Proteomic Signature of Mesenchymal Stromal Cell‐Derived Small Extracellular Vesicles

Small extracellular vesicles (EVs) are 50–200 nm vesicles secreted by most cells. They are considered as mediators of intercellular communication, and EVs from specific cell types, in particular mesenchymal stem/stromal cells (MSCs), offer powerful therapeutic potential, and can provide a novel therapeutic strategy. They appear promising and safe (as EVs are non‐self‐replicating), and eventually MSC‐derived EVs (MSC‐EVs) may be developed to standardized, off‐the‐shelf allogeneic regenerative and immunomodulatory therapeutics. Promising pre‐clinical data have been achieved using MSCs from different sources as EV‐producing cells. Similarly, a variety EV isolation and characterization methods have been applied. Interestingly, MSC‐EVs obtained from different sources and prepared with different methods show in vitro and in vivo therapeutic effects, indicating that isolated EVs share a common potential. Here, well‐characterized and controlled, publicly available proteome profiles of MSC‐EVs are compared to identify a common MSC‐EV protein signature that might be coupled to the MSC‐EVs’ common therapeutic potential. This protein signature may be helpful in developing MSC‐EV quality control platforms required to confirm the identity and test for the purity of potential therapeutic MSC‐EVs.     

Isolation of Extracellular Microvesicles from Cell Culture Medium: Comparative Evaluation of Methods

Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by “horizontal” exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional EV features are usually studied in vitro. Several methods of EV isolation from cell culture medium are currently used; however, selection of a particular method may have a significant impact on obtained results. The choice of the optimal method is usually determined by the amount of starting biomaterial and the aims of the research. We have performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with 30% sucrose/D₂O “cushion,” precipitation with plant proteins and latex-based immunoaffinity capturing. EV isolated from several human glial cell lines by different approaches were compared in terms of the following parameters: size, concentration, EV morphology, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, total RNA, and several glioma-associated miRNAs. The applied methods included nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. Based on the obtained results, we have developed practical recommendations that may help researchers to make the best choice of the EV isolation method.     

综述与专论: 基于核酸适配体的细胞外囊泡分离分析方法

细胞外囊泡通过参与细胞间通讯，在诸多生理病理过程中发挥着重要作用。因此，细胞外囊泡的分离分析对理解其生物学功能以及发展基于囊泡的疾病诊疗方法具有重要价值。细胞外囊泡的高效分离以及高灵敏可靠检测很大程度上取决于识别配体。核酸适配体是一类高效、特异结合其靶标分子的单链寡核苷酸。核酸适配体的易修饰和可程序化设计等特征，使其成为细胞外囊泡分离和分析的理想识别配体。为提高细胞外囊泡的分离效率，研究者们提出多种策略用于提升核酸适配体的亲和力，以及界面与细胞外囊泡的接触几率。此外，分离不同亚型的细胞外囊泡有助于理解细胞外囊泡的生物学意义。在细胞外囊泡分析方面，根据核酸适配体与细胞外囊泡识别信号的转导方式不同，分为电化学、可视化、表面增强拉曼光谱、荧光法等方法。本文综述了核酸适配体的筛选以及其在细胞外囊泡分离和分析中的最新进展、挑战及未来方向。     

Protein Composition Reflects Extracellular Vesicle Heterogeneity

Extracellular vesicles (EVs) are membrane‐enclosed particles that are released by virtually all cells from all living organisms. EVs shuttle biologically active cargo including protein, RNA, and DNA between cells. When shed by cancer cells, they function as potent intercellular messangers with important functional consequences. Cells produce a diverse spectrum of EVs, spanning from small vesicles of 40–150 nm in diameter, to large vesicles up to 10 μm in diameter. While this diversity was initially considered to be purely based on size, it is becoming evident that different classes of EVs, and different populations within one EV class may harbor distinct molecular cargo and play specific functions. Furthermore, there are considerable cell type‐dependent differences in the cargo and function of shed EVs. This review focuses on the most recent proteomic studies that have attempted to capture the EV heterogeneity by directly comparing the protein composition of different EV classes and EV populations derived from the same cell source. Recent studies comparing protein composition of the same EV class(es) derived from different cell types are also summarized. Emerging approaches to study EV heterogeneity and their important implications for future studies are also discussed.     

Assessment of an enterovirus sewage surveillance system by comparison of clinical isolates with sewage isolates from milwaukee, wisconsin, collected august 1994 to december 2002

The quantity and serotypes of enteroviruses (EVs) in the influent of a local sewage treatment plant were compared to local clinical EV cases to determine if testing of sewage is adequate for an EV surveillance system. The study was carried out from August 1994 to December 2002. Monthly influent specimens were processed by organic flocculation, and dilutions of concentrate were inoculated onto a number of different cell types for virus isolation. EVs were detected in 88 of 100 monthly influent samples. Sewage EV titers were calculated by using software provided by the U.S. Environmental Protection Agency for most-probable-number determination. All 1,068 sewage EV isolates were further grouped (echovirus, coxsackievirus B, coxsackievirus A, or poliovirus) by cell culture host range analysis (growth pattern of isolates on passage to seven cell lines), and 39.0% of the 1,022 EV isolates categorized as non-poliovirus EVs were specifically serotyped. For clinical cases, primary virus isolation tests were performed on specimens submitted by local hospitals and EV isolates submitted by hospitals were serotyped. Clinical EVs were documented for 81 of the 100 months studied. In all, 694 EV isolates from clinical cases were serotyped. Annually, between 4 and 11 different serotypes of non-poliovirus EVs were identified in sewage and from 9 to 19 different non-poliovirus EV serotypes were identified from clinical specimens. Usually, the most commonly detected sewage EV serotypes were similar to the most commonly detected clinical serotypes; e.g., for 1997, echovirus 6 accounted for 53.1% of the typed sewage isolates and 39.4% of the clinical infections, while in 1998, echovirus 30 accounted for 50.0 and 46.1%, respectively. In 1999, 60.3% of the EVs from clinical cases and 79.7% of the sewage isolates were echovirus 11; in 2000, 33.3% of the EVs from clinical cases and 40.7% of the sewage isolates were coxsackievirus B5; and in 2001, 44.1% of the EVs from clinical cases and 36.2% of the sewage isolates were echovirus 13. Annual peaks of both sewage EV titers and clinical cases occurred in late summer or early fall. In some years, early spring sewage EVs portended some of the EVs that would predominate clinically during the following summer.     

Assessment of an Enterovirus Sewage Surveillance System by Comparison of Clinical Isolates with Sewage Isolates from Milwaukee, Wisconsin, Collected August 1994 to December 2002

The quantity and serotypes of enteroviruses (EVs) in the influent of a local sewage treatment plant were compared to local clinical EV cases to determine if testing of sewage is adequate for an EV surveillance system. The study was carried out from August 1994 to December 2002. Monthly influent specimens were processed by organic flocculation, and dilutions of concentrate were inoculated onto a number of different cell types for virus isolation. EVs were detected in 88 of 100 monthly influent samples. Sewage EV titers were calculated by using software provided by the U.S. Environmental Protection Agency for most-probable-number determination. All 1,068 sewage EV isolates were further grouped (echovirus, coxsackievirus B, coxsackievirus A, or poliovirus) by cell culture host range analysis (growth pattern of isolates on passage to seven cell lines), and 39.0% of the 1,022 EV isolates categorized as non-poliovirus EVs were specifically serotyped. For clinical cases, primary virus isolation tests were performed on specimens submitted by local hospitals and EV isolates submitted by hospitals were serotyped. Clinical EVs were documented for 81 of the 100 months studied. In all, 694 EV isolates from clinical cases were serotyped. Annually, between 4 and 11 different serotypes of non-poliovirus EVs were identified in sewage and from 9 to 19 different non-poliovirus EV serotypes were identified from clinical specimens. Usually, the most commonly detected sewage EV serotypes were similar to the most commonly detected clinical serotypes; e.g., for 1997, echovirus 6 accounted for 53.1% of the typed sewage isolates and 39.4% of the clinical infections, while in 1998, echovirus 30 accounted for 50.0 and 46.1%, respectively. In 1999, 60.3% of the EVs from clinical cases and 79.7% of the sewage isolates were echovirus 11; in 2000, 33.3% of the EVs from clinical cases and 40.7% of the sewage isolates were coxsackievirus B5; and in 2001, 44.1% of the EVs from clinical cases and 36.2% of the sewage isolates were echovirus 13. Annual peaks of both sewage EV titers and clinical cases occurred in late summer or early fall. In some years, early spring sewage EVs portended some of the EVs that would predominate clinically during the following summer.     

Commonly used methods for extracellular vesicles’ enrichment: Implications in downstream analyses and use

Extracellular vesicles (EVs) are becoming promising tools for clinical application, either as sources of disease-specific molecular signatures for the unraveling of disease pathophysiology and establishment of novel biomarkers, or as platforms for cell-free nanotherapy. Yet, an unsolved issue is to define standardized techniques for EV isolation allowing data comparison across laboratories worldwide. Considering the difficulties to find this necessary consensus, it has to be stressed out that the outcome of the downstream analysis might be deeply biased by the isolation method, among other variables. Thus, it is crucial that the researcher is aware of the strengths and weaknesses of each method keeping their intended use in mind, and to sufficiently report the methodology details for the results to be comparable and solid. This review aims to present the most widely used EV isolation methods, from the initial differential ultracentrifugation (dUC) to newest approaches.     

Toward Standardization of Mesenchymal Stromal Cell‐Derived Extracellular Vesicles for Therapeutic Use: A Call for Action

Extracellular vesicles (EVs), which include a variety of nano‐sized membrane‐encapsulated particles, are released to the extracellular microenvironment by the vast majority of cells and carry lipids, proteins, mRNA, and miRNA or non‐coding RNA. Increasing evidence suggests the great versatility and potential of EV‐based applications in humans. In this issue, van Balkom et al. explore and compare the reported proteomic signature of mesenchymal stromal cell (MSC)‐derived small EVs. In particular, their paper offers a valuable approach and point of view on MSC‐EV manufacturing and therapeutic potential. Briefly, van Balkom et al. aimed to identify a common protein signature that may be useful in ensuring the homogeneity of therapeutic MSC‐EVs. In addition to excessive variability in EV‐producing cell sources and culture conditions, the harvesting time for the EV‐containing conditioned medium, and EV isolation procedure, the authors found a specific protein signature from the publicly available MSC‐EVs proteome. In light of their findings and those from the plentiful studies published in this continuously growing area of research, potential focus areas and issues are outlined for the more rational design and optimization of MSC‐EV production and potency for therapeutics.     

Effect of cell culture media on extracellular vesicle secretion from mesenchymal stromal cells and neurons

BackgroundExtracellular vesicles (EVs) secreted by neuronal cells in vitro have promising therapeutic potential for brain diseases. Optimization of cell culture conditions and methodologies for high-yield isolation of EVs for preclinical and clinical applications, however, remains a challenge.ObjectiveTo probe the cell culture conditions required for optimal EV secretion by human-derived neuronal cells.MethodologyFirst, we optimized the EV purification protocol using human mesenchymal stromal cell (MSC) cultures. Next, we compared the effects of different variables in human pluripotent stem cell (hPSC)-derived neuronal cultures on EV secretion. EVs were isolated from cell conditioned media (CCM) and control media with no cells (NCC) using ultrafiltration combined with size-exclusion chromatography (SEC). The hPSC neurons were cultured in 2 different media from which EVs were collected at 2 maturation time-points (days 46 and 60). Stimulation with 25 mM KCl was also evaluated as an activator of EV secretion by neurons. The collected SEC fractions were analyzed by nanoparticle tracking analysis (NTA), protein concentration assay, and blinded transmission electron microscopy (TEM).ResultsA peak in cup-shaped particles was observed in SEC fractions 7–10 of MSC samples, but not corresponding media controls, indicating successful isolation of EVs. Culture medium had no significant effect on EV yield. The EV yield of the samples did not differ significantly according to the culture media used or the cell maturation time-points. Stimulation of neurons with KCl for 3 h reduced rather than increased the EV yield.ConclusionsWe demonstrated successful EV isolation from MSC and neuronal cells using an ultrafiltration-SEC method. The EV yield from MSC and neuronal cultures exhibited a large batch effect, apparently related to the culture media used, highlighting the importance of including NCC as a negative control in all cell culture experiments.     

A method of separating extracellular vesicles from blood shows potential clinical translation,and reveals extracellular vesicle cargo gremlin-1 as a diagnostic biomarker

Extracellular vesicles (EVs) have potential as minimally invasive biomarkers. However, the methods most commonly used for EV retrieval rely on ultracentrifugation, are time-consuming, and unrealistic to translate to standard-of-care. We sought a method suitable for EV separation from blood that could be used in patient care. Sera from breast cancer patients and age-matched controls (n = 27 patients; n = 36 controls) were analysed to compare 6 proposed EV separation methods. The EVs were then characterised on 8 parameters. The selected method was subsequently applied to independent cohorts of sera (n = 20 patients; n = 20 controls), as proof-of-principle, investigating EVs’ gremlin-1 cargo. Three independent runs with each method were very reproducible, within each given method. All isolates contained EVs, although they varied in quantity and purity. Methods that require ultracentrifugation were not superior for low volumes of sera typically available in routine standard-of-care. A CD63/CD81/CD9-coated immunobead-based method was most suitable based on EV markers'' detection and minimal albumin and lipoprotein contamination. Applying this method to independent sera cohorts, EVs and their gremlin-1 cargo were at significantly higher amounts for breast cancer patients compared to controls. In conclusion, CD63/CD81/CD9-coated immunobeads may enable clinical utility of blood-based EVs as biomarkers.     

Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination

Extracellular vesicles (EVs) are widely implicated as novel diagnostic and therapeutic modalities for a wide range of diseases. Thus, optimization of EV biomanufacturing is of high interest. In the course of developing parameters for a human embryonic kidney cells (HEK293T) EV production platform, we examined the combinatorial effects of cell culture conditions (i.e., static versus dynamic) and isolation techniques (i.e., ultracentrifugation versus tangential flow filtration versus size-exclusion chromatography) on functional characteristics of HEK293T EVs, including anti-inflammatory bioactivity using a well-established lipopolysaccharide-stimulated mouse macrophage model. We unexpectedly found that, depending on culture condition and isolation strategy, HEK293T EVs appeared to significantly suppress the secretion of pro-inflammatory cytokines (i.e., interleukin-6, RANTES [regulated upon activation, normal T cell expressed and secreted]) in the stimulated mouse macrophages. Further examination revealed that these results were most likely due to non-EV fetal bovine serum components in HEK293T EV preparations. Thus, future research assessing the anti-inflammatory effects of EVs should be designed to account for this phenomenon.     

Proteomic Analysis of Extracellular Vesicles for Cancer Diagnostics

Extracellular vesicles (EVs) including exosomes and microvesicles are lipid bilayer‐encapsulated nanoparticles released by cells, ranging from 40 nm to several microns in diameter. Biological cargoes including proteins, RNAs, and DNAs can be ferried by EVs to neighboring and distant cells via biofluids, serving as a means of cell‐to‐cell communication under normal and pathological conditions, especially cancers. On the other hand, EVs have been investigated as a novel “information capsule” for early disease detection and monitoring via liquid biopsy. This review summarizes current advancements in EV subtype characterization, cancer EV capture, proteomic analysis technologies, as well as possible EV‐based multiomics for cancer diagnostics.     

Deep Sequencing of RNA from Three Different Extracellular Vesicle (EV) Subtypes Released from the Human LIM1863 Colon Cancer Cell Line Uncovers Distinct Mirna-Enrichment Signatures

Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863 – shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs - miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.     

Production cell analysis and compound-based boosting of small extracellular vesicle secretion using a generic and scalable production platform

Extracellular vesicles (EVs) are a novel format of advanced therapeutical medicinal products (ATMPs). They can act regenerative or immune-modulatory as cell therapy substitutes or as a platform for designer exosomes. The biotechnological production of therapeutic EVs is still very much uncharted territory so standardized host cells, production setups, and isolation methods are not yet implemented. In this work, we present a tangential flow filtration (TFF) and fast-performance liquid chromatography (FPLC)-based size exclusion chromatography (SEC) purification setup that is compatible for industry applications. Moreover, we evaluated a series of potential host cell lines regarding their EV productivity, characteristics, and biological functionality. It was found that telomerase-immortalized Wharton's jelly mesenchymal stromal cells (WJ-MSC/TERT273) secrete high amounts of EVs per cell with regenerative capabilities. On the other hand, Cevec's amniocyte producer cells® (CAP®) and human embryonic kidney (HEK293) suspension cells are suitable platforms for designer EVs with high yields. Finally, we aimed to boost the EV secretion of HEK293 cells via chemical adjuvants and verified four compounds that heighten cellular EV secretion in a presumably cAMP-dependent manner. A combination of fenoterol, iodoacetamide, and dinitrophenol increased the EV yield in HEK293 cells threefold and cellular secretion rate fivefold.     

Imaging flow cytometry challenges the usefulness of classically used extracellular vesicle labeling dyes and qualifies the novel dye Exoria for the labeling of mesenchymal stromal cell–extracellular vesicle preparations

Background aimsExtracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is assumed to be important for EVs in mediating intercellular communication processes, labeling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of EV target cells and tissues. However, the accuracy and specificity of commonly utilized labeling dyes have not been sufficiently analyzed.MethodsBy combining recent advances in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant mesenchymal stromal cell (MSC)-EV preparations, the authors explored the EV labeling efficacy of various fluorescent dyes, specifically carboxyfluorescein diacetate succinimidyl ester, calcein AM, PKH67, BODIPY TR ceramide (Thermo Fisher Scientific, Darmstadt, Germany) and a novel lipid dye called Exoria (Exopharm Limited, Melbourne, Australia).ResultsThe authors’ analyses qualified Exoria as the only dye that specifically labeled EVs within the MSC-EV preparations. Furthermore, the authors demonstrated that Exoria labeling did not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labeled EVs were differentially taken up by different immune cell types.ConclusionsOverall, the results qualify Exoria as an appropriate dye for the labeling of EVs derived from the authors’ MSC-EV preparations. This study also demonstrates the need for the development of next-generation EV characterization tools that are able to localize and confirm the specificity of EV labeling.     

What is the blood concentration of extracellular vesicles? Implications for the use of extracellular vesicles as blood-borne biomarkers of cancer

Circulating biomarkers have a great potential in diagnosing cancer diseases at early stages, where curative treatment is a realistic possibility. In the recent years, using extracellular vesicles (EVs) derived from blood as biomarkers has gained widespread popularity, mainly because they are thought to be easy to isolate and carry a vast variety of biological cargos that can be analyzed for biomarker purposes. However, our current knowledge on the plasma EV concentration in normophysiological states is sparse. Here, we provide the very first mean estimate of the plasma EV concentration based on values obtained from a thorough literature review. The different estimates obtained from the literature are correlated to the isolation techniques used to obtain them, illustrating how some methodologies may over- or underestimate the plasma EV concentration. We also show that the estimated plasma EV concentration (approximately 10¹⁰ EVs per mL) defines EVs as a minority population compared to other colloidal particles of the systemic circulation, namely the lipoproteins, which are known contaminants in EV isolates and carry biomarker molecules themselves. Lastly, we introduce the possibility of regarding EVs and lipoproteins as a continuum of lipid-containing particles to which biomarker molecules can be associated. Using such a holistic approach, increased strength of plasma-derived cancer biomarkers may soon be revealed.     

Extracellular vesicles concentration is a promising and important parameter for industrial bioprocess monitoring

Extracellular vesicles (EVs) are membrane vesicles that are produced by cells to be released into their microenvironment. In this study, we present the EV concentration as a new factor for optimization of industrial bioprocess control. The release of EVs depends on many cell properties, including cell activation and stress status, and cell death. Therefore, the EV concentration might provide a readout for identification of the cell state and the conditions during a bioprocess. Our data show that the EV concentration increased during the bioprocess, which indicated deteriorating conditions in the bioreactor. This increase in EV concentration in the fermentation broth was the consequence of two different processes: cell activation, and cell death. However, the release of EVs from activated living cells had a much weaker impact on EV concentration in the bioreactor than those released during cell death. EVs and cells in the bioprocess environment were quantified by flow cytometry. The most accurate data were obtained directly from unprocessed samples, making the monitoring of the EV concentration a rapid, easy, and cheap method. These EV concentrations reflect the conditions in the bioreactor and provide new information regarding the state of the bioprocess. Therefore, we suggest EV concentration as a new and important parameter for the monitoring of industrial bioprocesses.     

Optimizing Size Exclusion Chromatography for Extracellular Vesicle Enrichment and Proteomic Analysis from Clinically Relevant Samples

The field of extracellular vesicle (EV) research has rapidly expanded in recent years, with particular interest in their potential as circulating biomarkers. Proteomic analysis of EVs from clinical samples is complicated by the low abundance of EV proteins relative to highly abundant circulating proteins such as albumin and apolipoproteins. To overcome this, size exclusion chromatography (SEC) has been proposed as a method to enrich EVs whilst depleting protein contaminants; however, the optimal SEC parameters for EV proteomics have not been thoroughly investigated. Here, quantitative evaluation and optimization of SEC are reported for separating EVs from contaminating proteins. Using a synthetic model system followed by cell line‐derived EVs, it is found that a 10 mL Sepharose 4B column in PBS produces optimal resolution of EVs from background protein. By spiking‐in cancer cell‐derived EVs to healthy plasma, it is shown that some cancer EV‐associated proteins are detectable by nano‐LC‐MS/MS when as little as 1% of the total plasma EV number are derived from a cancer cell line. These results suggest that an optimized SEC and nanoLC‐MS/MS workflow may be sufficiently sensitive for disease EV protein biomarker discovery from patient‐derived clinical samples.     

A novel 3D heterotypic spheroid model for studying extracellular vesicle-mediated tumour and immune cell communication

Cancer-derived extracellular vesicles (EVs) have emerged as important mediators of tumour-host interactions, and they have been shown to exert various functional effects in immune cells. In most of the studies on human immune cells, EVs have been isolated from cancer cell culture medium or patients' body fluids and added to the immune cell cultures. In such a setting, the physiological relevance of the chosen EV concentration is unknown and the EV isolation method and the timing of EV administration may bias the results. In the current study we aimed to develop an experimental cell culture model to study EV-mediated effects in human T and B cells at conditions mimicking the tumour microenvironment. We constructed a human prostate cancer cell line PC3 producing GFP-tagged EVs (PC3-CD63-GFP cells) and developed a 3D heterotypic spheroid model composed of PC3-CD63-GFP cells and human peripheral blood mononuclear cells (PBMCs). The transfer of GFP-tagged EVs from PC3-CD63-GFP cells to the lymphocytes was analysed by flow cytometry and fluorescence imaging. The endocytic pathway was investigated using three endocytosis inhibitors. Our results showed that GFP-tagged EVs interacted with a large fraction of B cells, however, the majority of EVs were not internalised by B cells but rather remained bound at the cell surface. T cell subsets differed in their ability to interact with the EVs - 15.7–24.1% of the total CD3⁺ T cell population interacted with GFP-tagged EVs, while only 0.3–5.8% of CD8⁺ T were GFP positive. Furthermore, a fraction of EVs were internalised in CD3⁺ T cells via macropinocytosis. Taken together, the heterotypic PC3-CD63-GFP and PBMC spheroid model provides the opportunity to study the interactions and functional effects of cancer-derived EVs in human immune cells at conditions mimicking the tumour microenvironment.