Separation and isolation of CD9-positive extracellular vesicles from plasma using flow cytometry.

Extracellular vesicles (EVs) are nanosized (∼30-1000 nm) lipid-enclosed particles released by a variety of cell types. EVs are found in biological fluids and are considered a promising material for disease detection and monitoring. Given their nanosized properties, EVs are difficult to isolate and study. In complex biological samples, this difficulty is amplified by other small particles and contaminating proteins making the discovery and validation of EV-based biomarkers challenging. Developing new strategies to isolate EVs from complex biological samples is of significant interest. Here, we evaluate the utility of flow cytometry to isolate particles in the nanoscale size range. Flow cytometry calibration was performed and 100 nm nanoparticles and ∼124 nm virus were used to test sorting capabilities in the nanoscale size range. Next, using blood plasma, we assessed the capabilities of flow cytometry sorting for the isolation of CD9-positive EVs. Using flow cytometry, CD9-positive EVs could be sorted from pre-enriched EV fractions and directly from plasma without the need for any EV pre-enrichment isolation strategies. These results demonstrate that flow cytometry can be employed as a method to isolate subpopulations of EVs from biological samples.

Development of an in vivo system to model breast cancer metastatic organotropism and evaluate treatment response using the chick embryo.

Metastatic lesions produced through the process of systemic tumor cell dissemination and growth at distant sites are challenging to treat and the primary cause of patient mortality. Developing in vivo models of metastasis with utility in evaluating molecular targets and therapeutics in a timely manner would expedite the path to therapeutic discovery. Here, we evaluated breast cancer metastasis and metastatic organotropism using the chick embryo. We developed a method to evaluate metastasis using the MDA231 cell line. Then, using cell lines with demonstrated tropism for the bone, brain, and lung, we evaluated organotropism. Rapid and robust organ-specific metastasis was modeled in the chick embryo and, importantly, recapitulated metastatic organotropism congruent to what has been demonstrated in mice. Treatment response in the metastatic setting was also evaluated and quantified. This work establishes the chick embryo as a model for studies aimed at understanding organotropism and therapeutic response in the metastatic setting.

Engineered Cas9 extracellular vesicles as a novel gene editing tool.

Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.

Isolation and characterization of extracellular vesicles for clinical applications in cancer - time for standardization?

Extracellular vesicles (EVs) are nanometer sized lipid enclosed particles released by all cell types into the extracellular space and biological fluids in vivo, and into cell culture media in vitro. An important physiological role of EVs is cell-cell communication. EVs interact with, and deliver, their contents to recipient cells in a functional capacity; this makes EVs desirable vehicles for the delivery of therapeutic cargoes. In addition, as EVs contain proteins, lipids, glycans, and nucleic acids that reflect their cell of origin, their potential utility in disease diagnosis and prognostication is of great interest. The number of published studies analyzing EVs and their contents in the pre-clinical and clinical setting is rapidly expanding. However, there is little standardization as to what techniques should be used to isolate, purify and characterize EVs. Here we provide a comprehensive literature review encompassing the use of EVs as diagnostic and prognostic biomarkers in cancer. We also detail their use as therapeutic delivery vehicles to treat cancer in pre-clinical and clinical settings and assess the EV isolation and characterization strategies currently being employed. Our report details diverse isolation strategies which are often dependent upon multiple factors such as biofluid type, sample volume, and desired purity of EVs. As isolation strategies vary greatly between studies, thorough EV characterization would be of great importance. However, to date, EV characterization in pre-clinical and clinical studies is not consistently or routinely adhered to. Standardization of EV characterization so that all studies image EVs, quantitate protein concentration, identify the presence of EV protein markers and contaminants, and measure EV particle size and concentration is suggested. Additionally, the use of RNase, DNase and protease EV membrane protection control experiments is recommended to ensure that the cargo being investigated is truly EV associated. Overall, diverse methodology for EV isolation is advantageous as it can support different sample types and volumes. Nevertheless, EV characterization is crucial and should be performed in a rigorous manor.

Nanoscale flow cytometry for immunophenotyping and quantitating extracellular vesicles in blood plasma.

Extracellular vesicles (EVs) are lipid membrane enclosed nano-sized structures released into the extracellular environment by all cell types. EV constituents include proteins, lipids and nucleic acids that reflect the cell from which they originated. The molecular profile of cancer cells is distinct as compared to healthy cells of the same tissue type, and this distinct profile should be reflected by the EVs they release. This makes EVs desirable candidates for blood-based biopsy diagnosis of cancer. EVs can be time consuming to isolate therefore, a technology that can analyze EVs in complex biological samples in a high throughput manner is in demand. Here nanoscale flow cytometry is used to analyze EVs in whole, unpurified, plasma samples from healthy individuals and breast cancer patients. A known breast cancer marker, mammaglobin-a, was evaluated as a potential candidate for expression on EVs and increased levels in breast cancer. Mammaglobin-a particles were abundantly detected in plasma by nanoscale flow cytometry but only a portion of these particles were validated as bona fide EVs. EVs could be distinguish and characterized from small protein clusters and platelets based on size, marker composition, and detergent treatment. Mammaglobin-a positive EVs were characterized and found to be CD42a/CD41-positive platelet EVs, and the number of these EVs present was dependent upon plasma preparation protocol. Different plasma preparation protocols influenced the total number of platelet EVs and mammaglobin-a was found to associate with lipid membranes in plasma. When comparing plasma samples prepared by the same protocol, mammaglobin-a positive EVs were more abundant in estrogen receptor (ER) positive as compared to ER negative breast cancer patient plasma samples. This study demonstrates the capabilities of nanoscale flow cytometry for EV and small particle analysis in whole, unpurified, plasma samples, and highlights important technical challenges that need to be addressed when developing this technology as a liquid biopsy platform.

Clinical significance of STEAP1 extracellular vesicles in prostate cancer.

BACKGROUND: Extracellular vesicles (EVs) are cell-derived lipid bilayer enclosed structures shed from the plasma membrane by all cell types. Evidence of EV presence in biological fluids has led to considerable efforts focused on identifying their cargo and determining their utility as a non-invasive diagnostic platform for cancer. In this study, we identify circulating STEAP1 (six-transmembrane epithelial antigen of the prostate 1)-positive EVs in the plasma of healthy males and prostate cancer patients and evaluate its diagnostic and prognostic significance. METHODS: STEAP1 was identified on EVs in prostate cancer patient plasma. EVs were validated using electron microscopy, Western blot, nanoparticle tracking analysis, and nanoscale flow cytometry. STEAP1-positive EVs were quantified for 121 males with prostate cancer and 55 healthy age-matched control males. An evaluation of STEAP1 in prostate cancer tissue was also performed using established prostate cancer cohort data (TCGA, MSKCC, and SU2C/PCF Dream Team). RESULTS: Evaluation of STEAP1-positive EVs by nanoscale flow cytometry identified a significant increase in prostate cancer patient plasma compared to healthy males. However, no association was found between total STEAP1 EV levels and disease recurrence or overall survival. Cohort data from prostate cancer tissue also found STEAP1 to be elevated in prostate cancer while no significant association with recurrence or overall survival was identified. CONCLUSIONS: STEAP1 is known to be enriched on the cells of the prostate with potential clinical significance in prostate cancer. Our results identify and quantitate STEAP1-positive EVs in plasma and provide rationale for a STEAP1 EV-based liquid biopsy as a diagnostic strategy in prostate cancer.

Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution.

Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRß transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50-170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications.

Triple-Negative Primary Breast Tumors Induce Supportive Premetastatic Changes in the Extracellular Matrix and Soluble Components of the Lung Microenvironment.

The lung is one of the deadliest sites of breast cancer metastasis, particularly in patients with triple-negative (TN) disease. We hypothesized that the presence of a TN primary breast tumor induces changes in the extracellular matrix (ECM) and soluble components of the lung microenvironment that support metastatic behavior. SUM159 (TN) and MCF7 (luminal A) breast cancer cells were injected into mice, and primary breast tumors were established prior to assessing metastatic niche changes. We observed increased CD117+ hematopoietic progenitor cells in the bone marrow of SUM159 mice versus MCF7 or control mice (p < 0.05). Relative to mice bearing MCF7 tumors and non-tumor controls, mice bearing SUM159 tumors demonstrated enhanced expression of ECM proteins in the lung (fibronectin, tenascin-c and periostin), with similar changes observed in lung fibroblasts treated with extracellular vesicles (EVs) from TN breast cancer cells (p < 0.05). Exposure to lung-conditioned media (LCM) from SUM159 tumor-bearing mice resulted in increased migration/proliferation of both SUM159 and MCF7 cells relative to the control (p < 0.05). In contrast, LCM from MCF-7 tumor-bearing mice had no such effect. LCM from SUM159 tumor-bearing mice contained 16 unique proteins relative to other LCM conditions, including the metastasis-associated proteins CCL7, FGFR4, GM-CSF, MMP3, thrombospondin-1 and VEGF. These findings suggest for the first time that the TN breast cancer molecular subtype may be an important determinant of premetastatic changes to both the ECM and soluble components of the lung, potentially mediated via breast cancer-derived EVs.