Abstract
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.
Highlights
Extracellular vesicles (EVs) are lipid bilayer enclosed structures released by all cell types into the extracellular environment and biological fluids.[1,2] EVs are released either by budding from the plasma membrane to form microvesicles (∼50 nm– 1 μm) or are formed within the endosomal system and released as exosomes (∼30–100 nm).[1]
GFP labelled murine virus particles (122 nm) and silica Apogee beads with particle size range of 180 nm–1.3 μm were ran on the CytoFLEX S flow cytometer using violet side scatter (VSSC) trigger
A PBS wash between each sample and a 1% Contrad 70TM wash followed by a water wash at the end of each row was implemented to prevent buildup of plasma EVs and proteins during sample acquisition to minimize the risk of tubing blockages from arising (Fig. 1C)
Summary
Extracellular vesicles (EVs) are lipid bilayer enclosed structures released by all cell types into the extracellular environment and biological fluids.[1,2] EVs are released either by budding from the plasma membrane to form microvesicles (∼50 nm– 1 μm) or are formed within the endosomal system and released as exosomes (∼30–100 nm).[1]. Paper and cervical tissues.[12] Whilst the function of mammaglobin-a is unknown, mammaglobin-a expression is elevated in primary and metastatic breast tumors but is not conclusively predictive of cancer grade or subtype.[13,14,15] mammaglobin-a mRNA is elevated in the blood of breast cancer patients as detected by qPCR of circulating tumor cells. A meta-analysis of several studies revealed that higher levels of circulating mammaglobin-a in plasma correlates with lymph node metastasis and advanced tumor stage.[16,17,18,19] enzyme linked immunosorbent diagnostic assay (ELISA) has been used to show that secreted mammaglobin-a protein is elevated in breast cancer patient blood.[20] Predictive analysis of mammaglobin-a protein structure reveals a putative transmembrane domain suggesting that mammaglobin-a may be anchored at the plasma membrane on breast cells. Mammaglobin-a, is an interesting candidate to investigate for the development of an EV liquid blood biopsy in breast cancer
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