Study Design The discovery cohort was comprised of 124 individuals, including pancreatic ductal adenocarcinoma (PDAC, N=93), chronic pancreatitis (CP, N=12), intraductal papillary mucinous neoplasm (IPMN, N=8) and healthy controls (N=11). Plasma samples were processed for EV isolation using EVtrap and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Identification of cEV Proteins Differentially Expressed in Disease Groups

(A) Principal component analysis of cEV proteins differentially expressed in the plasma of patients with pancreatic diseases compared to controls. Each dot indicates one individual enrolled in the study: green, controls; blue, patients with intraductal papillary mucinous neoplasm (IPMN); purple, patients with chronic pancreatitis (CP); salmon, early stage (stages I and II) pancreatic ductal adenocarcinoma (PDAC); red, late stage (stages III and IV) PDAC. (B) Volcano plot of circulating EV proteins enriched in the plasma of patients with PDAC versus benign pancreatic diseases. X-axis, log base 2 of fold changes; Y-axis, negative of the log base 10 of p values (C) Heatmap of cEV proteins differentially expressed in the plasma of patients with pancreatic diseases compared to controls. Designations of clinical parameters were indicated at the top of the heatmap. (D) Expression of enriched cEV proteins in patients with PDAC versus benign pancreatic diseases. Each dot indicates the target protein signal from one patient. Y-axis, normalized log base 2 of protein signals detected by mass spectrometry; Error bars, min and max values; lines in boxes, median values. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Circulating EV Proteomics Reveal Markers Associated with Metastasis and Worse Prognosis.

(A) Heatmap showing EV proteins differentially expressed in the plasma of metastatic versus non-metastatic PDAC. Designations of clinical parameters are indicated at the top of the heatmap. (B) Expression patterns of cEV proteins associated with metastatic disease. Y-axis, normalized log base 2 of protein signals detected by mass spectrometry; N, non-metastatic PDAC group; M, metastatic PDAC group. Each dot indicated the target protein signal from one patient. Error bars, min and max values; lines in boxes, median values. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (C) As is (B), except for cEV markers with increased expression in non-metastatic PDAC. (D) Correlation of cEV marker expression with survival. Kaplan–Meier curves and log-rank test P values of representative survival cEV markers quantified in the discovery cohort.

Validation of cEV Markers and Identification of 7-EV Protein Signature for PDAC Diagnosis.

(A) Differences of cEV protein abundances between patients with PDAC (n=24) and benign pancreatic diseases (chronic pancreatitis and IPMN) (n=12). x axis, minus log p values of protein abundance differences between PDAC and benign groups; y axis, average fold changes of proteins in PDAC group compared to benign group. Size of bubbles indicate average protein abundances in PDAC group. Pink color, proteins that had at least two-fold enrichment in PDAC group (p<0.05). (B) ROC curves were calculated for individual cEV markers as well as for the 7-EV protein PDAC signature combination to determine optimum diagnostic performance. AUC, area under the curve.

Network Analyses of cEV Proteins Differentially Expressed in PDAC Compared to Benign Pancreatic Diseases.

(A) Functional association of proteins identified by STRING database. Red, cEV proteins enriched in PDAC patients as compared to benign pancreatic diseases. Green, cEV proteins decreased in patients with PDAC as compared to benign pancreatic diseases. Red, cEV proteins increased in PDAC as compared to benign pancreatic diseases. Thickness of lines indicate confidence of association. (B, C) Clustering of cEV protein pathways enriched (B) or downregulated (C) in PDAC cohorts. Pathways were identified using Gene Ontology database and REACTOME database.