A universal reagent for detection of emerging diseases using bioengineered multifunctional yeast nanofragments.

Accurate and early detection of biomarkers provides the molecular evidence for disease management, allowing prompt actions and timely treatments to save lives. Multivalent biomolecular interactions between the probe and biomarker as well as controlled probe orientation on material surfaces are keys for highly sensitive detection. Here we report the bioengineering of programmable and multifunctional nanoprobes, which can provide rapid, specific and highly sensitive detection of emerging diseases in a range of widely used diagnostic systems. These nanoprobes composed of nanosized cell wall fragments, termed as synthetic bionanofragments (SynBioNFs), are generated by the fragmentation of genetically programmed yeast cells. SynBioNFs display multiple copies of biomolecules for high-affinity target binding and molecular handles for the precisely orientated attachment on surfaces used in diagnostic platforms. SynBioNFs are demonstrated for the capture and detection of SARS-CoV-2 virions using multiple diagnostic platforms, including surface-enhanced Raman scattering, fluorescence, electrochemical and colorimetric-based lateral flow systems with sensitivity comparable with the gold-standard reverse-transcription quantitative polymerase chain reaction.

An epithelial-to-mesenchymal transition induced extracellular vesicle prognostic signature in non-small cell lung cancer.

Despite significant therapeutic advances, lung cancer remains the leading cause of cancer-related death worldwide1. Non-small cell lung cancer (NSCLC) patients have a very poor overall five-year survival rate of only 10-20%. Currently, TNM staging is the gold standard for predicting overall survival and selecting optimal initial treatment options for NSCLC patients, including those with curable stages of disease. However, many patients with locoregionally-confined NSCLC relapse and die despite curative-intent interventions, indicating a need for intensified, individualised therapies. Epithelial-to-mesenchymal transition (EMT), the phenotypic depolarisation of epithelial cells to elongated, mesenchymal cells, is associated with metastatic and treatment-refractive cancer. We demonstrate here that EMT-induced protein changes in small extracellular vesicles are detectable in NSCLC patients and have prognostic significance. Overall, this work describes a novel prognostic biomarker signature that identifies potentially-curable NSCLC patients at risk of developing metastatic NSCLC, thereby enabling implementation of personalised treatment decisions.

Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles.

Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.

Blood-Derived Extracellular Vesicle-Associated miR-3182 Detects Non-Small Cell Lung Cancer Patients.

With five-year survival rates as low as 3%, lung cancer is the most common cause of cancer-related mortality worldwide. The severity of the disease at presentation is accredited to the lack of early detection capacities, resulting in the reliance on low-throughput diagnostic measures, such as tissue biopsy and imaging. Interest in the development and use of liquid biopsies has risen, due to non-invasive sample collection, and the depth of information it can provide on a disease. Small extracellular vesicles (sEVs) as viable liquid biopsies are of particular interest due to their potential as cancer biomarkers. To validate the use of sEVs as cancer biomarkers, we characterised cancer sEVs using miRNA sequencing analysis. We found that miRNA-3182 was highly enriched in sEVs derived from the blood of patients with invasive breast carcinoma and NSCLC. The enrichment of sEV miR-3182 was confirmed in oncogenic, transformed lung cells in comparison to isogenic, untransformed lung cells. Most importantly, miR-3182 can successfully distinguish early-stage NSCLC patients from those with benign lung conditions. Therefore, miR-3182 provides potential to be used for the detection of NSCLC in blood samples, which could result in earlier therapy and thus improved outcomes and survival for patients.

Generation of Nanoyeast Single-Chain Variable Fragments as High-Avidity Biomaterials for Dengue Virus Detection.

Bioengineered yeast bio-nanomaterials termed nanoyeasts displaying antibody single-chain variable fragments (scFvs) against diagnostic targets are a promising alternative to monoclonal antibodies (mAbs). A potential limitation for translating nanoyeasts into diagnostic tools is batch-to-batch variability. Herein, we demonstrate a systematic approach for cost-efficient production of highly specific nanoyeasts that enabled accurate dengue virus (DENV) detection by immunoassay (2.5% CV). Yeasts bioengineered to surface express DENV-specific scFvs (up to 66% of the total cell population) were fragmented into nanoyeast fractions trialing sonication, bead beating, and high-pressure disruption methods. Nanoyeast fractions from sonication had optimal target binding, uniform particle size (±89 nm), were stable, and retained diagnostic activity for 7 days at 37 °C compared to traditional mAbs that lost activity after 1 day at 37 °C. We engineered a panel of nanoyeast scFvs targeting DENV nonstructural protein 1 (NS1): (i) specific for serotyping DENV 1-4 and (ii) cross-reactive anti-DENV scFvs that are suitable for "yes/no" diagnostic applications. We demonstrate highly specific nanoyeast scFvs for serotyping DENV. We show that nanoyeast scFvs specifically detect NS1 in simulated patient plasma with a limit of detection of 250 ng/mL, the concentration found in infected patients.

Characterizing the Heterogeneity of Small Extracellular Vesicle Populations in Multiple Cancer Types via an Ultrasensitive Chip.

Identifying small extracellular vesicle (sEV) subpopulations based on their different molecular signatures could potentially reveal the functional roles in physiology and pathology. However, it is a challenge to achieve this aim due to the nano-sized dimensions of sEVs, low quantities of biological cargo each sEV carries, and our incomplete knowledge of identifying features capable of separating heterogeneous sEV subpopulations. Here, a sensitive, multiplexed, and nano-mixing-enhanced sEV subpopulation characterization platform (ESCP) is proposed to precisely determine the sEV phenotypic heterogeneity and understand the role of sEV heterogeneity in cancer progression and metastasis. The ESCP utilizes spatially patterned anti-tetraspanin-functionalized micro-arrays for sEV subpopulation sorting and nanobarcode-based surface-enhanced Raman spectroscopy for multiplexed read-outs. An ESCP has been used for investigating sEV phenotypic heterogeneity in terms of canonical sEV tetraspanin molecules and cancer-associated protein biomarkers in both cancer cell line models and cancer patient samples. Our data explicitly demonstrate the selective enrichment of tetraspanins and cancer-associated protein biomarkers, in particular sEV subpopulations. Therefore, it is believed that the ESCP could enable the evaluation and broader application of sEV subpopulations as potential diagnostic disease biomarkers.

Amplification-Free SARS-CoV-2 Detection Using Nanoyeast-scFv and Ultrasensitive Plasmonic Nanobox-Integrated Nanomixing Microassay.

The implementation of accurate and sensitive molecular detection for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is paramount to effectively control the ongoing coronavirus disease 2019 (COVID-19) pandemic. In this regard, we herein propose the specific and highly sensitive SARS-CoV-2 detection based on nanoyeast single-chain-variable fragment (scFv) and ultrasensitive plasmonic nanobox-integrated nanomixing microassay. Importantly, this designed platform showcases the utility of nanoyeast-scFvs as specific capture reagents targeting the receptor-binding domain (RBD) of the virus and as monoclonal antibody alternatives suitable for cost-effective mass production and frequent testing. By capitalizing on single-particle active nanoboxes as plasmonic nanostructures for surface-enhanced Raman scattering (SERS), the microassay utilizes highly sensitive Raman signals to indicate virus infection. The developed microassay further integrated nanomixing for accelerating molecular collisions. Through the synergistic working of nanoyeast-scFv, plasmonic nanoboxes, and nanomixing, the highly specific and sensitive SARS-CoV-2 detection is achieved as low as 17 virus/µL without any molecular amplification. We successfully demonstrate SARS-CoV-2 detection in saliva samples of simulated patients at clinically relevant viral loads, suggesting the possibility of this platform for accurate and noninvasive patient screening.

Tumor microenvironmental cytokines bound to cancer exosomes determine uptake by cytokine receptor-expressing cells and biodistribution.

Metastatic spread of a cancer to secondary sites is a coordinated, non-random process. Cancer cell-secreted vesicles, especially exosomes, have recently been implicated in the guidance of metastatic dissemination, with specific surface composition determining some aspects of organ-specific localization. Nevertheless, whether the tumor microenvironment influences exosome biodistribution has yet to be investigated. Here, we show that microenvironmental cytokines, particularly CCL2, decorate cancer exosomes via binding to surface glycosaminoglycan side chains of proteoglycans, causing exosome accumulation in specific cell subsets and organs. Exosome retention results in changes in the immune landscape within these organs, coupled with a higher metastatic burden. Strikingly, CCL2-decorated exosomes are directed to a subset of cells that express the CCL2 receptor CCR2, demonstrating that exosome-bound cytokines are a crucial determinant of exosome-cell interactions. In addition to the finding that cytokine-conjugated exosomes are detected in the blood of cancer patients, we discovered that healthy subjects derived exosomes are also associated with cytokines. Although displaying a different profile from exosomes isolated from cancer patients, it further indicates that specific combinations of cytokines bound to exosomes could likewise affect other physiological and disease settings.

The evolving translational potential of small extracellular vesicles in cancer.

Cancer-derived extracellular vesicles (EVs) are regarded as having promising potential to be used as therapeutics and disease biomarkers. Mechanistically, EVs have been shown to function in most, if not all, steps of cancer progression. Cancer EVs, including small EVs (sEVs), contain unique biomolecular cargo, consisting of protein, nucleic acid and lipids. Through progress in the identification of this specific cargo, cancer biomarkers have been identified and developed, opening up novel and interesting opportunities for cancer diagnosis and prognosis. Intriguingly, we still lack a comprehensive understanding of the cancer-specific pathways that govern EV biogenesis in cancer cells. Filling this knowledge gap will rapidly improve cancer EV biomarkers, as it will also allow discrimination of the procancer and anticancer actions of those EVs. Even more promising is uncovering therapeutically targetable, tumour-specific EV pathways and content, which will generate novel classes of cancer therapies. This Review highlights the progress the cancer sEV field has made in the areas of biomarker discovery and validation as well as sEV-based therapeutics, highlights the challenges we are facing and identifies gaps in our knowledge, which currently prevent us from developing the full potential of sEVs in cancer diagnostic and therapy.

The role of exosomes in the promotion of epithelial-to-mesenchymal transition and metastasis.

The progression of a solid cancer from a localised disease to metastatic stages is a key reason for mortality in patients. Amongst the drivers of cancer progression, Epithelial-to-Mesenchymal Transition (EMT) has been shown to be of crucial importance. EMT results in the phenotypic shift of an immotile, treatment-sensitive epithelial cell into an elongated, metastatic and treatment-resistant mesenchymal cell. Depending on the cellular and molecular setting, a myriad of studies have demonstrated that EMT causes increased cancer cell motility, invasiveness, resistance to therapies, dormancy and cancer-stem cell phenotypes, all of which are prerequisites for metastasis. The alteration of non-canonical intercellular signalling events in cancer EMT is a phenomenon that is not completely understood. Recently, extracellular vesicles, especially small vesicles called exosomes, have shown to be involved in cancer cell EMT. Most intriguingly, across different cancer types, cancer-derived exosomes have demonstrated to be capable of transferring a mesenchymal phenotype upon recipient epithelial cells, including epithelial cancer cells. The uptake of EMT-inducing exosomes results in molecular changes, altering miRNA, mRNA, and protein levels, either through direct transfer of these components, or by altering gene expression networks involved in EMT. In this review, we are presenting the current state of research of exosomes in cancer EMT, highlight gaps in our current knowledge and propose strategies for future experiments in this area.

Breast Cancer-Derived Exosomes Reflect the Cell-of-Origin Phenotype.

A manner in which cells can communicate with each other is via secreted nanoparticles termed exosomes. These vesicles contain lipids, nucleic acids, and proteins, and are said to reflect the cell-of-origin. However, for the exosomal protein content, there is limited evidence in the literature to verify this statement. Here, proteomic assessment combined with pathway-enrichment analysis is used to demonstrate that the protein cargo of exosomes reflects the epithelial/mesenchymal phenotype of secreting breast cancer cells. Given that epithelial-mesenchymal plasticity is known to implicate various stages of cancer progression, the results suggest that breast cancer subtypes with distinct epithelial and mesenchymal phenotypes may be distinguished by directly assessing the protein content of exosomes. Additionally, the work is a substantial step toward verifying the statement that cell-derived exosomes reflect the phenotype of the cells-of-origin.

The Interaction of Selenium with Chemotherapy and Radiation on Normal and Malignant Human Mononuclear Blood Cells.

Selenium, a trace element with anticancer properties, can reduce harmful toxicities of chemotherapy and radiotherapy without compromising efficacy. However, the dose-response relationship in normal versus malignant human cells is unclear. We evaluated how methylseleninic acid (MSA) modulates the toxicity and efficacy of chemotherapy and radiation on malignant and non-malignant human mononuclear blood cells in vitro. We specifically investigated its effects on endoplasmic reticulum stress induction, intracellular glutathione concentration, DNA damage and viability of peripheral blood mononuclear cells and THP1 monocytic leukaemia cells in response to radiation, cytosine arabinoside or doxorubicin chemotherapy. MSA, at lower concentrations, induced protective responses in normal cells but cytotoxic effects in malignant cells, alone and in conjunction with chemotherapy or radiation. However, in normal cells higher concentrations of MSA were directly toxic and increased the cytotoxicity of radiation but not chemotherapy. In malignant cells higher MSA concentrations were generally more effective in combination with cancer treatments. Thus, optimal MSA concentrations differed between normal and malignant cells and treatments. This work supports clinical reports that selenium can significantly reduce dose-limiting toxicities of anticancer therapies and potentially improve efficacy of anticancer treatments. The optimal selenium compound and dose is not yet determined.

Breast Cancer-Derived Exosomes Alter Macrophage Polarization via gp130/STAT3 Signaling.

Tumor-derived exosomes are being recognized as essential mediators of intercellular communication between cancer and immune cells. It is well established that bone marrow-derived macrophages (BMDMs) take up tumor-derived exosomes. However, the functional impact of these exosomes on macrophage phenotypes is controversial and not well studied. Here, we show that breast cancer-derived exosomes alter the phenotype of macrophages through the interleukin-6 (IL-6) receptor beta (glycoprotein 130, gp130)-STAT3 signaling pathway. Addition of breast cancer-derived exosomes to macrophages results in the activation of the IL-6 response pathway, including phosphorylation of the key downstream transcription factor STAT3. Exosomal gp130, which is highly enriched in cancer exosomes, triggers the secretion of IL-6 from BMDMs. Moreover, the exposure of BMDMs to cancer-derived exosomes triggers changes from a conventional toward a polarized phenotype often observed in tumor-associated macrophages. All of these effects can be inhibited through the addition of a gp130 inhibitor to cancer-derived exosomes or by blocking BMDMs exosome uptake. Collectively, this work demonstrates that breast cancer-derived exosomes are capable of inducing IL-6 secretion and a pro-survival phenotype in macrophages, partially via gp130/STAT3 signaling.

Oncogenic transformation of lung cells results in distinct exosome protein profile similar to the cell of origin.

Lung cancer is responsible for the highest rate of cancer mortality worldwide. Lung cancer patients are often ineligible for tumor biopsies due to comorbidities. As a result, patients may not have the most effective treatment regimens administered. Patients with mutations in the epidermal growth factor receptor (EGFR) have improved survival in response to EGFR tyrosine kinase inhibitors. A noninvasive method of determining EGFR mutations in patients would have promising clinical applications. Exosomes have the potential to be noninvasive novel diagnostic markers in cancer. Using MS analysis, we identify differentially abundant cell and exosome proteins induced by mutations in p53 and EGFR in lung cells. Importantly, mutations in p53 and EGFR alter cell and exosome protein content compared to an isogenic normal lung epithelial cell. For some proteins, mutation had similar effects in the cell of origin and exosomes. Differences between the cells of origin and exosomes were also apparent, which may reflect specific packaging of proteins into exosomes. These findings that mutations alter protein abundance in exosomes suggest that analysis of exosomes may be beneficial in the diagnosis of oncogenic mutations.

Unique molecular profile of exosomes derived from primary human proximal tubular epithelial cells under diseased conditions.

Human proximal tubular epithelial cells (PTEC) of the kidney are known to respond to and mediate the disease process in a wide range of kidney diseases, yet their exosomal production and exosome molecular cargo remain a mystery. Here we investigate, for the first time, the production and molecular content of exosomes derived from primary human PTEC cultured under normal and diseased conditions representing a spectrum of in vivo disease severity from early inflammation, experienced in multiple initial kidney disease states, through to hypoxia, frequently seen in late stage chronic kidney disease (CKD) due to fibrosis and vascular compromise. We demonstrate a rapid reproducible methodology for the purification of PTEC-derived exosomes, identify increased numbers of exosomes from disease-state cultures and identify differential expression levels of both known and unique miRNA and protein species from exosomes derived from different disease-culture conditions. The validity of our approach is supported by the identification of miRNA, proteins and pathways with known CKD associations, providing a rationale to further evaluate these novel and known pathways as targets for therapeutic intervention.

Exosomes derived from mesenchymal non-small cell lung cancer cells promote chemoresistance.

Non-small cell lung cancer (NSCLC) is the most common lung cancer type and the most common cause of mortality in lung cancer patients. NSCLC is often associated with resistance to chemotherapeutics and together with rapid metastatic spread, results in limited treatment options and poor patient survival. NSCLCs are heterogeneous, and consist of epithelial and mesenchymal NSCLC cells. Mesenchymal NSCLC cells are thought to be responsible for the chemoresistance phenotype, but if and how this phenotype can be transferred to other NSCLC cells is currently not known. We hypothesised that small extracellular vesicles, exosomes, secreted by mesenchymal NSCLC cells could potentially transfer the chemoresistance phenotype to surrounding epithelial NSCLC cells. To explore this possibility, we used a unique human bronchial epithelial cell (HBEC) model in which the parental cells were transformed from an epithelial to mesenchymal phenotype by introducing oncogenic alterations common in NSCLC. We found that exosomes derived from the oncogenically transformed, mesenchymal HBECs could transfer chemoresistance to the parental, epithelial HBECs and increase ZEB1 mRNA, a master EMT transcription factor, in the recipient cells. Additionally, we demonstrate that exosomes from mesenchymal, but not epithelial HBECs contain the ZEB1 mRNA, thereby providing a potential mechanism for the induction of a mesenchymal phenotype in recipient cells. Together, this work demonstrates for the first time that exosomes derived from mesenchymal, oncogenically transformed lung cells can transfer chemoresistance and mesenchymal phenotypes to recipient cells, likely via the transfer of ZEB1 mRNA in exosomes.

Exosomes: Key mediators of metastasis and pre-metastatic niche formation.

While tumour cells are classically known to communicate via direct cell-to-cell contact and the secretion of soluble protein-based factors such as cytokines and growth factors, alternative novel mechanisms that promote tumour progression have recently emerged. Now, new critical components of the secretome thought to be involved in tumour progression are exosomes, small vesicles of endocytic origin that carry a variety of bioactive molecules, including proteins, lipids, RNA, as well as DNA molecules. Cancer cell-derived exosomes have been shown to participate in crucial steps of metastatic spread of a primary tumour, ranging from oncogenic reprogramming of malignant cells to formation of pre-metastatic niches. These effects are achieved through the mediation of intercellular cross-talk and subsequent modification of both local and distant microenvironments in an autocrine and paracrine fashion. Here, we summarise the recent findings that implicate this non-canonical signalling within the tumour as a critical driver of metastatic disease progression, and discuss how understanding the molecular mechanisms involved in exosome-mediated metastasis is of great value for the development of new therapeutic strategies to prevent cancer progression.

The Biodistribution and Immune Suppressive Effects of Breast Cancer-Derived Exosomes.

Small membranous secretions from tumor cells, termed exosomes, contribute significantly to intercellular communication and subsequent reprogramming of the tumor microenvironment. Here, we use optical imaging to determine that exogenously administered fluorescently labeled exosomes derived from highly metastatic murine breast cancer cells distributed predominantly to the lung of syngeneic mice, a frequent site of breast cancer metastasis. At the sites of accumulation, exosomes were taken up by CD45+ bone marrow-derived cells. Subsequent long-term conditioning of naïve mice with exosomes from highly metastatic breast cancer cells revealed the accumulation of myeloid-derived suppressor cells in the lung and liver. This favorable immune suppressive microenvironment was capable of promoting metastatic colonization in the lung and liver, an effect not observed from exosomes derived from nonmetastatic cells and liposome control vesicles. Furthermore, we determined that breast cancer exosomes directly suppressed T-cell proliferation and inhibited NK cell cytotoxicity, and hence likely suppressed the anticancer immune response in premetastatic organs. Together, our findings provide novel insight into the tissue-specific outcomes of breast cancer-derived exosome accumulation and their contribution to immune suppression and promotion of metastases. Cancer Res; 76(23); 6816-27. ©2016 AACR.

Radiotherapy for Non-Small Cell Lung Cancer Induces DNA Damage Response in Both Irradiated and Out-of-field Normal Tissues.

PURPOSE: To study the response of irradiated and out-of-field normal tissues during localized curative intent radiotherapy. EXPERIMENTAL DESIGN: Sixteen patients with non-small cell lung carcinoma (NSCLC) received 60 Gy in 30 fractions of definitive thoracic radiotherapy with or without concurrent chemotherapy. Peripheral blood lymphocytes (PBL) and eyebrow hairs were sampled prior, during, and after radiotherapy. Clinical variables of radiotherapy dose/volume, patient age, and use of chemoradiotherapy were tested for association with γ-H2AX foci, a biomarker of DNA damage that underlies cellular response to irradiation. RESULTS: Radiotherapy induced an elevation of γ-H2AX foci in PBL, representing normal tissues in the irradiated volume, 1 hour after fraction one. The changes correlated directly with mean lung dose and inversely with age. γ-H2AX foci numbers returned to near baseline values in 24 hours and were not significantly different from controls at 4 weeks during radiotherapy or 12 weeks after treatment completion. In contrast, unirradiated hair follicles, a surrogate model for out-of-field normal tissues, exhibited delayed "abscopal" DNA damage response. γ-H2AX foci significantly increased at 24 hours post-fraction one and remained elevated during treatment, in a dose-independent manner. This observed abscopal effect was associated with changes in plasma levels of MDC/CCL22 and MIP-1α/CCL3 cytokines. No concordant changes in size and concentration of circulating plasma exosomes were observed. CONCLUSIONS: Both localized thoracic radiotherapy and chemoradiotherapy induce pronounced systemic DNA damage in normal tissues. Individual assessment of biologic response to dose delivered during radiotherapy may allow for therapeutic personalization for patients with NSCLC. Clin Cancer Res; 22(19); 4817-26. ©2016 AACRSee related commentary by Verma and Lin, p. 4763.

Optimized exosome isolation protocol for cell culture supernatant and human plasma.

Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.