Extracellular vesicle-based biosensors for Alzheimer's disease: A new frontier in precision diagnostics.

Extracellular Vesicles: How a Circulating Biomarker Can Double As a Regulator of Blood Pressure.

Author response: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

MP18-13 IDENTIFICATION OF TUMOR-SPECIFIC MARKERS ON EXTRACELLULAR VESICLES IN PATIENTS WITH RENAL CELL CARCINOMA

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

Extracellular vesicles (EVs) are a heterogeneous group of nanoparticles possessing a lipid bilayer membrane that plays a significant role in intercellular communication by transferring their cargoes, consisting of peptides, proteins, fatty acids, DNA, and RNA, to receiver cells. Isolation of EVs is cumbersome and time-consuming due to their nano size and the co-isolation of small molecules along with EVs. This is why current protocols for the isolation of EVs are unable to provide high purity. So far, studies have focused on EVs derived from cell supernatants or body fluids but are associated with a number of limitations. Cell lines with a high passage number cannot be considered as representative of the original cell type, and EVs isolated from those can present distinct properties and characteristics. Additionally, cultured cells only have a single cell type and do not possess any cellular interactions with other types of cells, which normally exist in the tissue microenvironment. Therefore, studies involving the direct EVs isolation from whole tissues can provide a better understanding of intercellular communication in vivo. This underscores the critical need to standardize and optimize protocols for isolating and characterizing EVs from tissues. We have developed a differential centrifugation-based technique to isolate and characterize EVs from whole adipose tissue, which can be potentially applied to other types of tissues. This may help us to better understand the role of EVs in the tissue microenvironment in both diseased and normal conditions. Key features • Isolation of tissue-derived extracellular vesicles from ex vivo culture of visceral adipose tissue or any whole tissue. • Microscopic visualization of extracellular vesicles' morphology without dehydration steps, with minimum effect on their shape. • Flow cytometry approach to characterize the extracellular vesicles using specific protein markers, as an alternative to the time-consuming western blot.

BackgroundLipid dyshomeostasis is associated with various disorders including Alzheimer’s disease (AD). The work from our laboratory (Su et al, JEV 2021), and others, suggests that extracellular vesicles (EVs) contain lipid biomarkers that could aid in the diagnosis of AD and diseases associated with impaired lipid metabolism.Knowing the lipid content of EVs is a key first step to discovering EV‐based lipid biomarkers in blood. Some studies have reported the lipid content of ‘’EVs’’ in blood. However, used EV isolation techniques that are known to co‐isolate free lipid and lipoproteins.Here we: 1) demonstrate the importance of size exclusion resin size to isolating highly enriched EVs for lipidomic analysis, 2) have developed a reference EV plasma lipidome for the research community and 3) determining if plasma EVs report on lipid dyshomeostasis in Alzheimer’s disease.MethodHuman plasma EVs were isolated using an assortment of commercially available kits or by density fractionation and size exclusion chromatography and characterised by western blot, transmission electron microscopy, and quantitative mass spectrometry based proteomic and lipidome analysis.ResultOf the high‐throughput EV isolation kits tested, the majority were unable to separate lipoproteins from EVs for the purposes of EV lipidomics. We then relied on comparing labor intensive methods (density gradient and size exclusion) to identify a combination of techniques that isolate the analytes of interest, EV proteins and lipids. Quantitative proteome and lipidome analysis strategies are being applied to highly enriched EVs with the goal of providing the reference lipidome of EVs in plasma and in Alzheimer’s disease.ConclusionTo enhance the confidence of EV lipid identification and reveal the lipidome of EVs in plasma, our study compared and identified techniques to enrich EVs and deplete non‐EV associated lipid and protein from human plasma. These findings serve as a reference resource and the foundation for research investigating plasma EV lipids as Alzheimer’s disease biomarkers.

Objective: Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since they are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, EVs represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation (UC-EV) are laborious, time-consuming, and require high inputs. Method: A recently described isolation method, which can be performed at small ‘miniprep’ scale, utilizes specific Heat Shock Protein (HSP)-binding peptides to aggregate HSP-decorated EVs (Ghosh et al. (2014), PLoS ONE 9:e110443). The authors showed comparable results for their method (abbreviated HSP-EV here) and UC-EV, but a detailed proteomic comparison was lacking. Therefore, we compared both methods using label-free proteomics of replicate EV isolations from HT-29 cancer cell-conditioned medium. Subsequently we applied this technique on secretomes of fresh human colorectal cancer (CRC) (n=17) and colon adenoma (n=4) tissue as well as patient-matched normal colon tissue. Results: Despite a 30-fold different input scale (UC-EV: 60 ml versus HSP-EV: 2 ml), both methods yielded comparable numbers of identified proteins (3115 versus 3085), with reproducible identifications (72.5% versus 75.5%) and spectral count-based quantification (average CV 31% versus 27%). EVs obtained by either method contained established EV markers and proteins linked to vesicle-related gene ontologies. In the EV fraction of the tissue secretomes 6390 proteins were identified, of which 471 proteins were significantly 5-fold more present in CRC samples than in normal tissue EVs. Gene ontology analysis revealed enrichment of nuclear proteins involved in DNA damage response, chromosome organization and RNA processing in the CRC EVs. Conclusions: The HSP-EV method provides an advantageous, simple and rapid approach for EV isolation from small amounts of biological samples, enabling high-throughput analysis in a biomarker discovery setting. Citation Format: Meike De Wit, Jaco Knol, Inge de Reus, Tim Tim Schelfhorst, Logan Bishop-Currey, Nicole van Grieken, Sander Piersma, Thang V. Pham, Remond J. Fijneman, Gerrit A. Meijer, Henk Verheul, Connie R. Jimenez. Peptide-mediated 'miniprep' isolation of extracellular vesicles is suitable for high-throughput proteomics; method evaluation and application in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2208. doi:10.1158/1538-7445.AM2017-2208

Assessing the role of extracellular vesicles in renin-angiotensin system signalling in cardiomyocyte hypertrophy

Introduction: Extracellular vesicles (EVs) contain protein and nucleic cargo that has been shown to be reflective of physiological and pathological states, or their originating cells, in many diseases including cancer. Specifically, these EV-associated biomarkers have been shown to fluctuate with the change in pathological processes. As a result, characterization of blood-based circulating EVs have been widely investigated for diagnostic applications such as disease monitoring and treatment selection. The heterogeneity of blood-based EVs present significant challenges to current rapid isolation methods due to enrichment with unwanted sub-populations, e.g. apoptotic bodies, or contamination with lipids, e.g. APOB, for both capture-based chemistry-based isolation methods. We have developed a novel lab-on-a-chip technology for isolation and on-chip characterization of EVs from blood-based matrices using an AC electrokinetics (ACE) methodology. Methods: Blood samples were collected from 10 healthy volunteers and 10 donors with known cancer diagnosis into K2EDTA tubes under IRB approved protocols. Plasma was processed from the blood and stored at -80C. 120 µL of thawed plasma was applied to the microelectrode array flow cell (ExoVerita Flex, Biological Dynamics, San Diego, CA) and the EVs were isolated on the microelectrodes. Following isolation, the flow cell was washed, the isolated material was released from the array and eluted from the flow cell. For comparison, isolation of EVs from the same amount of plasma was performed using a chemistry-based method (ExoQuick Ultra, System Biosciences, Palo Alto, CA) according to manufacturer's instructions. Following isolation, the eluates were evaluated for the presence of EV-associated biomarkers using Western blotting capillary electrophoresis and mass spectrometry (Orbitrap Fusion Lumos, TFS). Nanoparticle tracking analysis (qNano Gold, Izon Science, New Zealand) was performed to determine EV concentration and size distribution. Quantitative qRT-PCR analysis of the purified plasma Evs was performed to confirm presence of EV-bound mRNA. Results: Western Blotting demonstrated that eluates from both ACE-based methodology and chemistry-based isolation contain the expected CD9 and CD63. The ACE-isolated EVs showed a single prominent band for CD63, whereas chemistry-based isolated EVs display multiple smaller CD63-reactive species, which was suggestive of proteolysis. Mass spectrometry analysis confirmed presence of CD9 and CD81 in EVs isolated by ACE method; but not in EVs purified by chemistry-based method. APOB contamination was present in chemistry-based EVs, but not in ACE-isolated EVs. For RT-PCR experiments, EVs were purified from 5 subjects with lung cancer and 3 subjects with melanoma patient plasma using ACE method. Quantitative PCR confirmed presence of mRNA, via successful amplification of the housekeeping genes PGK1 and β-actin, in all ACE-isolated EVs. Conclusions: The novel ACE-based platform successfully demonstrated direct isolation of EVs from plasma while preserving the integrity of protein and mRNA biomarkers. The compatibility of the eluted EVs with multiple downstream technologies, such as mass spectrometry, Western blotting and qRT-PCR, may enable novel biomarker discovery and use of EV-associated biomarkers in diagnostic assays. Citation Format: Rajaram Krishnan, Jean Lewis, David Searson, Orlando Perrera, Alfred Kinana, Heath Balcer, Iryna Clark, Juan Pablo Hinestrosa. Characterization of circulating extracellular vesicles isolated from plasma of cancer subjects using novel AC electrokinetics platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2850.

Extracellular vesicles (EVs) are gaining increased importance in fundamental research as key players in disease pathogenic mechanisms, but also in translational and clinical research due to their value in biomarker discovery, either for diagnostics and/or therapeutics. In the first research scenario, the study of EVs isolated from neuronal models mimicking neurodegenerative diseases can open new avenues to better understand the pathological mechanisms underlying these conditions or to identify novel molecular targets for diagnosis and/or therapeutics. In the second research scenario, the easy availability of EVs in body fluids and the specificity of their cargo, which can reflect the cell of origin or disease profiles, turn these into attractive diagnostic tools. EVs with exosome‐like characteristics, circulating in the bloodstream and other peripheral biofluids, constitute a non‐invasive and rapid alternative to study several conditions, including brain‐related disorders. In both cases, several EVs isolation methods are already available, but each neuronal model or biofluid presents its own challenges. Herein, a literature overview on EVs isolation methodologies from distinct neuronal models (cellular culture and brain tissue) and body fluids (serum, plasma, cerebrospinal fluid, urine and saliva) was carried out. Focus was given to approaches employed in the context of Alzheimer's and Parkinson's diseases, and the main research findings discussed. The topics here revised will facilitate the choice of EVs isolation methodologies and potentially prompt new discoveries in EVs research and in the neurodegenerative diseases field.

BackgroundInfection and graft-versus-host disease (GvHD) are the major causes for mortality and morbidity of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Plasma-derived extracellular vesicles (EVs) contain disease-related proteins, DNAs and RNAs, and have recently been suggested as potential biomarker candidates for transplantation complications. However, EV isolation from small plasma volumes in clinical biomarker studies using conventional methods is challenging. We therefore investigated if EVs isolated by novel automated acoustic trapping could be developed as potential biomarkers for allo-HSCT complications by performing a clinical proof-of-principle study.ResultsPlasma samples were collected from twenty consecutive patients with high-risk/relapsed hematologic malignancies undergoing allo-HSCT before transplantation and post-transplant up to 12 weeks. EVs were isolated from small plasma sample volumes (150 μl) by an automated, acoustofluidic-based particle trapping device, which utilizes a local λ/2 ultrasonic standing wave in a borosilicate glass capillary to capture plasma EVs among pre-seeded polystyrene microbeads through sound scatter interactions. We found that EVs could be reliably isolated from all plasma samples (n = 173) and that EV numbers increased more than 2-fold in the majority of patients after transplantation. Also, sufficient quantities of RNA for downstream microRNA (miRNA) analysis were obtained from all samples and EV miRNA profiles were found to differ from whole plasma profiles. As a proof of principle, expression of platelet-specific miR-142-3p in EVs was shown to correlate with platelet count kinetics after transplantation as expected. Importantly, we identified plasma EV miRNAs that were consistently positively correlated with infection and GvHD, respectively, as well as miRNAs that were consistently negatively correlated with these complications.ConclusionsThis study demonstrates that acoustic enrichment of EVs in a clinical biomarker study setting is feasible and that downstream analysis of acoustically-enriched EVs presents a promising tool for biomarker development in allo-HSCT. Certainly, these findings warrant further exploration in larger studies, which will have significant implications not only for biomarker studies in transplantation but also for the broad field of EV-based biomarker discovery.

Emergence of Extracellular Vesicles as “Liquid Biopsy” for Neurological Disorders: Boom or Bust

Isolation and digital counting of extracellular vesicles from blood via membrane-integrated microfluidics

Background: Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since they are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, EVs represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation (UC-EV) are laborious, time-consuming, and require high inputs. Recently a novel isolation method was described, which can be performed at small ‘miniprep’ scale, utilizes specific Heat Shock Protein (HSP)-binding peptides to aggregate HSP-decorated EVs (Ghosh et al. (2014), PLoS ONE 9:e110443). Using cancer secretome and biofluid samples, the authors showed enrichment of exosome markers for their method (abbreviated HSP-EV here) but a detailed description of the captured EV proteomes in comparison to the gold-standard ultracentrifugation (UC) method and application to small tumor proximal fluid samples is lacking. Approach: Here we used label-free proteomics of replicate EV isolations from HT-29 cancer cell-conditioned medium to compare EV fractions captured using the new HSP peptide method and UC. Subsequently we applied this novel method to profile EVs released from fresh human colorectal tumors (CRC) (n = 17) and colon adenoma (n = 4) tissue as well as patient-matched normal colon tissue. Results: Despite a 30-fold different input scale (UC-EV: 60 ml versus HSP-EV: 2 ml), both methods yielded comparable numbers of identified proteins (3115 versus 3085), with reproducible identifications (72.5% versus 75.5%) and spectral count-based quantification (average CV 31% versus 27%). EVs obtained by either method contained established EV markers and proteins linked to vesicle-related gene ontologies. In the EV fraction of the tissue secretomes 6390 proteins were identified, of which 471 proteins were significantly 5-fold more present in CRC samples than in normal tissue EVs. Gene ontology analysis revealed enrichment of nuclear proteins involved in DNA damage response, chromosome organization and RNA processing in the CRC EVs. Conclusion: The HSP-EV method provides an advantageous, simple and rapid approach for EV isolation from small amounts of biological samples, enabling high-throughput analysis in a biomarker discovery setting. Citation Format: Connie R. Jimenez, Meike de Wit, Jaco C. Knol, Inge de Reus, Tim Schelfhorst, Logan Bishop-Currey, Valerie Dusseldorp, Nicole van Grieken, Robin Beekhof, Sander R. Piersma, Thang V. Pham, Egbert F. Smit, Remond JA Fijneman, Gerrit Meijer, Henk MW Verheul. Peptide-mediated ‘miniprep’ isolation of extracellular vesicles for high-throughput proteomics; method evaluation and application in colon cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3885.

Response of Serum-Isolated Extracellular Vesicles to Focused Ultrasound-Mediated Blood-Brain Barrier Opening.