Nano Pom-poms Prepared Highly Specific Extracellular Vesicles Expand the Detectable Cancer Biomarkers

AbstractExtracellular vesicles (EVs), particularly exosomes, are emerging biomarker sources. However, due to heterogeneous populations secreted from diverse cell types, mapping EV multi-omic molecular information specifically to their pathogenesis origin for cancer biomarker identification is still extraordinary challenging. Herein, we introduced a novel 3D-structured nanographene immunomagnetic particles (NanoPoms) with unique flower pom-poms morphology and photo-click chemistry for specific marker-defined capture and release of intact small EVs. This specific EV isolation approach leads to the expanded identification of targetable cancer biomarkers with enhanced specificity and sensitivity, as demonstrated by multi-omic EV analysis of bladder cancer patient tissue fluids using the next generation sequencing of somatic DNA mutations, miRNAs, and the global proteome. The NanoPoms prepared sEVs also exhibit distinctive in vivo biodistribution patterns, highlighting the highly viable and integral quality. The developed method is simple and straightforward, and is applicable to nearly all types of biological fluids and amenable for scale up and high-throughput EV isolation.

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Nano Pom-Poms Prepared Exosomes for Highly Specific Cancer Biomarker Detection

10.21203/rs.3.rs-977580/v1 ◽

2021 ◽

Author(s):

Nan He ◽

Sirisha Thippabhotla ◽

Cuncong Zhong ◽

Zachary Greenberg ◽

Liang Xu ◽

...

Keyword(s):

Biological Fluids ◽

Scale Up ◽

Cell Types ◽

Cancer Biomarker ◽

Specific Marker ◽

Bladder Cancer Patient ◽

Dna Mutations ◽

Specificity And Sensitivity ◽

Tissue Fluids

Abstract Extracellular vesicles (EVs), particularly nano-sized small EV exosomes, are emerging biomarker sources. However, due to heterogeneous populations secreted from diverse cell types, mapping exosome multi-omic molecular information specifically to their pathogenesis origin for cancer biomarker identification is still extraordinarily challenging. Herein, we introduced a novel 3D-structured nanographene immunomagnetic particles (NanoPoms) with unique flower pom-poms morphology and photo-click chemistry for specific marker-defined capture and release of intact exosome. This specific exosome isolation approach leads to the expanded identification of targetable cancer biomarkers with enhanced specificity and sensitivity, as demonstrated by multi-omic exosome analysis of bladder cancer patient tissue fluids using the next generation sequencing of somatic DNA mutations, miRNAs, and the global proteome. The NanoPoms prepared exosomes also exhibit distinctive in vivo biodistribution patterns, highlighting the highly viable and integral quality. The developed method is simple and straightforward, which is applicable to nearly all types of biological fluids and amenable for enrichment, scale up, and high-throughput exosome isolation.

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NANOmetric BIO-Banked MSC-Derived Exosome (NANOBIOME) as a Novel Approach to Regenerative Medicine

Journal of Clinical Medicine ◽

10.3390/jcm7100357 ◽

2018 ◽

Vol 7 (10) ◽

pp. 357 ◽

Cited By ~ 7

Author(s):

Bruna Codispoti ◽

Massimo Marrelli ◽

Francesco Paduano ◽

Marco Tatullo

Keyword(s):

Plasma Membrane ◽

Regenerative Medicine ◽

Extracellular Vesicles ◽

Biological Fluids ◽

Cell Types ◽

Endogenous Factors ◽

Cell Lineages ◽

Novel Approach ◽

Therapeutic Uses ◽

Technical Issues

Mesenchymal stem cells (MSCs) are well known for their great potential in clinical applications. In fact, MSCs can differentiate into several cell lineages and show paracrine behavior by releasing endogenous factors that stimulate tissue repair and modulate local immune response. Each MSC type is affected by specific biobanking issues—technical issues as well as regulatory and ethical concerns—thus making it quite tricky to safely and commonly use MSC banking for swift regenerative applications. Extracellular vesicles (EVs) include a group of 150–1000 nm vesicles that are released by budding from the plasma membrane into biological fluids and/or in the culture medium from varied and heterogenic cell types. EVs consist of various vesicle types that are defined with different nomenclature such as exosomes, shedding vesicles, nanoparticles, microvesicles and apoptotic bodies. Ectosomes, micro- and nanoparticles generally refer to the direct release of single vesicles from the plasma membrane. While many studies describe exosomes as deriving from multivesicular bodies, solid evidence about the origin of EVs is often lacking. Extracellular vesicles represent an important portion of the cell secretome. Their numerous properties can be used for diagnostic, prognostic, and therapeutic uses, so EVs are considered to be innovative and smart theranostic tools. The aim of this review is to investigate the usefulness of exosomes as carriers of the whole information panel characterizing the use of MSCs in regenerative medicine. Our purpose is to make a step forward in the development of the NANOmetric BIO-banked MSC-derived Exosome (NANOBIOME).

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Tetraspanin immunocapture phenotypes extracellular vesicles according to biofluid source but may limit identification of multiplexed cancer biomarkers

10.1101/2021.03.02.433595 ◽

2021 ◽

Author(s):

Rachel R. Mizenko ◽

Terza Brostoff ◽

Tatu Rojalin ◽

Hanna J. Koster ◽

Hila S. Swindell ◽

...

Keyword(s):

Flow Cytometry ◽

Extracellular Vesicles ◽

Fluorescence Detection ◽

Single Particle ◽

Cancer Biomarkers ◽

Surface Markers ◽

Isolation Method ◽

Standard Methods

AbstractTetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their general detection and classification from background contaminants. This common practice typically assumes a consistent expression of tetraspanins across EV sources, thus obscuring subpopulations of variable or limited tetraspanin expression. While some recent studies indicate differential expression of tetraspanins across bulk isolated EVs, here we present analysis of single EVs isolated using various field-standard methods from a variety of in vitro and in vivo sources to identify distinct patterns in colocalization of tetraspanin expression. We report an optimized method for the use of antibodycapture single particle interferometric reflectance imaging sensing (SP-IRIS) and fluorescence detection to identify subpopulations according to tetraspanin expression and compare our findings with nanoscale flow cytometry. Using SP-IRIS and immunofluorescence, we report that tetraspanin profile is consistent from a given EV source regardless of isolation method, but that tetraspanin profiles are distinct across various sources. Tetraspanin profiles as measured by flow cytometry do not share similar trends, suggesting that limitations in subpopulation detection significantly impact apparent protein expression. We further analyzed tetraspanin expression of single EVs captured non-specifically, revealing that tetraspanin capture can bias the apparent multiplexed tetraspanin profile. Finally, we demonstrate that this bias can have significant impact on diagnostic sensitivity for tumor-associated EV surface markers. Our findings may reveal key insights into the complexities of the EV biogenesis and signaling pathways and better inform EV capture and detection platforms for diagnostic or other downstream use.

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Postprandial transfer of colostral extracellular vesicles and their protein and miRNA cargo in neonatal calves

10.21203/rs.2.10032/v1 ◽

2019 ◽

Author(s):

Benedikt Kirchner ◽

Dominik Buschmann ◽

Vijay Paul ◽

Michael W. Pfaffl

Keyword(s):

Extracellular Vesicles ◽

Expression Profiles ◽

Bovine Milk ◽

Cell Types ◽

Specific Protein ◽

In Vivo Experiments ◽

Neonatal Calves ◽

Almost All

Abstract Background Extracellular vesicles (EVs) such as exosomes are key regulators of intercellular communication that can be found in almost all bio fluids. Although studies in the last decade have made great headway in discerning the role of EVs in many physiological and pathophysiological processes, the bioavailability and impact of dietary EVs and their cargo still remain to be elucidated. Due to its widespread consumption and high content of EV-associated microRNAs and proteins, a major focus in this field has been set on EVs in bovine milk and colostrum. Despite promising in vitro studies in recent years that show high resiliency of milk EVs to degradation and uptake of milk EV cargo in a variety of intestinal and blood cell types, in vivo experiments continue to be inconclusive and sometimes outright contradictive. Results To resolve this discrepancy, we assessed the potential postprandial transfer of colostral EVs to the circulation of newborn calves by analysing colostrum-specific protein and miRNAs, including specific isoforms (isomiRs) in cells, EV isolations and unfractionated samples from blood and colostrum. Our findings reveal distinct populations of EVs in colostrum and blood from cows that can be clearly separated by density, particle concentration and protein content (BTN1A1, MFGE8). Postprandial blood samples of calves show a time-dependent increase in EVs that share morphological and protein characteristics of colostral EVs. Analysis of miRNA expression profiles by Next-Generation Sequencing gave a different picture however. Although significant postprandial expression changes could only be detected for calf EV samples, expression profiles show very limited overlap with highly expressed miRNAs in colostral EVs or colostrum in general. Conclusions Taken together our results indicate a selective uptake of membrane-associated protein cargo but not luminal miRNAs from colostral EVs into the circulation of neonatal calves.

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Abstract 897: Alliance of Blood Derived Endothelial Cells and Adipose Stromal Cells in Human Vasculogenesis

Circulation ◽

10.1161/circ.116.suppl_16.ii_175-d ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Dmitry O Traktuev ◽

Daniel N Prater ◽

Aravind R Sanjeevaiah ◽

Stephanie Merfeld-Clauss ◽

Brian H Johnstone ◽

...

Keyword(s):

Endothelial Cells ◽

Stromal Cells ◽

De Novo ◽

Functional Integration ◽

Cell Types ◽

Specific Marker ◽

Pronounced Difference ◽

Adipose Stromal Cells ◽

Small Vessels

Introduction Both Endothelial progenitor cells (EPC) and adipose stromal cells (ASC) are under investigation as therapies for cardiovascular diseases. Both cell types are capable of modulating vascular assembly and are, thereby, capable of directly promoting revascularization of ischemic tissues. We have shown that EPC differentiate into endothelial cells to form small vessels, whereas ASC have pericytic properties and naturally stabilize vessels. In this study we tested the possibility that ASC would interact with EPC to assemble de novo vessels in collagen in an in vivo chimeric implant. Methods and Results Collagen implants embedded with either umbilical cord blood EPC or adult ASC or a 4:1 mixture of both (2x10 6 cells/ml) were implanted subcutaneously into NOD/SCID mice. After 14 d implants were harvested and evaluated by immunohistochemistry. There was a pronounced difference among the groups in vascular network assembly. The majority of vessels formed in the EPC and ASC monocultures were small capillaries bounded by a single endothelial layer. Conversely, 100% of the plugs embedded with both cell types were highly invaded with multilayered arteriolar vessels. The density of the CD31 + vessels in the EPC and co-culture plugs was 26.6 ± 5.8 and 122.4 ± 9.8 per mm 2 , respectively. No CD31 + cells of human origin were detected in the ASC monocultures, indicating that ASC, which do not express this EC-specific marker, engage murine EC or form pseudovessels in this system. The density of α-SMA + vessels with lumens per mm 2 was 13.1 ± 3.6 (EPC), 10.2 ± 3.5 (ASC) and 124.7 ± 19.7 (co-culture). The total overlap of CD31 + and SMA + vessels demonstrates that mature, multilayered conduits were formed with the co-culture. Moreover, the majority of these vessels were filled with erythrocytes (92.5 ± 16.2 per mm 2 ), indicating inosculation with the native vasculature, which was confirmed by ultrasound with echogenic microbubbles and persisted to at least 4 months. Conclusion This study is the first to demonstrate that non-transformed human EPC and ASC cooperatively form mature and stable vasculature with subsequent functional integration into a host vasculature system.

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Extracellular Vesicles in Prostate Cancer: New Future Clinical Strategies?

BioMed Research International ◽

10.1155/2014/561571 ◽

2014 ◽

Vol 2014 ◽

pp. 1-14 ◽

Cited By ~ 14

Author(s):

Ilaria Giusti ◽

Vincenza Dolo

Keyword(s):

Prostate Cancer ◽

Extracellular Vesicles ◽

Specific Antigen ◽

Specific Marker ◽

Cellular Origin ◽

Organ Specific ◽

Potential Use

Prostate cancer (PCa) is the most common cancer—excluding skin tumors—in men older than 50 years of age. Over time, the ability to diagnose PCa has improved considerably, mainly due to the introduction of prostate-specific antigen (PSA) in the clinical routine. However, it is important to take into account that although PSA is a highly organ-specific marker, it is not cancer-specific. This shortcoming suggests the need to find new and more specific molecular markers. Several emerging PCa biomarkers have been evaluated or are being assessed for their potential use. There is increasing interest in the prospective use of extracellular vesicles as specific markers; it is well known that the content of vesicles is dependent on their cellular origin and is strongly related to the stimulus that triggers the release of the vesicles. Consequently, the identification of a disease-specific molecule (protein, lipid or RNA) associated with vesicles could facilitate their use as novel biological markers. The present review describes severalin vitrostudies that demonstrate the role of vesicles in PCa progression and severalin vivostudies that highlight the potential use of vesicles as PCa biomarkers.

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Skeletal muscle releases extracellular vesicles with distinct protein and miRNA signatures that accumulate and function within the muscle microenvironment

10.1101/2021.11.30.470551 ◽

2021 ◽

Author(s):

Sho Watanabe ◽

Yuri Sudo ◽

Satoshi Kimura ◽

Kenji Tomita ◽

Makoto Noguchi ◽

...

Keyword(s):

Skeletal Muscle ◽

Extracellular Vesicles ◽

Cell Types ◽

The Body ◽

C2c12 Cells ◽

Ips Cell ◽

Potential Marker ◽

And Function ◽

Intercellular Communications

Extracellular vesicles (EVs) contain various regulatory molecules and mediate intercellular communications. Although EVs are secreted from various cell types, including skeletal muscle cells, and present in the blood, their identity is poorly characterized in vivo, limiting the identification of their origin in the blood. Since the skeletal muscle is the largest organ in the body, it could substantially contribute to circulating EVs as their source. However, due to the lack of defined markers that distinguish SkM-EVs from others, whether the skeletal muscle releases EVs in vivo and how much the skeletal muscle-derived EVs (SkM-EVs) account for plasma EVs remain poorly understood. In this work, we perform quantitative proteomic analyses on EVs released from C2C12 cells and human iPS cell-derived myocytes and identify potential marker proteins that mark SkM-EVs. These markers we identified apply to in vivo tracking of SkM-EVs. The results show that skeletal muscle makes only a subtle contribution to plasma EVs as their source in both control and exercise conditions in mice. On the other hand, we demonstrate that SkM-EVs are concentrated in the skeletal muscle interstitium. Furthermore, we show that interstitium EVs are highly enriched with the muscle-specific miRNAs and repress the expression of the paired box transcription factor Pax7, a master regulator for myogenesis. Taken together, our findings reveal that the skeletal muscle releases exosome-like small EVs with distinct protein and miRNA profiles in vivo and that SkM-EVs mainly play a role within the muscle microenvironment where they accumulate.

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Recent Advances in Extracellular Vesicles as Drug Delivery Systems and Their Potential in Precision Medicine

Pharmaceutics ◽

10.3390/pharmaceutics12111006 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1006 ◽

Cited By ~ 2

Author(s):

Bart de Jong ◽

Eric Raul Barros ◽

Joost G. J. Hoenderop ◽

Juan Pablo Rigalli

Keyword(s):

Drug Delivery ◽

Extracellular Vesicles ◽

Current Knowledge ◽

Early Stage ◽

Treatment Options ◽

Cell Types ◽

Administration Routes ◽

The Individual ◽

Key Steps

Extracellular vesicles (EVs) are membrane-bilayered nanoparticles released by most cell types. Recently, an enormous number of studies have been published on the potential of EVs as carriers of therapeutic agents. In contrast to systems such as liposomes, EVs exhibit less immunogenicity and higher engineering potential. Here, we review the most relevant publications addressing the potential and use of EVs as a drug delivery system (DDS). The information is divided based on the key steps for designing an EV-mediated delivery strategy. We discuss possible sources and isolation methods of EVs. We address the administration routes that have been tested in vivo and the tissue distribution observed. We describe the current knowledge on EV clearance, a significant challenge towards enhancing bioavailability. Also, EV-engineering approaches are described as alternatives to improve tissue and cell-specificity. Finally, a summary of the ongoing clinical trials is performed. Although the application of EVs in the clinical practice is still at an early stage, a high number of studies in animals support their potential as DDS. Thus, better treatment options could be designed to precisely increase target specificity and therapeutic efficacy while reducing off-target effects and toxicity according to the individual requirements of each patient.

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Automated cell-type classification in intact tissues by single-cell molecular profiling

eLife ◽

10.7554/elife.30510 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 46

Author(s):

Monica Nagendran ◽

Daniel P Riordan ◽

Pehr B Harbury ◽

Tushar J Desai

Keyword(s):

Single Cell ◽

Spatial Information ◽

Housekeeping Genes ◽

Cell Types ◽

Molecular Profiling ◽

Mouse Lung ◽

Intact Mouse ◽

Specificity And Sensitivity

A major challenge in biology is identifying distinct cell classes and mapping their interactions in vivo. Tissue-dissociative technologies enable deep single cell molecular profiling but do not provide spatial information. We developed a proximity ligation in situ hybridization technology (PLISH) with exceptional signal strength, specificity, and sensitivity in tissue. Multiplexed data sets can be acquired using barcoded probes and rapid label-image-erase cycles, with automated calculation of single cell profiles, enabling clustering and anatomical re-mapping of cells. We apply PLISH to expression profile ~2900 cells in intact mouse lung, which identifies and localizes known cell types, including rare ones. Unsupervised classification of the cells indicates differential expression of ‘housekeeping’ genes between cell types, and re-mapping of two sub-classes of Club cells highlights their segregated spatial domains in terminal airways. By enabling single cell profiling of various RNA species in situ, PLISH can impact many areas of basic and medical research.

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Asymmetric depth-filtration - a versatile and scalable approach for isolation and purification of extracellular vesicles

10.1101/2021.08.23.457333 ◽

2021 ◽

Author(s):

Vasiliy S. Chernyshev ◽

Roman N. Chuprov-Netochin ◽

Ekaterina Tsydenzhapova ◽

Elena Victorovna Svirshchevskaya ◽

Rimma A. Poltavtseva ◽

...

Keyword(s):

Extracellular Vesicles ◽

Biological Samples ◽

Biological Fluids ◽

Scale Up ◽

Size Exclusion ◽

Standard Laboratory ◽

Laboratory Equipment ◽

Isolation And Purification ◽

Depth Filtration ◽

Exclusion Chromatography

A novel asymmetric depth filtration (DF) approach for isolation of extracellular vesicles (EVs) from biological fluids is presented, and its performance is compared with established methods. The developed workflow is simple, inexpensive, and relatively fast. Compared with ultracentrifugation and size-exclusion chromatography, the developed method isolates EVs with higher purity and yield. Only standard laboratory equipment is needed for its implementation, which makes it suitable for low-resource locations. The described implementation of the method is suitable for EV isolation from small biological samples in diagnostic and treatment guidance applications. Following the scale-up routes adopted in the biomanufacturing of therapeutics, which routinely rely on DF as one of the product purification steps, the developed method may be scaled up to harvesting therapeutic EVs from large volumes of growth medium.

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