of neutralizing antibodies against integrin α5 and integrin αV significantly decreased the exosome uptake by 11.8%, while application of CD46 siRNA reduced it by 39.0%. Immunohistochemical analysis confirmed the presence of CD46 in human brain capillary endothelial cells. These results suggest that CD46 is a major receptor for the uptake of SK-Mel-28-derived exosomes by human blood-brain barrier endothelial cells (hCMEC/D3 cells).

PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.

Exosomes isolated from sera of mice fed Lactobacillus strains affect inflammatory cytokine production in macrophages in vitro

Orally administered Lactobacillus strains, including L. plantarum No.14 and L. rhamnosus GG, reportedly reduce inflammatory cytokine production in mice. The present study tested our idea that circulating exosomes mediate the action of Lactobacillus strains. The lipopolysaccharide-induced production of TNF-α and IL-6 in vitro was attenuated in peritoneal exudate cells (PECs) isolated from C57BL/6N mice that had been fed L. plantarum No.14. When PECs were cultured for 24 h with exosomes isolated from the serum of mice fed L. plantarum No.14 or L. rhamnosus GG, accumulation of both TNF-α and of the corresponding mRNA was lowered. Growth in the presence of these exosomes also decreased the production of TNF-α and IL-6 by the murine macrophage cell line RAW264.7. In contrast, supplementation with exosome-depleted serum of mice fed L. plantarum No.14 or L. rhamnosus GG failed to affect the production of TNF-α and IL-6 by RAW264.7 cells. When PECs and RAW264.7 cells were cultured for 24 h with PKH67-labeled exosomes isolated from murine serum, fluorescent signal was observed inside the cells, suggesting that these cells incorporate serum exosomes. We propose that the anti-inflammatory activity of orally administered L. plantarum No.14 and L. rhamnosus GG is mediated, at least in part, by circulating exosomes.

Uptake of lymphoma-derived exosomes by peripheral blood leukocytes.

Exosomes are nanosized lipid vesicles secreted into blood and other body fluids and serve as vehicles for intercellular communication. Despite being an important component of the tumor microenvironment (TME), exosomal targeting and uptake into recipient cells are still not fully understood. Few studies have looked at lymphoma exosomes and their interactions with circulating blood cells. In this study, we examine the exosomal uptake distribution among peripheral blood leukocytes (PBLs) using vesicles derived from a diffuse large B cell lymphoma cell line, WSU-DLCL2. Lymphoma cells survive, proliferate, and are protected from the cytotoxic effects of chemotherapeutic agents by soluble factors or by direct contact with inflammatory and stromal cells within the TME. In an attempt to close the gap in knowledge concerning lymphoma TME immunosuppression, we have treated normal human PBLs with PKH67-labeled lymphoma exosomes and monitored the uptake by measuring fluorescence at different time points using flow cytometry and fluorescent microscopy. Our results show that of the four populations examined, B cells and monocytes demonstrated uptake of PKH67-labeled exosomes, while T cells and NK cells displayed significantly less uptake.

Exosomes from iPSCs Delivering siRNA Attenuate Intracellular Adhesion Molecule-1 Expression and Neutrophils Adhesion in Pulmonary Microvascular Endothelial Cells.

The pro-inflammatory activation of pulmonary microvascular endothelial cells resulting in continuous expression of cellular adhesion molecules, and subsequently recruiting primed neutrophils to form a firm neutrophils-endothelium (PMN-EC) adhesion, has been examined and found to play a vital role in acute lung injury (ALI). RNA interference (RNAi) is a cellular process through harnessing a natural pathway silencing target gene based on recognition and subsequent degradation of specific mRNA sequences. It opens a promising approach for precision medicine. However, this application was hampered by many obstacles, such as immunogenicity, instability, toxicity problems, and difficulty in across the biological membrane. In this study, we reprogrammed urine exfoliated renal epithelial cells into human induced pluripotent stem cells (huiPSCs) and purified the exosomes (Exo) from huiPSCs as RNAi delivery system. Through choosing the episomal system to deliver transcription factors, we obtained a non-integrating huiPSCs. Experiments in both vitro and vivo demonstrated that these huiPSCs possess the pluripotent properties. The exosomes of huiPSCs isolated by differential centrifugation were visualized by transmission electron microscopy (TEM) showing a typical exosomal appearance with an average diameter of 122nm. Immunoblotting confirmed the presence of the typical exosomal markers, including CD63, TSG 101, and Alix. Co-cultured PKH26-labeled exosomes with human primary pulmonary microvascular endothelial cells (HMVECs) confirmed that they could be internalized by recipient cells at a time-dependent manner. Then, electroporation was used to introduce siRNA against intercellular adhesion molecule-1 (ICAM-1) into exosomes to form an Exo/siRNA compound. The Exo/siRNA compound efficiently delivered the target siRNA into HMVECs causing selective gene silencing, inhibiting the ICAM-1 protein expression, and PMN-EC adhesion induced by lipopolysaccharide (LPS). These data suggest that huiPSCs exosomes could be used as a natural gene delivery vector to transport therapeutic siRNAs for alleviating inflammatory responses in recipient cells.

Circulating Exosomes Carrying an Immunosuppressive Cargo Interfere with Adoptive Cell Therapy in Acute Myeloid Leukemia

Introduction: Adoptive immunotherapy, including transfer of activated NK cells (aNK), is currently under active investigation for patients with refractory/relapsed acute myeloid leukemia (AML). However, the highly immunosuppressive microenvironment in patients with AML may create a major barrier for adoptive immunotherapy. Exosomes, virus-size (30-100 nm) membrane-bound vesicles found in all body fluids, are produced in abundance by leukemic blasts and are strongly immunosuppressive. The presence of exosomes in the plasma of relapsed/refractory AML patients could impair anti-leukemia activity of adoptively transferred aNK cells. To test this hypothesis, we evaluated ex vivo effects of exosomes isolated from plasma of AML patients on the activity of aNK cells which were used for adoptive immunotherapy in a Phase 1 clinical trial (NCT00900809) for patients with relapsed/refractory AML performed at our institution.Methods: Venous blood (20-50 mL) was obtained from patients with refractory/relapsed AML (n=7) prior to and after therapy with adoptively-transferred aNK cells. Exosomes were isolated from the patients’ plasma as well as normal controls’ (NC) plasma by size exclusion chromatography on Sepharose 2B. Exosome numbers and size (qNano), morphology (TEM) and protein levels were determined. The presence of exosome-related markers (Tsg101, CD81), myeloid cell-surface markers associated with AML (CD123, CLL-1, CD44, CD96) and of immunosuppressive proteins (CD39, CD73, PD-1/ PDL-1, Fas/FasL, TGF-β1) in exosomes was studied by Western blots. The AML plasma-derived exosomes were co-incubated with normal human NK cells or aNK cells used for therapy. Up-take of PKH26-labeled exosomes by recipient aNK cells was monitored by confocal microscopy and changes in NKG2D expression levels on aNK cells or in NK-cell cytotoxicity by multiparameter flow cytometry. Exosome-induced signaling via receptors (A2AR, TGFβR, CD95 or PD-1) on aNK cells was studied by Western blots ± inhibitors of each relevant pathway.Results: The protein levels of exosome fractions in plasma of refractory/relapsed AML patients prior to adoptive therapy with aNK cells were significantly higher (70 ± 15 μg protein/mL) than those isolated from plasma of NCs (35±2.7 μg protein/mL) (P<0.05). Exosome protein levels remained persistently elevated after therapy with the aNK cells. The molecular profile of exosomes in AML patients’ plasma was distinct from that of exosomes in plasma of NCs; in addition to leukemia-associated antigens (LAA), AML exosomes were enriched in TGF-β1 (LAP), CD39 and CD73 ectoenzymes, PD-L1 and FasL. Co-incubation of these AML exosomes with the aNK cells that express cognate surface receptors for the above listed ligands or ectoenzymes down-regulated survival and lytic functions of aNK cells. Pharmacologic inhibitors or blocking anti-PD1 mAbs and anti-TGF-β1 mAbs prevented apoptosis and partly abrogated dysfunction of aNK cells. Inhibitory in vivo effects of the high plasma exosome levels in plasma of AML patients was demonstrated by the absence of evidence for up-regulation of NK cytotoxicity in the peripheral blood of the patients receiving adoptive therapy with aNK cells.Conclusions: Persistently elevated levels of biologically-active immunosuppressive exosomes carrying leukemia-associated antigens and various immunosuppressive proteins in plasma of patients with relapsed/refractory AML interfere with anti-leukemia activity of adoptively transferred aNK cells. Blast-derived exosomes emerge as a significant deterrent to a success of adoptive immunotherapies with activated immune cells. DisclosuresNo relevant conflicts of interest to declare.

Immunosuppressive effect of mesenchymal stem cell-derived exosomes on a concanavalin A-induced liver injury model.

BackgroundThis study aimed to evaluate the effect of mesenchymal stem cell (MSC)-derived exosomes on an immune-induced liver injury model. MSCs show a unique function to modulate immune reaction although the molecular mechanisms are still under investigation. Exosomes are a nanoparticle containing microRNA and many ligands and are recognized as important factors secreted from MSC to express their function. This research is undertaken to evaluate the effect of MSC-derived exosome on concanavalin-A (con-A)-induced liver injury.MethodsExosomes were collected from the supernatant of MSC from the bone marrow of C57B6 mice with ultracentrifugation. The collected exosomes or MSCs were injected intravenously into liver injury mice that had been prepared by the intravenous con-A injection. Liver and serum samples were collected 24 h later to evaluate the macro- and microscopic images, the alanine aminotransferase (ALT), and cytokine messenger RNA (mRNA) expression levels. Phenotypical change of non-parenchymal liver cells was also evaluated by flow cytometry. Liver localization of PKH26 after the injection of PKH26-labeled exosomes or MSCs was observed by microscope. Each result was statistically analyzed with Student’s t test.ResultsPKH was observed in the liver after PKH-labeled exosomes were injected into mouse, whereas it was only observed in the lung in a mouse group receiving PKH-leveled MSC. There were decreases in ALT, liver necrotic areas, and the extent of apoptosis indicated by the single-stranded DNA index of groups that received multiple injections of MSC-derived exosomes, but an increase in the Ki-67 index. The mRNA expression of anti-inflammatory cytokines was enhanced. The number of Treg was increased among NPCs in a group receiving exosomes multiple times.ConclusionsSuppression of con-A-induced liver injury by injection of exosomes was observed as same extent as MSC. Considering the advantage of exosomes as its non-living nature and dosing adjustability over MSC, exosome will be one alternative of MSC transplantation.

Regulation of exosomes released from normal ovarian epithelial cells and ovarian cancer cells.

As important cell to cell communicator, exosomes carry a range of bioactive molecules which can significantly influence phenotype of recipient cells. Inhibiting or removing cancer cell-derived exosomes are of therapeutic interest. However, regulation of secretion and release mechanism of exosomes is still unclear. To explore the regulation of exosomes released from normal ovarian epithelial cells and ovarian cancer cells, a normal ovarian epithelial cell line and three ovarian epithelial cancer cell lines were utilized to investigate their exosomes' release and regulation. A cervical cancer cell SiHa was used for identifying tissue specificity. NanoSight NS500 was used to quantify exosome numbers. Exosomes were labeled and observed by confocal microscopy to investigate their interaction with different ovarian cell lines. Exosomes released from normal or ovarian cancer cells were regulated by the extracellular exosomes. Exosome release was inhibited with the extracellular exosome concentration increase. Exosomes from normal ovarian cell and cervical cancer cell also inhibited ovarian cancer cell-derived exosome release, and there was no tissue specificity. PKH26-labeled exosomes from normal ovarian cell and cervical cancer cell were uptaken by ovarian cancer cells. Release of exosomes from ovarian cancer cell is regulated by a feedback mechanism without tissue specificity. This may provide a therapeutic approach to control the release of exosomes from ovarian cancer cells.

Role of Phosphatidylserine-Derived Negative Surface Charges in the Recognition and Uptake of Intravenously Injected B16BL6-Derived Exosomes by Macrophages

Exosomes are cell-derived extracellular vesicles that function as intercellular delivery carriers. Our previous study demonstrated that macrophages in the liver contributed to the rapid clearance of intravenously administered B16BL6-derived exosomes from the systemic circulation in mice. Phosphatidylserine (PS) may be responsible for this clearance because it is exposed on the surface of exosomes and is recognized by macrophages. In this study, the role of PS exposed on the membranes of exosomes in the uptake of B16BL6-derived exosomes by macrophages was investigated. Negatively charged PS- or phosphatidylglycerol-loaded liposomes suppressed the cellular uptake of PKH67-labeled exosomes by macrophages, whereas phosphatidylcholine-containing liposome did not affect uptake. Subsequently, for the in vivo analysis, exosomes were labeled with Gaussia luciferase, a reporter protein, or (3-125I-iodobenzoyl)norbiotinamide using exosome-tropic fusion proteins comprising the exosome-tropic protein lactadherin. The blood clearance of Gaussia luciferase-labeled exosomes after intravenous injection into mice was significantly delayed by the preinjection of PS- or phosphatidylglycerol-containing liposomes. Moreover, the accumulation of (3-125I-iodobenzoyl)norbiotinamide-labeled exosomes in the liver was decreased by the preinjection of PS-containing liposomes. These results indicate that the negative charge of PS in exosomal membranes is involved in the recognition and clearance of intravenously injected exosomes by macrophages.

Abstract 357: Exosome-mediated transfer of alphaV integrins promotes prostate cancer cell-cell communication

Abstract Intercellular communications have been recently shown to be mediated by exosomes, exocytosed vesicles of endosomal origin, which are thought to be involved in cancer progression. We have investigated whether integrins, transmembrane receptors that are known to be deregulated in cancer progression, are transferred among different subsets of prostate cancer cells through exosomes and have the ability to support functional aberrations in the recipient cells. Recent studies have characterized integrin expression in exosomes but have not investigated whether integrins are actually shuttled to recipient cells and whether they modulate the phenotype of these cells. We have focused on the alphaVbeta3 and alphaVbeta6 integrins since they are highly up-regulated in cancer and metastasis. We show here that these alphaV integrins are present in exosomes of several prostate cancer cells and are transferred from donor to recipient cells. The quality of our exosome preparations was tested by electron microscopy, which confirms the size range (40-100 nm) and the typical cup shape of these vesicles; by continuous sucrose gradient, which shows a density range of 1.15-1.17g/ml characteristic of exosomes, and by biochemical characterization using antibodies to CD63 and CD81. After exosome internalization was determined by confocal microscopy of red PKH26-labeled exosomes followed by Z-stack image analysis, these integrins are shown to be transferred as proteins to recipient cells by immunoblotting. Furthermore, FACS analysis demonstrated that these integrins are localized to the cell surface indicating they are functional. The active state of alphaVbeta6 was confirmed in cell migration assays which show that the alphaVbeta6 integrin transferred through exosomes mediates cell migration of recipient cells on an alphaVbeta6 specific ligand, latency-associated peptide-TGFBeta. To evaluate the relevance of our findings, we purified exosomes from sera of TRAMP mice which had developed prostate cancer. The results show that the alphaVbeta3 integrin is expressed in these exosomes and is a potential biomarker for prostate cancer diagnostic purposes. Overall, this study shows that alphaV integrins are transferred through exosomes to recipient cells and promote cell migration on specific ligands, thus, suggesting that this novel pathway may lead to increased prostate cancer metastasis in distinct distant sites. Supported by NIH-CA089720 and P01CA140043 to LRL; an American-Italian Cancer Foundation Post-Doctoral Research Fellowship to CF; a JGSBS Alumni Association Graduate Student Travel Fellowship to AS. Citation Format: Amrita Singh, Carmine Fedele, Renato V. Iozzo, Lucia R. Languino. Exosome-mediated transfer of alphaV integrins promotes prostate cancer cell-cell communication. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 357. doi:10.1158/1538-7445.AM2015-357

Mast cell exosomes promote lung adenocarcinoma cell proliferation - role of KIT-stem cell factor signaling.

BackgroundHuman cells release nano-sized vesicles called exosomes, containing mRNA, miRNA and specific proteins. Exosomes from one cell can be taken up by another cell, which is a recently discovered cell-to-cell communication mechanism. Also, exosomes can be taken up by different types of cancer cells, but the potential functional effects of mast cell exosomes on tumor cells remain unknown.Methods and resultsExosomes were isolated from the human mast cell line, HMC-1, and uptake of PKH67-labelled exosomes by the lung epithelial cell line, A549, was examined using flow cytometry and fluorescence microscopy. The RNA cargo of the exosomes was analyzed with a Bioanalyzer and absence or presence of the c-KIT mRNA was determined by RT-PCR. The cell proliferation was determined in a BrdU incorporation assay, and proteins in the KIT-SCF signaling pathway were detected by Western blot. Our result demonstrates that exosomes from mast cells can be taken up by lung cancer cells. Furthermore, HMC-1 exosomes contain and transfer KIT protein, but not the c-KIT mRNA to A549 cells and subsequently activate KIT-SCF signal transduction, which increase cyclin D1 expression and accelerate the proliferation in the human lung adenocarcinoma cells.ConclusionsOur results indicate that exosomes can transfer KIT as a protein to tumor cells, which can affect recipient cell signaling events through receptor-ligand interactions.

Monocyte Exosomes Stimulate the Osteogenic Gene Expression of Mesenchymal Stem Cells

Inflammation and regeneration at the implant-bone interface are intimately coupled via cell-cell communication. In contrast to the prevailing view that monocytes/macrophages orchestrate mesenchymal stem cells (MSCs) and progenitor cells via the secretion of soluble factors, we examined whether communication between these different cell types also occurs via exosomes. LPS-stimulated human monocytes released exosomes, positive for CD9, CD63, CD81, Tsg101 and Hsp70, as determined by flow cytometry and Western blot. These exosomes also contained wide size distribution of RNA, including RNA in the size of microRNAs. The exosomes were shown to interact with human mesenchymal stem cells. After 24 h of culture, a considerable portion of the MSCs had internalised PKH67-labelled exosomes. Furthermore, after 72 h, the gene expression of the osteogenic markers runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein-2 (BMP-2) had increased in comparison with control medium, whereas no significant difference in osteocalcin (OC) expression was demonstrated. The present results show that, under given experimental conditions, monocytes communicate with MSCs via exosomes, resulting in the uptake of exosomes in MSCs and the stimulation of osteogenic differentiation. The present observations suggest that exosomes constitute an additional mode of cell-cell signalling with an effect on MSC differentiation during the transition from injury and inflammation to bone regeneration.

A translational study of urine miRNAs in acute myocardial infarction

The currently used biomarkers for acute myocardial infarction (AMI) are blood creatinine phosphokinase-muscle band (CPK-MB), troponin-T (TnT), and troponin I (TnI). However, no good biomarkers are identified in urine after AMI, because these blood protein biomarkers are difficult to be filtered into urine. In this study, the role of urine microRNAs in the diagnosis of AMI and the mechanism involved were determined. We found that urine miR-1 was quickly increased in rats after AMI with peak at 24h after AMI, in which an over 50-fold increase was demonstrated. At 7days after AMI, the urine miR-1 level was returned to the basal level. No miR-208 was found in normal urine. In urine from rats with AMI, miR-208 was easily detected. To determine the mechanism involved, we determined the levels of heart-released miR-1 in the liver, spleen and kidney after AMI in rats and found that the kidney was an important metabolic organ. To determine the renal elimination of blood miRNAs, we isolated serum exosomes from rats after AMI and injected these exosomes into the circulating blood of normal rats. We found that the urine miR-1 was significantly increased in exosome-injected animals. Moreover, PKH67-labeled exosomes injected into circulating blood could enter into the kidney tissues and cells, as well as urine. Furthermore, the levels of urine miR-1 were significantly increased in patients with AMI. The results suggest that urine miRNAs such as miR-1 could be novel urine biomarkers for AMI.

Exosomes secreted from human colorectal cancer cell lines contain mRNAs, microRNAs and natural antisense RNAs, that can transfer into the human hepatoma HepG2 and lung cancer A549 cell lines

Exosomes are microvesicles that are released from various cells into the extracellular space. It has been reported that the components within exosomes vary according to the type of secreted cell. In the present study, we investigated the tetraspanin family proteins CD63, CD9 and CD81 as useful collection markers of exosomes derived from the three colorectal cancer (CRC) cell lines HCT-15, SW480 and WiDr. In addition, we aimed to detect the mRNAs, microRNAs and natural antisense RNAs within the exosomes secreted from the three CRC cell lines. Furthermore, we examined whether exosomes containing their RNAs were transferred into the hepatoma cell line HepG2 and lung cancer cell line A549. CD81 was detected in exosomes secreted from the three CRC cell lines. This result indicates that CD81 can be a collection marker of exosomes derived from the three CRC cell lines. When the RNA species within exosomes derived from the three CRC cell lines were examined, the mRNAs of housekeeping genes such as ACTB and GAPDH, the microRNAs such as miR-21, miR-192 and miR-221, and the natural antisense RNAs of LRRC24, MDM2 and CDKN1A genes, were detected. We discovered their natural antisense RNAs within exosomes for the first time in the present study. Furthermore, PKH67-labeled exosomes derived from the CRC cell lines were taken up into HepG2 and A549 cells. These findings indicate that the intracellular RNAs enclosed within exosomes are secreted to the outside, and exosomes derived from the CRC cell lines are transferred into HepG2 and A549 cells. In conclusion, we reveal that exosomes derived from the CRC cell lines contain mRNAs, microRNAs and natural antisense RNAs, and can be delivered into HepG2 and A549 cells. These findings indicate that exosomal RNAs can shuttle between cells, and may be involved in the regulation of gene expression in recipient cells.