Cellular Uptake Of Exosomes Research Articles

Fluorescent Chiral Quantum Dots to Unveil Origin-Dependent Exosome Uptake and Cargo Release.

Exosomes are promising nanocarriers for drug delivery. Yet, it is challenging to apply exosomes in clinical use due to the limited understanding of their physiological functions. While cellular uptake of exosomes is generally known through endocytosis and/or membrane fusion, the mechanisms of origin-dependent cellular uptake and subsequent cargo release of exosomes into recipient cells are still unclear. Herein, we investigated the intricate mechanisms of exosome entry into recipient cells and intracellular cargo release. In this study, we utilized chiral graphene quantum dots (GQDs) as representatives of exosomal cargo, taking advantage of the superior permeability of chiral GQDs into lipid membranes as well as their excellent optical properties for tracking analysis. We observed that the preferential cellular uptake of exosomes derived from the same cell-of-origin (intraspecies exosomes) is higher than that of exosomes derived from different cell-of-origin (cross-species exosomes). This uptake enhancement was attributed to receptor-ligand interaction-mediated endocytosis, as we identified the expression of specific ligands on exosomes that favorably interact with their parental cells and confirmed the higher lysosomal entrapment of intraspecies exosomes (intraspecies endocytic uptake). On the other hand, we found that the uptake of cross-species exosomes primarily occurred through membrane fusion, followed by direct cargo release into the cytosol (cross-species direct fusion uptake). We revealed the underlying mechanisms involved in the cellular uptake and subsequent cargo release of exosomes depending on their cell-of-origin and recipient cell types. Overall, this study envisions valuable insights into further advancements in effective drug delivery using exosomes, as well as a comprehensive understanding of cellular communication, including disease pathogenesis.

Low‑intensity pulsed ultrasound accelerates diabetic wound healing by ADSC‑derived exosomes via promoting theuptake of exosomes and enhancing angiogenesis.

Diabetic wounds remain a great challenge for clinicians globally as a lack of effective radical treatment often results in poor prognosis. Exosomes derived from adipose‑derived stem cells (ADSC‑Exos) have been explored as an appealing nanodrug delivery system in the treatment of diabetic wounds. However, the short half‑life and low utilization efficiency of exosomes limit their therapeutic effects. Low‑intensity pulsed ultrasound (LIPUS) provides a non‑invasive mechanical stimulus to cells and exerts a number of biological effects such as cavitation and thermal effects. In the present study, whether LIPUS could enhance ADSC‑Exo‑mediated diabetic wound repair was investigated and its possible mechanism of action was explored. After isolation and characterization, ADSC‑Exos were injected into mice with diabetic wounds, then the mice were exposed to LIPUS irradiation. The control mice were subcutaneously injected with PBS. Wound healing assays, laser Doppler perfusion, Masson's staining and angiogenesis assays were used to assess treatment efficiency. Then, ADSC‑Exos were cocultured with human umbilical vein endothelial cells (HUVECs), and the proliferation, migration and tube formation of HUVECs were assessed. Moreover, the cellular uptake of ADSC‑Exos invitro and invivo was assessed to explore the synergistic mechanisms underlying the effects of LIPUS. The invivo results demonstrated that LIPUS increased the uptake of exosomes and prolonged the residence of exosomes in the wound area, thus enhancing angiogenesis and accelerating wound repair in diabetic mice. The invitro results further confirmed that LIPUS enhanced the uptake efficiency of ADSC‑Exos by 10.93‑fold and significantly increased the proliferation, migration and tubular formation of HUVECs. Therefore, the present study indicates that LIPUS is a promising strategy to improve the therapeutic effects of ADSC‑Exos in diabetic wounds by promoting the cellular uptake of exosomes and enhancing angiogenesis.

Exosomal miR-105-5p derived from bladder cancer stem cells targets for GPR12 to promote the malignancy of bladder cancer

Bladder cancer stem cells (BCSCs) are considered as the root cause of BC initiation and recurrence, and exosomes derived from BCSCs (CSCs-exo) are the vital tool for establishing a stable tumor microenvironment. miR-105-5p has been revealed to promote tumor growth in a variety of cancers, but the effects on BC are still not included.Characteristics of CSCs-exo were examined by transmission electron microscope and nanoparticle tracking analysis. PKH67 dye was used to observe the cellular uptake of exosomes. Cell viability, migration and invasion were detected by CCK-8, wound healing and transwell invasion assays, respectively. The interaction between miR-105-5p and GPR12 was verified by luciferase activity assay. Xenografts were induced in the nude mice, and H&E staining method was applied to analyze the histological changes of xenografts. CSCs-exo efficiently promoted BC cell viability, migration and invasion. miR-105-5p was highly expressed in CSCs and CSCs-exo treatment significantly upregulated the expression of miR-105-5p in BC cells.GPR12 was subsequently verified to be the target gene of miR-105-5p, and overexpression of GPR12 abrogated the effects of miR-105-5p on BC cell growth and metastasis. Reversely, the anti-tumor function of miR-105-5p antagomir was observed in the xenograft mice.CSCs aggravated the malignancy of BC partly through transmitting exosomal miR-105-5p to BC cells to inhibit the expression of GPR12, which developed a novel aspect for CSC-targeted therapies.

Role of tear exosomes in the spread of herpes simplex virus type 1 in recurrent herpes simplex keratitis.

Herpes simplex keratitis (HSK) is the most common but serious infectious keratitis with high recurrence. It is predominantly caused by herpes simplex virus type 1 (HSV-1). The spread mechanism of HSV-1 in HSK is not entirely clear. Multiple publications indicate that exosomes participate in the intercellular communication process during viral infections. However, there is rare evidence that HSV-1 spreads in HSK by exosomal pathway. This study aims to investigate the relationship between the spread of HSV-1 and tear exosomes in recurrent HSK. Tear fluids collected from total 59 participants were included in this study. Tear exosomes were isolated by ultracentrifugation, then identified by silver staining and western blot. The size was determined by dynamic light scattering (DLS). The viral biomarkers were identified by western blot. The cellular uptake of exosomes was studied using labelled exosomes. Tear exosomes were indeed enriched in tear fluids. Collected exosomes own normal diameters consistent with related reports. The exosomal biomarkers existed in tear exosomes. Labelled exosomes were successfully taken up by human corneal epithelial cells (HCEC) in large numbers in a short time. After cellular uptake, HSK biomarkers were detectable by western blot in infected cells. Tear exosomes should be the latent sites of HSV-1 in recurrent HSK and might be involved in the spread of HSV-1. Besides, this study verifies HSV-1 genes can be indeed transferred between cells by exosomal pathway, providing new inspiration for the clinical intervention and treatment as well as the drug discovery of recurrent HSK.

Fluorescence Spectroscopic Analysis of Lateral and Transbilayer Fluidity of Exosome Membranes.

Exosomes are small extracellular vesicles (sEVs) involved in distal cell-cell communication and cancer migration by transferring functional cargo molecules. Membrane domains similar to lipid rafts are assumed to occur in exosome membranes and are involved in interactions with target cells. However, the bilayer membrane properties of these small vesicles have not been fully investigated. Therefore, we examined the fluidity, lateral domain separation, and transbilayer asymmetry of exosome membranes using fluorescence spectroscopy. Although there were some differences between the exosomes, TMA-DPH anisotropy showing moderate lipid chain order indicated that ordered phases comprised a significant proportion of exosome membranes. Selective TEMPO quenching of the TMA-DPH fluorescence in the liquid-disordered phase indicated that 40-50% of the exosome membrane area belonged to the ordered phase based on a phase-separated model. Furthermore, NBD-PC in the outer leaflet showed longer fluorescence lifetimes than those in the inner leaflets. Therefore, the exosome membranes maintained transbilayer asymmetry with a topology similar to that of the plasma membranes. In addition, the lateral and transbilayer orders of exosome membranes obtained from different cell lines varied, probably depending on the different membrane lipid components and compositions partially derived from donor cells. As these higher membrane orders and asymmetric topologies are similar to those of cell membranes with lipid rafts, raft-like functional domains are possibly enriched on exosome membranes. These domains likely play key roles in the biological functions and cellular uptake of exosomes by facilitating selective membrane interactions with target organs.

Role of tumour derived exosomes in manuring the metastatic niche in patients with lung cancer liver metastasis: Beyond seed soil hypothesis

Metastatic lung cancer is often diagnosed at a late stage and is widely known to metastasize to the liver in the Asian population, but the underlying mechanism still remains unclear. Tumour derived exosomes (TDEs) play an important role in metastasis and its contributions to the development of the pre-metastatic niche formation is of utmost importance. In this study serum, derived tumour exosomes form lung cancer liver metastatic patients showed active incorporation by A549 cells in a concentration and time-dependent manner and induced migratory/invasive properties. Moreover, it was observed that cellular uptake of exosomes was increased during G2/M phase stimulating the cells to enter cell cycle phases leading to cell proliferation. Further, we observed that E-cadherin, beta catenin, VEGFA, CDKN2A and TGFBR2 were differentially expressed in treated A549 cells demonstrating an important role of these TDEs in altering the tumour microenvironment. In vivo model demonstrated an increase in serum SGPT and SGOT levels whereas the histopathological examination showed patches of pneumonitis in lungs and advanced inflammation in the liver. Conclusively, our results depict an undisputable role of these exosomes as key modulators in the formation of the pre-metastatic niche required for the colonization of circulating tumour cells (CTCs) ultimately leading to distant metastasis.

Oil Red O based method for exosome labelling and detection

With the realization of the role of exosomes in diseases, especially cancer, exosome research is gaining popularity in biomedical sciences. To understand exosome biology, their labelling and tracking studies are important. New and improved methods of exosome labelling for detection and tracking of exosomes need to be developed to harness their therapeutic and diagnostic potential. In this paper, we report a novel, simple and effective method of labelling and detecting exosomes using Oil Red O (ORO), a dye commonly used for lipid staining. Using ORO is a cost effective and easy approach with an intense red coloration of exosomes. Further, the issues faced with commonly used lipophilic dyes for exosome labelling like long-term persistence of dyes, aggregation and micelle formation of dyes, difficulty in distinguishing dye particles from labelled exosomes, and detection of large aggregates of dye or dye-exosome, are also resolved with ORO dye. This method shows good labelling efficacy with very sensitive detection and real-time tracking of the cellular uptake of exosomes.

Epidermal growth factor/epidermal growth factor receptor signaling blockage inhibits tumor cell-derived exosome uptake by oral squamous cell carcinoma through macropinocytosis.

Various cell types secrete exosomes into their surrounding extracellular space, which consequently affect the function and activity of recipient cells. Numerous studies have showed that tumor cell‐derived exosomes play important roles in tumor growth and progression. Although a variety of endocytic pathways are reportedly involved in the cellular uptake of exosomes, detailed mechanisms remain unknown. The present study demonstrated that treatment with recombinant epidermal growth factor (EGF) time‐ and dose‐dependently promoted cellular uptake of oral squamous cell carcinoma (OSCC) cell‐derived exosomes into OSCC cells themselves. Conversely, EGF receptor (EGFR) knockdown and treatment with EGFR inhibitors, including erlotinib and cetuximab, abrogated OSCC cell uptake of exosomes. The macropinocytosis inhibitor 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA) blocked the effects of active EGF/EGFR signaling on uptake of OSCC cell‐derived exosomes. These EGFR inhibitors also suppressed OSCC cell‐derived exosome‐induced proliferation, migration, invasion, stemness, and chemoresistance of OSCC cells. Taken together, the data presented herein suggest that EGFR inhibitors might inhibit the malignant potential of OSCC cells through direct inhibition of not only EGFR downstream signaling pathway but also cellular uptake of OSCC cell‐derived exosomes through macropinocytosis.

Human Bone Mesenchymal Stem Cell-Derived Exosomes Inhibit IL-1β-Induced Inflammation in Osteoarthritis Chondrocytes.

ObjectiveHuman bone marrow mesenchymal stem cell (hBMSC)-derived exosomes exhibit protective effects against inflammatory diseases. This study aimed to explore the effects of hBMSC-derived exosomes on osteoarthritis (OA) in vitro and its related mechanisms.Materials and Methods In this experimental study, we characterised exosomes derived from hBMSCs by transmission electron microscopy, nanoparticle tracking and Western blot analysis. Cellular uptake of exosomes was observed by fluorescent microscopy. Cell viability of chondrocytes exposed to interleukin-1 beta (IL-1β) was determined by the Cell Counting Kit-8 (CCK-8). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine expression levels of genes related to apoptosis, inflammation, cartilage collagen metabolism and mitogen-activated protein kinases. Results Fluorescence microscopy revealed that hBMSC-derived exosomes could be taken up by chondrocytes. hBMSC-derived exosomes could significantly enhance cell viability of chondrocytes in response to IL-1β treatment. RT-qPCR showed significant up-regulation of Survivin, Versican, IL-1β, IL-6, NF-κB, MMP-13, MAPK p38, JNK, ERK, Aggrecan and SOX9 expression levels by IL-1β treatment, while their mRNA expression levels decreased after co- culture with exosomes. The anti-inflammatory gene TGF-β was markedly suppressed by IL-1β treatment; however, we observed its expression after co-culture with exosomes. Additionally, the pro-inflammatory genes IL-1β, IL-6, NF-κB, TNF-α and TNF-β displayed significantly elevated expression levels in the IL-1β group and reduced expression levels after co-culture with exosomes. ConclusionhBMSC-derived exosomes may play a protective role in chondrocytes through inhibiting cell apoptosis and the inflammatory response. These results will provide a novel therapeutic strategy for OA.

Platelet membrane and stem cell exosome hybrids enhance cellular uptake and targeting to heart injury

Exosomes from mesenchymal stem cells have been widely studied as therapeutics to treat myocardial infarction. However, exosomes injected for therapeutic purposes face a number of challenges, including competition from endogenous exosomes, and the internalization/clearance by the mononuclear phagocyte system. There is also a lack of targeting. In this study, we hybridized stem cell-derived exosomes with platelet membranes to enhance their ability to target the injured heart and to reduce uptake by macrophages. Furthermore, we found that hybridization with platelet membranes induces macropinocytosis, enhancing the cellular uptake of exosomes by endothelial cells and cardiomyocytes drastically. In vivo studies showed the cardiac targeting ability of hybrid exosomes in a mouse model of myocardial infarction injury. Lastly, we determined cardiac functions and performed immunohistochemistry to confirm an enahnced therapeutic potency of platelet membrane modified exosomes as compared to non-modified exosomes. Our studies provide proof-of-concept data and a universal approach to enhance the binding and accumulation of exosomes in injured tissues.

Polyethylene Glycol 35 (PEG35) Modulates Exosomal Uptake and Function.

Polyethylene glycols (PEGs) are neutral polymers widely used in biomedical applications due to its hydrophilicity and biocompatibility. Exosomes are small vesicles secreted by nearly all cell types and play an important role in normal and pathological conditions. The purpose of this study was to evaluate the role of a 35-kDa molecular weight PEG (PEG35) on the modulation of exosome-mediated inflammation. Human macrophage-like cells THP-1, epithelial BICR-18, and CAPAN-2 cells were exposed to PEG35 prior to incubation with exosomes of different cellular origins. Exosome internalization was evaluated by confocal microscopy and flow cytometry. In another set of experiments, macrophages were treated with increasing concentrations of PEG35 prior to exposure with the appropriate stimuli: lipopolysaccharide, BICR-18-derived exosomes, or exosomes from acute pancreatitis-induced rats. Nuclear Factor Kappa B (NFκB) and Signal transducer and activator of transcription 3 (STAT3) activation and the expression levels of pro-inflammatory Interleukin 1β (IL1β) were determined. PEG35 administration significantly enhanced the internalization of exosomes in both macrophages and epithelial cells. Further, PEG35 ameliorated the inflammatory response induced by acute pancreatitis-derived exosomes by reducing the expression of IL1β and p65 nuclear translocation. Our results revealed that PEG35 promotes the cellular uptake of exosomes and modulates the pro-inflammatory effect of acute pancreatitis-derived vesicles through inhibition of NFκB, thus emphasizing the potential value of PEG35 as an anti-inflammatory agent for biomedical purposes.

Scavenger receptor MARCO contributes to cellular internalization of exosomes by dynamin-dependent endocytosis and macropinocytosis

Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor class-A protein that is expressed on the cell surface of macrophages. MARCO mediates binding and ingestion of unopsonized environmental particles, including nano-sized materials. Exosomes are cell-derived, nano-sized vesicles (40–150 nm) that can contain lipids, RNA, DNA, and various proteins. Exosomes play an essential role in cell-to-cell communication via body fluids. However, mechanisms for the recognition and internalization of exosomes by recipient cells remain poorly characterized. In this study, cellular association of serum-derived fluorescent exosomes and 20-nm fluorescent nanoparticles (positive control) was compared between MARCO-expressing (CHO-MARCO) and control (CHO-CT) CHO-K1 cells to examine whether MARCO expression by recipient cells mediates the cellular uptake of exosomes and environmental nanoparticles. Fluorescence microscopic studies and quantitative analyses revealed that the cellular associations of both exosomes and 20-nm nanoparticles were greater in CHO-MARCO cells than in CHO-CT cells. Exosomes and nanoparticles colocalized with green fluorescent protein (GFP)-MARCO in cells transfected with GFP-MARCO-encoding constructs . Furthermore, inhibitory studies showed that actin reorganization and dynamin are involved in the MARCO-mediated cellular internalization of exosomes. These results indicated that MARCO plays a role in the uptake of exosomes.

Exosome Characterization Utilizing the Immune System Based on the Interrupting Currents by Solid State Nanopore

Exosomes, which releases from all living cells, are nano-sized vesicles. They are well known to distribute between the cell communication because they circulate through a living organism as a carrier containing information derived from secretory and are incorporated into recipient cells. Exosomes derivative tumor cells are the main topic as a trigger because of containing its cell-specific intercellular protein and µRNA. Furthermore, cellular uptake of exosomes from tumor cells indicates that tumor cells are more accessible uptake into recipient cells depends on membrane protein and lipid composition than healthy cells.

Rapid On-Demand Extracellular Vesicle Augmentation with Versatile Oligonucleotide Tethers.

Exosomes show potential as ideal vehicles for drug delivery because of their natural role in transferring biological cargo between cells. However, current methods to engineer exosomes without negatively impacting their function remain challenging. Manipulating exosome-secreting cells is complex and time-consuming, while direct functionalization of exosome surface proteins suffers from low specificity and low efficiency. We demonstrate a rapid, versatile, and scalable method with oligonucleotide tethers to enable diverse surface functionalization on both human and murine exosomes. These exosome surface modifiers, which range from reactive functional groups and small molecules to aptamers and large proteins, can readily and efficiently enhance native exosome properties. We show that cellular uptake of exosomes can be specifically altered with a tethered AS1411 aptamer, and targeting specificity can be altered with a tethered protein. We functionalize exosomes with an immunomodulatory protein, FasL, and demonstrate their biological activity both in vitro and in vivo. FasL-functionalized exosomes, when bioprinted on a collagen matrix, allows spatial induction of apoptosis in tumor cells and, when injected in mice, suppresses proliferation of alloreactive T cells. This oligonucleotide tethering strategy is independent of the exosome source and further circumvents the need to genetically modify exosome-secreting cells.

Calcium chloride enhances the delivery of exosomes.

Exosomes might have an unimproved potential to serve as effective delivery vehicles. However, when exosomes are developed for therapeutic applications, a method to enhance their delivery is important. This study aimed to evaluate wheather calcium chloride (CaCl2) or other chloride compounds could enhance exosome delivery to various cells without causing toxicity. Exosomes were purified from human serum by using the ExoQuick exosome precipitation kit. Isolated exosomes were mixed with CaCl2 at concentrations ranging from 100 μM to 1 mM, and then washed using Amicon filter for treating the cells. The delivery efficiency of exosomes and the viability of the cells [HEK 293 (human kidney cells) and H9C2 (rat cardiomyocytes)] were evaluated. Cellular uptake of exosomes was observed using a confocal microscope based on PKH26 labeling of exosomes. CaCl2 increased the delivery of exosomes in a dose- and treatment time-dependent manner. In HEK 293 cells, a CaCl2 concentration of 400 μM and exposure time of 12 h increased the delivery of exosomes by >20 times compared with controls. In H9C2 cells, a CaCl2 concentration of 400 μM and exposure time of >24 h increased the delivery of exosomes by >400 times compared with controls. The viability of both cell lines was maintained up to a CaCl2 concentration of 1 mM. However, cobalt chloride, cupric chloride, and magnesium chloride did not change the delivery of exosomes in both cell lines. These results suggest that the use of CaCl2 treatment might be a useful method for enhancing the delivery of exosomes.

The Effect of Triptolide-Loaded Exosomes on the Proliferation and Apoptosis of Human Ovarian Cancer SKOV3 Cells.

Triptolide has been proven to possess anticancer efficacy; however, its application in the clinical practice was limited by poor water solubility, hepatotoxicity, and nephrotoxicity. In this study, a triptolide-loaded exosomes delivery system (TP-Exos) was constructed and its effects on the proliferation and apoptosis of SKOV3 cells in vitro and in vivo were observed. SKOV3-exosomes (SK-Exos) were collected by ultracentrifugation and ultrafiltration centrifugation. TP-Exos was constructed by sonication and ultrafiltration centrifugation. SK-Exos and TP-Exos were characterized by transmission electron microscopy, western blotting, nanoparticle-tracking analysis, and high-performance liquid chromatography. Cellular uptake of exosomes, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, bromodeoxyuridine (BrdU) cell proliferation assay, and cell apoptosis experiment were used to study the effect of TP-Exos on ovarian cancer in vitro. Tumor-targeting study of exosomes, monitoring the tumor volume of mice, and TdT-mediated dUTP Nick-End labeling (TUNEL) assay were used to evaluate the effect of TP-Exos on ovarian cancer in vivo. The toxicity of TP-Exos in vivo was evaluated by liver and kidney function and histopathology of major organs (heart, liver, spleen, lung, kidney, and ovary). The results revealed that TP-Exos not only have the general characteristics of exosomes but also have high drug encapsulation efficiency. Besides, PKH26 labeled exosomes (PKH26-Exos) could be uptaken by SKOV3 cells, and Dir labeled exosomes (Dir-Exos) could be enriched to the tumor site of tumor bearing mice. Furthermore, the cytotoxic and apoptotic effects on SKOV3 cells of TP-Exos were weaker than those of free TP, and tumor cell proliferation inhibition and tumor growth inhibition were stronger than that of free TP. Moreover, TP-Exos have toxic effect on liver and spleen. In conclusion, the TP-Exos could be a promising strategy for ovarian cancer, but they need to be further optimized to attenuate the damage to liver and spleen.

Effects of Localization of Antigen Proteins in Antigen-Loaded Exosomes on Efficiency of Antigen Presentation.

Exosomes are considered to be vehicles of antigen delivery. The localization of antigen proteins, i.e., whether they lie on the outer surface or inner surface of exosomes, might affect antigen presentation after exosomes are taken up by antigen-presenting cells; however, little is known about the importance of this phenomenon. In this study, lactadherin (LA) and group-specific antigen (Gag), exosome-tropic proteins that had previously been shown to cause the localization of luciferase to the outer surface and inner surface of exosomes, respectively [ Takahashi , Y. ; J. Biotechnol. 2013 ; Charoenviriyakul , C. ; Mol. Pharm. 2018 ], were used to examine the importance of the localization of antigen proteins in antigen presentation. Human embryonic kidney cells 293 (HEK293) were selected as exosomes producing cells. First, green fluorescent protein (GFP) was used to trace intracellular behavior of antigen proteins after uptake by murine dendritic DC2.4 cells. GFP-derived fluorescence signals were detected in cells only when GFP-inner-loaded (Gag-GFP) exosomes were added to them. Then, ovalbumin (OVA) was used as a model antigen protein, and OVA-loaded exosomes were added to bone marrow-derived dendritic cells. OVA-inner-loaded (Gag-OVA) exosomes showed enhanced class I antigen presentation capacity as compared with OVA-outer-loaded (OVA-LA) exosomes. Using PKH-labeled exosomes, it was found that the localization of OVA had very little effect on the cellular uptake of exosomes. These results indicate that the loading of antigen proteins inside exosomes helps in efficient antigen presentation.

Effect of Human Corneal Mesenchymal Stromal Cell-derived Exosomes on Corneal Epithelial Wound Healing.

PurposeMesenchymal stromal cells (MSCs) have been used therapeutically to modulate inflammation and promote repair. Extracellular vesicles, including exosomes, have been identified as one of the important mediators. This study investigated the effect of human corneal MSC-derived exosomes on corneal epithelial wound healing.MethodsCorneal MSCs (cMSCs) were isolated from human cadaver corneas. The secretome was collected after 72 hours and exosomes were isolated using differential ultracentrifugation. Morphology and size of exosomes were examined by electron microscopy and dynamic light scattering. Expression of CD9, CD63, and CD81 by cMSC exosomes was evaluated by western blotting. Cellular uptake of exosomes was studied using calcein-stained exosomes. The effect of exosome on wound healing was measured in vitro using a scratch assay and in vivo after 2-mm epithelial debridement wounds in mice.ResultscMSC exosomes were morphologically round and main population ranged between 40 and 100 nm in diameter. They expressed CD9, CD63, and CD81, and did not express GM130, Calnexin, and Cytochrome-C. Stained cMSC exosomes were successfully taken up by human cMSCs, human corneal epithelial cells (HCECs), and human macrophages in vitro and by corneal epithelium in vivo. In scratch assay, after 16 hours, cMSC exosome treated HCECs had 30.1% ± 14% remaining wound area compared to 72.9% ± 8% in control (P < 0.005). In vivo, after 72 hours, cMSC exosome-treated corneas had 77.5% ± 3% corneal wound healing compared to 41.6% ± 7% in the control group (P < 0.05).ConclusionsHuman cMSC exosomes can accelerate corneal epithelial wound healing, and thus, may provide a therapeutic approach for ocular surface injuries.

Abstract 5187: Metastatic lung cancer-derived exosomes stimulate premetastatic niche in liver: Beyond seed soil hypothesis

Abstract Background: Tumor-derived exosomes (TDEs) are small membrane bound extracellular vesicles (EVs) that have the ability to alter their immediate microenvironment (ME) through cell-cell interaction and are known to be critical modulators of pre-metastatic niche. Lung cancer is a highly metastatic disease, with metastases often occurring when the tumor is in a clinically early stage. It is known to metastasize to the liver in the Asian population, but the underlying mechanism still remains unclear. Thus, understanding the role of TDEs in development of liver metastasis and its contributions to improvise the prognostic and therapeutic implications is of great importance. Methodology: Exosomes isolated from serum of lung cancer liver metastasis patients were characterized by nanoparticle tracking analysis, electron microscopy and flow cytometry with antibody directed at CD63. These exosomes were further co-cultured with A549 cells to check for their uptake at various time intervals. Moreover, effect of isolated exosomes was tested on: 1. Cell motility and invasiveness of A549 cells determined by transwell chamber migration assay and scratch assay; 2. Gene expression profiles and cell cycle regulation studies; and 3. Histopathological changes and liver function tests after exosome were intraperitoneally injected in Wistar rats to investigate the early events involved in the metastatic spread. Results: We found that CD63 - FITC labelled exosomes isolated from serum of liver metastatic patients were actively incorporated by A549 cells. The exosome uptake by A549 cells induced migratory/invasive phenotypic and morphologic changes in a concentration and time- dependent manner. Moreover, on further evaluating the effect of TDEs on different phases of cell cycle it was observed that cellular uptake of exosomes was increased during G2/M phase stimulating the cells to enter these cell cycle phases leading to cell proliferation. Further, we observed that E-cadherin, beta catenin, VEGFA, CDKN2A and TGFBR2 were differentially expressed when A549 cells were treated with exosomes demonstrating an important role of these TDEs in altering the tumor microenvironment. Liver function test demonstrated an increase in serum SGPT and SGOT levels whereas when histopathological examination was done the lungs showed patches of pneumonitis and the liver was highly inflamed. Conclusion: Collectively this study indicated that metastatic tumor derived exosomes are capable of increasing the migratory and invasive property of the primary lung cancer cells. Furthermore, our results depict an undisputable role of these exosomes as key modulators in the formation of the pre-metastatic niche required for the colonization of circulating tumor cells (CTCs) ultimately leading to distant metastasis. Thus understanding and targeting tumor derived exosomes could lead to better and novel therapeutic strategies. Citation Format: Kanisha Shah, Dr. Rakesh M. Rawal. Metastatic lung cancer-derived exosomes stimulate premetastatic niche in liver: Beyond seed soil hypothesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5187.

Regeneration of Anti-Hypoxic Myocardial Cells by Transduction of Mesenchymal Stem Cell-Derived Exosomes Containing Tat-Metallothionein Fusion Proteins

Stem cells secrete many extracellular vesicles such as micro vesicles, exosomes and membrane particles. Exosomes represent characteristics similar to their native cells and exosomes secreted from human mesenchymal stem cells (hMSCs) have demonstrated cardio protective effects. In this study, we examined the synergistic effects of exosomes derived from hMSCs expressing metallothionein (MT), a well-known therapeutic protein to treat myocardial infarction, for recovery of cell viability in vitro in hypoxic conditions. Tat-metallothionein (Tat-MT) recombinant fusion proteins were prepared by a recombinant method to increase the transduction of metallothionein into exosomes via Tat’s transduction characteristic. Exosomes from hMSCs were transduced with Tat-MT, and characterized by transmission electron microscopy and immunoblotting. Cellular uptake of exosomes and protein was analyzed by confocal microscopy. The cytoprotective effects of exosomes transfected with Tat-MT (Exo/Tat-MT) on cardiomyocytes were evaluated by accessing cell viability. Exo/Tat-MT significantly upregulated cell viability and downregulated apoptosis in cardiomyocytes. The therapeutic potential of exosome-mediated therapeutic protein delivery was demonstrated by strong cell viability (70-80%) under in vitro hypoxic conditions. This study reveals the dual benefits of exosomes derived from hMSCs and highlights a new method of intercellular stem cells mediation for the stem cell-derived treatment of myocardial infarction.