Exosome Subpopulations Research Articles

Exosomes and Tumor Progression: Our Current Knowledge

Exosomes are tiny vesicles that cells secrete into the extracellular environment. They are crucial in cellular communication and have wide-ranging physiological and pathological ramifications. Cargo sorting, MVB development and maturation, MVB transport, and MVB fusion with the plasma membrane are the four essential steps in exosome biogenesis. The high heterogeneity of exosomes is due to the fact that each process is modulated by the competition or coordination of multiple mechanisms, resulting in the sorting of diverse compositions of molecular cargos into different subpopulations of exosomes. In cancer, exosomes have been shown to play a crucial role in tumor growth, metastasis, and pre-metastatic niche formation. In this mini-review, we briefly compile what we know about exosomes at present, including how they are made, what they carry, and how they promote tumor growth. Exosomes' potential as diagnostic and prognostic biomarkers is discussed. We also take a look at the research that hasn't been done and the challenges that have been overlooked

Macrophage-derived CD36 + exosome subpopulations as novel biomarkers of Candida albicans infection

Invasive candidiasis (IC) is a notable healthcare-associated fungal infection, characterized by high morbidity, mortality, and substantial treatment costs. Candida albicans emerges as a principal pathogen in this context. Recent academic advancements have shed light on the critical role of exosomes in key biological processes, such as immune responses and antigen presentation. This burgeoning body of research underscores the potential of exosomes in the realm of medical diagnostics and therapeutics, particularly in relation to fungal infections like IC. The exploration of exosomal functions in the pathophysiology of IC not only enhances our understanding of the disease but also opens new avenues for innovative therapeutic interventions. In this investigation, we focus on exosomes (Exos) secreted by macrophages, both uninfected and those infected with C. albicans. Our objective is to extract and analyze these exosomes, delving into the nuances of their protein compositions and subgroups. To achieve this, we employ an innovative technique known as Proximity Barcoding Assay (PBA). This methodology is pivotal in our quest to identify novel biological targets, which could significantly enhance the diagnostic and therapeutic approaches for C. albicans infection. The comparative analysis of exosomal contents from these two distinct cellular states promises to yield insightful data, potentially leading to breakthroughs in understanding and treating this invasive fungal infection. In our study, we analyzed differentially expressed proteins in exosomes from macrophages and C. albicans -infected macrophages, focusing on proteins such as ACE2, CD36, CAV1, LAMP2, CD27, and MPO. We also examined exosome subpopulations, finding a dominant expression of MPO in the most prevalent subgroup, and a distinct expression of CD36 in cluster14. These findings are crucial for understanding the host response to C. albicans and may inform targeted diagnostic and therapeutic approaches. Our study leads us to infer that MPO and CD36 proteins may play roles in the immune escape mechanisms of C. albicans. Additionally, the CD36 exosome subpopulations, identified through our analysis, could serve as potential biomarkers and therapeutic targets for C. albicans infection. This insight opens new avenues for understanding the infection's pathology and developing targeted treatments.

Metabolic labelling of a subpopulation of small extracellular vesicles using a fluorescent palmitic acid analogue.

Exosomes are among the most puzzling vehicles of intercellular communication, but several crucial aspects of their biogenesis remain elusive, primarily due to the difficulty in purifying vesicles with similar sizes and densities. Here we report an effective methodology for labelling small extracellular vesicles (sEV) using Bodipy FL C16, a fluorescent palmitic acid analogue. In this study, we present compelling evidence that the fluorescent sEV population derived from Bodipy C16-labelled cells represents a discrete subpopulation of small exosomes following an intracellular pathway. Rapid cellular uptake and metabolism of Bodipy C16 resulted in the incorporation of fluorescent phospholipids into intracellular organelles specifically excluding the plasma membrane and ultimately becoming part of the exosomal membrane. Importantly, our fluorescence labelling method facilitated accurate quantification and characterization of exosomes, overcoming the limitations of nonspecific dye incorporation into heterogeneous vesicle populations. The characterization of Bodipy-labelled exosomes reveals their enrichment in tetraspanin markers, particularly CD63 and CD81, and in minor proportion CD9. Moreover, we employed nanoFACS sorting and electron microscopy to confirm the exosomal nature of Bodipy-labelled vesicles. This innovative metabolic labelling approach, based on the fate of a fatty acid, offers new avenues for investigating exosome biogenesis and functional properties in various physiological and pathological contexts.

BIOM-50. EXOSOMAL MICRORNA EXPRESSION SIGNATURE IN BLOOD AND CEREBROSPINAL FLUID OF GLIOBLASTOMA PATIENTS

Abstract Analysis of exosomes derived from plasma or cerebrospinal fluid (CSF) has emerged as a promising biomarker platform for therapeutic monitoring in glioblastoma patients. However, the contents of the various subpopulations of exosomes in these clinical specimens remain poorly defined. Here we characterize the relative abundance of miRNA species in exosomes derived from the plasma and CSF of glioblastoma patients. To this end, we first employed miRNA arrays to measure the expression of exosomal miRNAs in the plasma from glioblastoma patients (n = 24) and healthy volunteers (n = 7) as control. In addition, we performed global miRNA profiling of exosomal miRNAs in the CSF from glioblastoma patients (n = 5) and non-tumoral patients (n = 3; hydrocephalus patients) as control. In plasma derived exosomes, 80 miRNAs were altered by >2-fold in glioblastoma patients compared to controls. In CSF, 92 miRNAs were altered by >2-fold in glioblastoma patients compared to controls. Combined analysis of plasma and CSF revealed a similar fold difference in eight miRNAs. Next, we measured these eight miRNAs expression in in the plasma from pre- and post-operative glioblastoma patients (n = 9). Among these eight miRNAs, we identified only one miRNA (miR-34b-3p) that was upregulated in exosomes from pre-operative glioblastoma patients. Our results suggest that miR-34b-3p might have a potential as a novel diagnostic marker or a therapeutic tool for glioblastoma patients.

Targeted Delivery of RGD-CD146+CD271+ Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Promotes Blood-Spinal Cord Barrier Repair after Spinal Cord Injury.

Spinal cord injury (SCI) disrupts the blood-spinal cord barrier (BSCB), potentially exacerbating nerve damage and emphasizing the criticality of preserving the BSCB integrity during SCI treatment. This study explores an alternative therapeutic approach for SCI by identifying a subpopulation of exosomes with stable BSCB function and achieving a specific targeted delivery. Specific subpopulations of CD146+CD271+ umbilical cord mesenchymal stem cells (UCMSCs) were isolated, from which engineered exosomes (RGD-CD146+CD271+ UCMSC-Exos) with targeted neovascularization function were obtained through gene transfection. In vivo and in vitro experiments were performed to explore the targeting and therapeutic effects of RGD-CD146+CD271+ UCMSC-Exos and the potential mechanisms underlying BSCB stabilization and neural function recovery. The results demonstrated that RGD-CD146+CD271+ UCMSC-Exos exhibited physical and chemical properties similar to those of regular exosomes. Notably, following intranasal administration, RGD-CD146+CD271+ UCMSC-Exos exhibited enhanced aggregation at the SCI center and demonstrated the specific targeting of neovascular endothelial cells. In the SCI model, intranasal administration of RGD-CD146+CD271+ UCMSC-Exos reduced Evans blue dye leakage, increased tight junction protein expression, and improved neurological function recovery. In vitro testing revealed that RGD-CD146+CD271+ UCMSC-Exos treatment significantly reduced the permeability of bEnd.3 cells subjected to oxygen-glucose deprivation, thereby restoring the integrity of tight junctions. Moreover, further exploration of the molecular mechanism underlying BSCB stabilization by CD146+CD271+ UCMSC-Exos identified the crucial role of the miR-501-5p/MLCK axis in this process. In conclusion, targeted delivery of RGD-CD146+CD271+ UCMSC-Exos presents a promising and effective treatment option for SCI.

Decoding of exosome heterogeneity for cancer theranostics.

Extracellular Vesicles (EVs) discovery establishes a new milestone in cancer research. It explains cancer biology in a new way. EVs classified some significant subclasses such as macrovesicle (originated from plasma membrane), apoptotic bodies (originated from the plasma membrane and endoplasmic reticulum), and exosomes (originated from endosomes).1 The theranostic signature of exosomes in cancer healing is impressive, but its heterogeneity leads most complicated domain of research. Therefore we comment on decoding exosome heterogeneity for future efficiency and effective cancer theranostics development. EVs are the cell-secreted one of the most important entities. The exosome attracted much attention due to its versatile uses. Overall, its participation in intercellular communication and its inner cargos (DNA, RNA, proteins, lipids and glycan)2 can reprogram the recipient cells. In addition, it also shows the healthy or pathologic complication status of the parent cells. In cancer, tumor and exosomes interlink a complex chapter of cancer biology. Exosomes play an important role in accelerated tumor growth, immune cell reprogramming,2 angiogenesis,3 extracellular matrix remodelling,4, 5 metastasis,4, 5 epithelial-mesenchymal transition (EMT),4, 5 organ-specific metastasis,5, 6 drug resistance and cancer stem cell development.3, 4 Clinically aspect, the exosome is the source of the cancer biomarkers.7-9 Exosome heterogeneity1 is evolving into a major conflict in cancer biomarker and therapeutic research.10 The exosome heterogeneity (Figure 1) is based on several facts such as origin, size, quantity, and internal molecular diversity.1 Exosome size distribution is the most complicated side of tumor-derived exosomes (TEXs). Scientific evidence shows that the same cells release differently-sized exosomes. This size-oriented biodiversity is reflected in using different isolation methods.9, 10 The tumor cells release more exosomes compared to healthy cells. The higher release of exosomes from tumor cells depends on several factors, such as drug and therapeutic exposures, hypoxic conditions, and senescence. The functional diversity of exosomes is related to the surface molecular expression and inner cargos of exosomes. All these complicated/complex parameters lead to a challenging ecosystem development of exosome-based cancer theragnostics (combination of biomarkers and therapeutics research). This scenario requires a solution to solve these challenges. Based on several innovative nano platform-based approaches transform these challenges into an opportunity for exosome researcher in a detailed exploration of exosome.11 Exosomes isolation, size and quantity related complication solved via microfluidic device, fluorescence antibody based exosome tracking, Nanoparticle tracking analysis (NTA) (above 60 nm exosome size challenging for its application),12, 13 magnetic and surface plasma resonance principle based single exosome screening,1 advanced sensitive flow cytometry (it is capable of the separation of exosome subpopulation) and electrophoresis chip. Molecular profiling of exosomes is one of the most critical tasks revealing the functional diversity of bioactive cargo molecules of exosomes. Droplet digital polymerase chain reaction (it is sensitive to the detection of rear mutations in tumor-derived exosomes),1 microfluidic technology combined with a chip system, and an electrochemical principle-based micro RNA profiling of exosomes explain the cancer complication of a new dimension.1 The downstream process of exosome profiling needs a muti-omics approach for proper molecular diversity to understand.1 The single cells exosome profiling approach combines with machine learning for more precision cancer marker development.14 All these technological advancements support constructing the next-generation promising cancer theranostic era based on exosomes.15, 16 Finally, decoding exosome heterogeneity opens a new door for exosome-based precision medicine17 and vaccines18 for cancer. We hope this article encourages large-scale EV research to explore a single exosome profiling approach and future exosome-based cancer precision medicine development. Not applicable. The authors declare no conflict of interest. There is no funding for this study.

Do different exosome biogenesis pathways and selective cargo enrichment contribute to exosomal heterogeneity?

Exosomes are emerging intercellular communicators essential for cellular homeostasis during development and differentiation. The dysregulation in exosome-mediated communication alters cellular networking leads to developmental defects and chronic diseases. Exosomes are heterogeneous in nature depending on differences in size, membrane protein abundance, and differential cargo load. In this review, we have highlighted the latest developments in exosome biogenesis pathways, heterogeneity, and selective enrichment of various exosomal cargoes including proteins, nucleic acids, and mitochondrial DNA. Furthermore, the recent developments in the isolation techniques of exosome subpopulations have also been discussed. The comprehensive knowledge of extracellular vesicle (EV) heterogeneity and selective cargo enrichment during specific pathology may provide a clue for disease severity and early prognosis possibilities. The release of specific exosome subtypes is associated with the progression of specific disease type and hence a probable tool for therapeutics and biomarker development.

Alternating Magnetic Field-Promoted Nanoparticle Mixing: The On-Chip Immunocapture of Serum Neuronal Exosomes for Parkinson's Disease Diagnostics.

The analysis of cargo proteins in exosome subpopulations has considerable value in diagnostics but a translatable impact has been limited by lengthy or complex exosome extraction protocols. We describe herein a scalable, fast, and low-cost exosome extraction using an alternating (AC) magnetic field to support the dynamic mixing of antibody-coated magnetic beads (MBs) with serum samples within 3D-printed microfluidic chips. Zwitterionic polymer-coated MBs are, specifically, magnetically agitated and support ultraclean exosome capture efficiencies >70% from <50 μL of neat serum in 30 min. Applied herein to the immunocapture of neuronal exosomes using anti-L1CAM antibodies, prior to the array-based assaying of α-synuclein (α-syn) content by a standard duplex electrochemical sandwich ELISA, sub pg/mL detection was possible with an excellent coefficient of variation and a sample-to-answer time of ∼75 min. The high performance and semiautomation of this approach hold promise in underpinning low-cost Parkinson's disease diagnostics and is of value in exosomal biomarker analyses more generally.

SARS CoV-2 spike protein-guided exosome isolation facilitates detection of potential miRNA biomarkers in COVID-19 infections.

Nearly three years into the pandemic, SARS-CoV-2 infections are occurring in vaccinated and naturally infected populations. While humoral and cellular responses in COVID-19 are being characterized, novel immune biomarkers also being identified. Recently, an increase in angiotensin-converting enzyme 2 expressing (aka, ACE2 positive) circulating exosomes (ExoACE2) were identified in the plasma of COVID-19 patients (El-Shennawy etal.). In this pilot study, we describe a method to characterize the exosome-associated microRNA (exo-miRNA) signature in ACE2-positive and ACE2-negative exosomal populations (non-ExoACE2). We performed a sorting protocol using the recombinant biotin-conjugated SARS CoV-2 spike protein containing the receptor binding domain (RBD) on plasma samples from six patients. Following purification, exo-miRNA were characterized for ACE2-positive and ACE2-negative exosome subpopulations by RT-PCR. We identified differential expression of several miRNA. Specifically let-7g-5p and hsa-miR-4454+miR-7975 were upregulated, while hsa-miR-208a-3p and has-miR-323-3p were downregulated in ExoACE2 vs. non-ExoACE2. The SARS CoV-2 spike-protein guided exosome isolation permits isolation of ExoACE2 exosomes. Such purification facilitates detailed characterization of potential biomarkers (e.g. exo-miRNA) for COVID-19 patients. This method could be used for future studies to further the understanding mechanisms of host response against SARS CoV-2.

ImmunoInertial microfluidics: A novel strategy for isolation of small EV subpopulations

Cancer-specific small extracellular vesicles (sEVs), known as exosomes, have shown a great promise to serve as novel biomarkers for cancer diagnosis and prognosis in liquid biopsies. However, the high heterogeneity of sEVs posed great technical challenges to acquiring their molecular information. A simple and reproducible method for isolating subpopulations of sEVs can significantly enhance the detection and stratification of these circulating biomarkers and their function. This study used the synergic effects of the immunoaffinity-based approach and inertial microfluidics (ImmunoInertial microfluidics) to isolate specific subpopulations of sEVs. At first, the cancer cell-derived sEVs were captured on microbeads of varying sizes which were functionalized with specific capture antibodies such as epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), and the programmed death-ligand 1 (PD-L1), facilitating the selective capture of sEVs. The sEVs-bearing microbeads were subsequently introduced to a series of inertial microfluidic channels, called iZExoSub (inertial zigzag microfluidics for exosome subpopulation separation), for size-based bead separation. Results revealed more than 90% efficiency in sEVs subpopulation separation, further proved via flow cytometry analysis data. Our approach is capable of selective isolation and quantitative detection of important biomarkers from sEVs subpopulations with high sensitivity and low cost and has the capacity to process samples of varying volumes, ranging from µL up to mL continuously. This system can outperform FACS machines in terms of sample throughput by orders of magnitudes. In addition, this study emphasized the necessity of using a consistent sEV marker (as capture and detector) across different samples for accurate assessment of subpopulations.

Single-Exosome Profiling Identifies ITGB3+ and ITGAM+ Exosome Subpopulations as Promising Early Diagnostic Biomarkers and Therapeutic Targets for Colorectal Cancer.

Tumor metastasis is a hallmark of colorectal cancer (CRC), in which exosome plays a crucial role with its function in intercellular communication. Plasma exosomes were collected from healthy control (HC) donors, localized primary CRC and liver-metastatic CRC patients. We performed proximity barcoding assay (PBA) for single-exosome analysis, which enabled us to identify the alteration in exosome subpopulations associated with CRC progression. By in vitro and in vivo experiments, the biological impact of these subpopulations on cancer proliferation, migration, invasion, and metastasis was investigated. The potential application of exosomes as diagnostic biomarkers was evaluated in 2 independent validation cohorts by PBA. Twelve distinct exosome subpopulations were determined. We found 2 distinctly abundant subpopulations: one ITGB3-positive and the other ITGAM-positive. The ITGB3-positive cluster is rich in liver-metastatic CRC, compared to both HC group and primary CRC group. On the contrary, ITGAM-positive exosomes show a large-scale increase in plasma of HC group, compared to both primary CRC and metastatic CRC groups. Notably, both discovery cohort and validation cohort verified ITGB3+ exosomes as potential diagnostic biomarker. ITGB3+ exosomes promote proliferation, migration, and invasion capability of CRC. In contrast, ITGAM+ exosomes suppress CRC development. Moreover, we also provide evidence that one of the sources of ITGAM+ exosomes is macrophage. ITGB3+ exosomes and ITGAM+ exosomes are proven 2 potential diagnostic, prognostic, and therapeutic biomarkers for management of CRC.

Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues.

Extracellular vesicles (EVs) are nanoscale vesicles secreted into the extracellular space by all cell types, including neurons and astrocytes in the brain. EVs play pivotal roles in physiological and pathophysiological processes such as waste removal, cell-to-cell communication and transport of either protective or pathogenic material into the extracellular space. Here we describe a detailed protocol for the reliable and consistent isolation of EVs from both murine and human brains, intended for anyone with basic laboratory experience and performed in a total time of 27 h. The method includes a mild extracellular matrix digestion of the brain tissue, a series of filtration and centrifugation steps to purify EVs and an iodixanol-based high-resolution density step gradient that fractionates different EV populations, including mitovesicles, a newly identified type of EV of mitochondrial origin. We also report detailed downstream protocols for the characterization and analysis of brain EV preparations using nanotrack analysis, electron microscopy and western blotting, as well as for measuring mitovesicular ATP kinetics. Furthermore, we compared this novel iodixanol-based high-resolution density step gradient to the previously described sucrose-based gradient. Although the yield of total EVs recovered was similar, the iodixanol-based gradient better separated distinct EV species as compared with the sucrose-based gradient, including subpopulations of microvesicles, exosomes and mitovesicles. This technique allows quantitative, highly reproducible analyses of brain EV subtypes under normal physiological processes and pathological brain conditions, including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

Tackling CD147 exosome-based cell-cell signaling by electrochemical biosensing for early colorectal cancer detection

The great opportunities represented by exosomes in liquid biopsy diagnostics and the relevance of CD147 protein as diagnostic and prognostic cancer biomarker led us to develop the first bio-electroanalytical platform for the determination of exosomal CD147 (exoCD147) by exploiting micro-sized magnetic beads coated with specific anti-CD147 antibodies. The captured exosomal target protein was sandwiched by specific biotin functionalized detector antibodies followed by attaching streptavidin-HRP conjugate to perform the amperometric reading using screen-printed carbon electrodes (SPCEs) as electrode transducers in the presence of hydroquinone (HQ) and H2O2. The analytical and operational characteristics achieved by implementing this simple methodology allowed the sensitive (LOD 29 pg mL−1) and selective determination of CD147 and the analysis of exoCD147 in different but inter-related real clinical scenarios including lysed and entire exosomes previously isolated from CRC cell lines with different metastatic potential. The obtained results, in agreement with those provided by ELISA and WB, proved the reliability of the developed immunosensor and its potential to isolate or identify specific subpopulations of exosomes based on the differential expression of characteristic surface biomarkers.

Abstract 3417: Effects of plasma exosome isolation methods on detectable multiomic profiles in cancer patients and healthy controls

Abstract Introduction: Circulating exosomes in blood have been considered as a treasure chest for biomarker discovery in cancer. Due to their heterogeneity, different isolation methods may enrich distinct exosome cargos generating different omic profiles. In this study, we evaluated the effects of plasma exosome isolation methods on detectable multiomic profiles in cancer patients and healthy controls. Methods: Plasma exosomes were isolated from prostate cancer patients (PC; N = 11), lung cancer patients (LC; N = 13), and matched healthy controls (HC; N = 10) using three methods [SBI (size exclusion), Takara (lectin binding), and Wako (Tim4 binding)]. Mass spectrometry was performed to determine exosome lipidome and metabolome profiles. To evaluate group-specific exosome enrichment, we developed an exosome enrichment index (EEI) by summing 50 smallest p-values after log2 transformation. Higher EEI indicates prefrential capture of significant exosome cargos from patients over controls. Results: Our data showed that the SBI method generated the most identified lipids from exosome across all groups (LC = 935, PC = 939, and HC = 943), followed by Takara (245, 245 and 245) and Wako (205, 208 and 213). Compared to controls, LC patients had 31 higher and 5 lower lipid levels (FDR &amp;lt; 0.1) using the SBI and 3 higher and 1 lower level (FDR &amp;lt; 0.1) using Takara. We did not observe any differences between cancer and normal samples using Wako. The most detectable known metabolites were found in exosomes from SBI (LC = 188, PC = 191, and HC = 100), followed by Wako (LC = 107, PC = 107, and HC = 107) and Takara (LC = 99, PC = 100, and HC = 100). Of the metabolites from SBI, 5 were significantly higher (FDR&amp;lt;0.1) in PC patients than in controls. The most significant metabolite in PC was L-Cystathionine, which has been shown to be upregulated in bone-metastatic PC3 cells. Neither Takara nor Wako exosome fractions showed differently expressed metabolites between any groups. Our EEI analysis showed the highest lipid EEI from SBI in PC (474.4) and LC (906.74), while a lower lipid EEI from Takara in PC (359.9), LC (495.58), and Wako in PC (307.5) and LC (148.1). Metabolite EEI showed an EEI range from 590.3 in SBI PC to 185.6 in Takara LC. SBI, again, achieved the highest EEI for both PC and LC (357.3). Conclusion: These results support that the SBI method can capture more heterogeneous exosome populations, possibly because this size exclusion-based method does not discriminate exosomes with different protein components. In contrast, Takara and Wako methods target exosome surface proteins, enriching specific exosome subpopulations resulting in less diversity of detectable lipids and metabolites. Overall, these findings suggest that exosome isolation methods can select subpopulations and determine the exosome content, which will significantly impact exosome-based biomarker discovery. Citation Format: Alex C. Soupir, Paul A. Stewart, Yury Nunez Lopez, Brandon J. Manley, Bruna Pellini, Jingsong Zhang, Qianxing Mo, Douglas C. Marchion, Min Lui, John M. Koomen, Erin M. Siegel, Liang Wang. Effects of plasma exosome isolation methods on detectable multiomic profiles in cancer patients and healthy controls [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3417.

Ascorbate peroxidase-mediated in situ labelling of proteins in secreted exosomes.

The extracellular vesicle exosome mediates intercellular communication by transporting macromolecules such as proteins and ribonucleic acids (RNAs). Determining cargo contents with high accuracy will help decipher the biological processes that exosomes mediate in various contexts. Existing methods for probing exosome cargo molecules rely on a prior exosome isolation procedure. Here we report an in situ labelling approach for exosome cargo identification, which bypasses the exosome isolation steps. In this methodology, a variant of the engineered ascorbate peroxidase APEX, fused to an exosome cargo protein such as CD63, is expressed specifically in exosome‐generating vesicles in live cells or in secreted exosomes in the conditioned medium, to induce biotinylation of the proteins in the vicinity of the APEX variant for a short period of time. Mass spectrometry analysis of the proteins biotinylated by this approach in exosomes secreted by kidney proximal tubule‐derived cells reveals that oxidative stress can cause ribosomal proteins to accumulate in an exosome subpopulation that contains the CD63‐fused APEX variant.

370-OR: Proinflammatory Stress Activates Neutral Sphingomyelinase 2 Based Generation of Ceramide-Enriched ß-Cell Exosomes

Data suggest that β cell exosomes, extracellular vesicles released by exocytosis of multivesicular endosomes (MVEs) , may act as paracrine effectors in islet health. However, mechanisms linking β cell stress to changes in exosomes and whether activation of these pathways can impact diabetes remain less explored. In other cells, the enzyme neutral spingomyelinase 2 (nSMase2) induces ceramide dependent MVE vesicle formation and release of ceramide-enriched exosomes. We hypothesized that β cell inflammatory stress engages ceramide-dependent exosomal formation pathways, and that ceramide-enriched exosomes could then impact surrounding β cells. To test this we treated INS-1 β cells with 24 hrs of 5 ng/mL of IL1β. nSMase 2 and ceramide expression were quantified with immunoblot and/or flow cytometry and exosomes were isolated using a tetraspanin bead-based pulldown. IL1β increased β cell nSMase2 expression and ceramide expression in concert. Direct nSMase2 activation with 24 hrs of 5-ng/mL Caffeic acid phenethyl ester (CAPE) also increased β cell ceramide staining. IL1β and CAPE treatments also yielded increased ceramide expression on β cell exosomes. Human islets exhibited similar increases in cellular nSMase2 and ceramide staining after 24-hr cytokine mix treatment (5ng/mL IL1β, 10ng/mL TNFα, and 100ng/mL IFNγ) . To test the potential for transfer of β cell exosomes to surrounding cells, we generated INS-1 cells with GFP tagged CD9 (exosome transmembrane protein) , which release GFP labeled exosomes. Flow cytometry staining in wild-type INS-1 cells after 24-hr transwell coculture with CD9-GFP cells showed that cocultured wild-type cells exhibited increased green fluorescence, suggesting transfer of CD9-GFP exosomes. Our findings suggest that nSMase2 activity may regulate β cell exosome subpopulations under conditions of inflammatory stress. Future work will interrogate impacts on exosome cargo and physiologic impacts of transfer on recipient cells. Disclosure J.Xu: None. E.K.Sims: Other Relationship; Medscape, Patent. Funding 5 R01DK121929

Microprobe‐based technology for antigen‐specific purification of exosomes for OMICS analysis

Exosomes encapsulate genomic and proteomic biomarkers for non‐invasive diagnosis and disease monitoring. However, exosome surface‐markers heterogeneity is a major drawback of current isolation methods. Here, we report a direct, one‐step exosome sampling technology, ExoPRIME, for selective capture of CD63+ exosome subpopulations using an immune‐affinity protocol. Microneedles (300μm × 30 mm), functionalized with anti‐ CD63 antibodies, were incubated under various experimental conditions in conditioned astrocyte medium and astrocyte‐derived exosome suspension. The probe’s capture efficiency and specificity were validated using Flu‐oroCet assay, immunofluorescent imaging, and OMICS analyses. Significantly higher exosomes were captured by probes incubated for 16 h at 4 0C in enriched exosomal suspension (23 × 10 6 exosomes per probe) vis‐'a‐vis 2 h at 4 0 C (12 × 10 6) and 16 h at 22 0C (3 × 10 6) in conditioned cell media. Our results demonstrate the application of ExoPRIME over a broad dynamic range of temperature and incubation parameters, offering flexibility for any desired application. ExoPRIME permits the use and re‐use of minimal sample volumes (≤200 μL), can be multiplexed in arrays, and integrated into a lab‐on‐a‐chip platform to achieve parallel, high‐throughput isolation of different exosome classes in a semi‐automated workstation. This platform could provide direct exosomal analysis of biological fluids since it can elegantly interface with existing room‐temperature, picomolar‐range nucleic acid assays to provide a clinical diagnostic tool at the point of care.

Impact of Obesity on Exercise‐Induced Skeletal Muscle Exosomes

Exosomes are small extracellular vesicles (EVs) that can function as effective delivery vehicles through systemic circulation due to the protective effects of the phospholipid bilayer. Our previous work in mice indicated that resistance exercise induces the release of skeletal muscle EVs to promote adipose lipolysis through the action of microRNA‐1. To better understand the effect of resistance exercise on exosomes in humans, we collected blood samples as well as skeletal muscle and adipose tissue biopsies before and after a single bout of resistance exercise. Using the ExoView platform, we were able to analyze subpopulations of exosomes based on their surface tetraspanin markers (CD63, CD81, and CD9). Our preliminary findings show that serum CD81/CD9 exosome abundance is significantly lower in high BMI (&gt;30) subjects, and serum CD63/CD81 exosome abundance may be differentially affected by exercise in subjects with high BMI. Further studies are necessary to define the nucleic acid and protein contents of the exercise‐induced exosomes. To this end, we have generated a transgenic mouse with an inducible skeletal muscle specific exosome surface protein reporter, which will allow definitive tracking of exosomes released from skeletal muscle that are delivered to other organ systems.

LAMP2A regulates the loading of proteins into exosomes.

Exosomes are extracellular vesicles of endosomal origin that are released by practically all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs, and proteins between different cells, tissues, or organs. Here, we describe a mechanism whereby proteins containing a KFERQ motif pentapeptide are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, we demonstrate that this mechanism is independent of the ESCRT machinery but dependent on HSC70, CD63, Alix, Syntenin-1, Rab31, and ceramides. We show that the master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. In addition, by tagging fluorescent proteins with KFERQ-like sequences, we were able to follow the interorgan transfer of exosomes. Our findings open new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs.

Comparison of the yield and purity of plasma exosomes extracted by ultracentrifugation, precipitation, and membrane-based approaches

Abstract Exosomes were enriched from plasma by ultracentrifugation, precipitation, and membrane-based approaches for yield and purity. Using the four isolation approaches, particles with mode sizes within the expected range (50–200 nm) can be isolated. By protein estimation, polymer precipitation resulted in a maximum yield (5610.59 ± 51.189 µg/mL), followed by membrane affinity (471.57 ± 12.16 µg/mL), ultracentrifugation (440.22 ± 11.71 µg/mL) and filter + ultracentrifugation (235.47 ± 13.27 µg/mL). By total RNA estimation, the yield of polymer precipitation (3.26 ± 0.42 ng/mL) was higher than that of ultracentrifugation (1.52 ± 0.06 ng/mL), filter + ultracentrifugation (1.21 ± 0.25 ng/mL) and membrane affinity (1.44 ± 0.14 ng/mL). The purity of exosomal preparations was determined as the ratio of the particle number to protein and of protein to RNA. According to the ratio of the particle number to protein concentration, the “purity” of the polymer precipitation method was similar to that of the membrane affinity method and higher than that of ultracentrifugation and filter + ultracentrifugation. When the ratio of RNA to protein was used, the “purity” of the polymer precipitation method was lower than that of the membrane affinity method. Differential methods can be employed to enrich specific exosome subpopulations. The steps of the methods affect the particle number, protein content, and even exosomal purity. The best extraction and evaluation methods for exosomes need to be selected in the laboratory according to their experimental needs.