Abstract
Extracellular vesicles (EVs) are produced and released by both healthy and malignant cells and bear markers indicative of ongoing biological processes. In the present study we utilized high resolution flow cytometry to detect EVs in the plasma of patients with pancreatic ductal adenocarcinoma (PDAC) and in the supernatants of PDAC and healthy control (HC) pancreatic organoid cultures. Using ultrafiltration and size exclusion chromatography, PDAC and HC pancreatic organoid EVs were isolated for mass spectrometry analysis. Proteomic and functional protein network analysis showed a striking distinction in that EV proteins profiled in pancreatic cancer organoids were involved in vesicular transport and tumorigenesis while EV proteins in healthy organoids were involved in cellular homeostasis. Thus, the most abundant proteins identified in either case represented non-overlapping cellular programs. Tumor-promoting candidates LAMA5, SDCBP and TENA were consistently upregulated in PDAC EVs. Validation of specific markers for PDAC EVs versus healthy pancreatic EVs will provide the biomarkers and enhanced sensitivity necessary to monitor early disease or disease progression, with or without treatment. Moreover, disease-associated changes in EV protein profiles provide an opportunity to investigate alterations in cellular programming with disease progression.
Highlights
Extracellular vesicles (EVs) are produced and released by both healthy and malignant cells and bear markers indicative of ongoing biological processes
Analyzed EVs include both exosomes, which are 30–150 nm diameter vesicles generated as endosomal-derived multivesicular bodies (MVB), and 100 to > 500 nm microvesicles derived from the plasma membrane
Because the refractive and reflective properties of beads are different from that of EVs49,50, reference beads were not used to determine absolute size of the EV population, but rather as a relative reference to establish consistency in the detected size range over the course of this study
Summary
Extracellular vesicles (EVs) are produced and released by both healthy and malignant cells and bear markers indicative of ongoing biological processes. Abbreviations EV Extracellular vesicles MVB Multivesicular body PPP Platelet-poor plasma PDAC Pancreatic ductal adenocarcinoma HC Healthy control HRFC High resolution flow cytometry SEC Size exclusion chromatography PE Phycoerythrin APC Allophycocyanin AF647 Alexa Fluor 647. Analyzed EVs include both exosomes, which are 30–150 nm diameter vesicles generated as endosomal-derived multivesicular bodies (MVB), and 100 to > 500 nm microvesicles derived from the plasma membrane Both vesicle types are known to carry functional biomolecules including protein, nucleic acid and lipid as cargo. Efforts to detect pancreatic cancer markers at earlier disease stages include the investigation of EVs produced by the tumor cells themselves[15], and by the cellular microenvironment influenced by the tumor[16–19]. Screening with a panel of EV biomarkers would significantly improve the sensitivity and specificity of detecting pancreatic cancer at an early s tage[28]
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