Proteomic analysis distinguishes extracellular vesicles produced by cancerous versus healthy pancreatic organoids

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.

Figure 1

Flow cytometry detection of EVs in PDAC patient plasma. (A) Megamix beads (left panel, 100–500 nm in size) were detected with 488 nm fluorescence and were used to standardize the flow cytometer for acquisition of labeled EVs. Additional panels compare acquisition of PBS alone or PBS with 200 nm Fluoresbrite beads (fl-200 nm) or platelet-poor plasma (PPP) with fl-200 nm beads. (B) Detection of CD9 and CD45 antibody staining of plasma EVs is reduced to background by the addition of 0.1% Triton X-100. (C) CD9, CD45 or CD41 positive EVs were assessed in PPP samples from PDAC patients receiving neoadjuvant treatment for 0 to 4 cycles.

Figure 2

Isolation of EVs from organoids established from PDAC patients and healthy controls (HC). (A) Image of representative pancreatic organoid culture (PDAC). (B) Fractions obtained after size exclusion chromatography (SEC) of PDAC organoid supernatants were analyzed for CD9⁺ EVs by flow cytometry and protein levels by measurement at 280 nm. (C) Representative EM images of EVs isolated after SEC on supernatants collected from two healthy control (HC-1 and HC-2) and two PDAC (PDAC-2 and PDAC-4) organoid cultures.

Figure 3

Statistical comparison of proteins identified in EVs isolated from healthy control (HC) and PDAC pancreatic organoids. (A) Multidimensional scaling (MDS) demonstrates clustering of samples by condition (HC versus PDAC) with PDAC-4 further removed from other PDAC samples. (B) Distribution of the candidate proteins with significant change (Up or Down) or without differential expression (non-DE) between the HC and PDAC sample sets are visualized by plotting fold change vs CPM.

Figure 4

Proteomic analysis of EV samples isolated from four healthy control (HC) and four pancreatic cancer (PDAC) organoid cultures (4 × 4 study). (A) Fold change of spectral count averages for top 10 proteins upregulated in HC (top panel) and top 10 proteins upregulated in PDAC (bottom panel). (B) Spectral count averages in the HC or PDAC group for each corresponding protein identified in (A).

Figure 5

Protein interaction analysis. The STRING database (https://string-db.org) was used to analyze protein–protein interactions among the candidates found to be upregulated in pancreatic organoid EVs from (A) healthy controls or (B) PDAC patients. Nodes highlighted with a red ring designate proteins within the top ten highest fold change in each group (p < 0.01, see Fig. 4).

Figure 6

Upregulated proteins analyzed in additional PDAC organoid EVs. Comparison of average spectral counts of top proteins identified in the 4 × 4 study (see Fig. 4) to normalized spectral counts for each corresponding protein in six additional PDAC organoid EV samples. PDAC stage is shown for each PDAC organoid (met metastatic tumor sample).