Proteomic Profiling of Small Extracellular Vesicles Secreted by Human Pancreatic Cancer Cells Implicated in Cellular Transformation

Abstract

Extracellular vesicles secreted from tumor cells are functional vehicles capable of contributing to intercellular communication and metastasis. A growing number of studies have focused on elucidating the role that tumor-derived extracellular vesicles play in spreading pancreatic cancer to other organs, due to the highly metastatic nature of the disease. We recently showed that small extracellular vesicles secreted from pancreatic cancer cells could initiate malignant transformation of healthy cells. Here, we analyzed the protein cargo contained within these vesicles using mass spectrometry-based proteomics to better understand their makeup and biological characteristics. Three different human pancreatic cancer cell lines were compared to normal pancreatic epithelial cells revealing distinct differences in protein cargo between cancer and normal vesicles. Vesicles from cancer cells contain an enrichment of proteins that function in the endosomal compartment of cells responsible for vesicle formation and secretion in addition to proteins that have been shown to contribute to oncogenic cell transformation. Conversely, vesicles from normal pancreatic cells were shown to be enriched for immune response proteins. Collectively, results contribute to what we know about the cargo contained within or excluded from cancer cell-derived extracellular vesicles, supporting their role in biological processes including metastasis and cancer progression.

Figure 1

Isolation and characterization of small extracellular vesicles (sEVs) from four human pancreatic cell lines: Capan-2, MIA PaCa-2, Panc-1, and HPDE. (A) Isolation protocol and workflow for LC-MS/MS analysis of sEVs. Detailed protocol included in Methods. (B) Table of common sEV marker proteins found by MS: average peptide spectral matches (PSMs) were calculated from three biological replicates for each of the four types of sEVs. (C) TEM images of Capan-2, MIA PaCa-2, Panc-1, and HPDE sEVs (data reprinted from ref. ).

Figure 2

Comparison of proteins found in sEVs from human pancreatic cell lines. (A) Venn diagram showing the overlap of proteins found in Capan-2, MIA PaCa-2, Panc-1, and HPDE sEVs, n = 3 for each type of sEV. (B) PANTHER classification of the molecular functions, biological processes, and cellular components associated with the complete protein profile of sEVs from each of the four human pancreatic cell lines. (C) PANTHER classification of only the unique proteins found in each type of sEV; analysis was performed on 444, 593, 527, and 313 unique proteins found in Capan-2, MIA PaCa-2, Panc-1, and HPDE, respectively.

Figure 3

Gene ontology (GO) enrichment analysis on the complete protein profiles for Capan-2, MIA PaCa-2, Panc-1, and HPDE sEVs. Only biological processes found to be significantly enriched in all four types of sEVs are shown. P-values <0.05 were considered as statistically significant. Enriched biological processes are grouped based on parent GO terms including cellular process, metabolic process, cellular component organization or biogenesis, and localization.

Figure 4

Comparative analysis of unique cancer versus unique normal sEV proteins. (A) GO enrichment analysis of 348 common pancreatic cancer sEV proteins that are not found in normal HPDE sEVs. Enriched biological processes found are labeled as “Cancer Processes 1–6.” (B) GO enrichment analysis of 313 unique HPDE proteins that are not found in the three pancreatic cancer sEVs. Enriched biological processes found are labeled as “Normal Processes 1–12”.

Figure 5

Significantly enriched Reactome Pathways associated with (A) the set of 348 common pancreatic cancer sEV proteins and (B) the set of 313 unique HPDE proteins from Fig. 4. Enriched Reactome pathways were identified via GO enrichment analysis on the two subsets of proteins.

Figure 6

Characterization of Capan-2 sEVs after sucrose density gradient purification. (A) Number of proteins found in Fractions 1–6 and the corresponding Crude population of sEVs, two biological replicates shown. The dashed black lines shown on Fraction 3, Fraction 4, and Crude represent the number of proteins found in common between the two biological replicates for each corresponding sample. Crude sEVs were isolated using the ultrafiltration-ultracentrifugation method and then further purified using a sucrose density gradient to produce the six fractions. (B) Venn diagrams show the overlap of proteins found in the crude sEVs with the corresponding Fraction 3 samples containing purified sEVs. (C) Proteins identified in Fraction 3 Capan-2 sEVs are compared with the 348 common cancer sEV proteins that were analyzed in Fig. 4. (D) Significantly enriched Reactome Pathways associated with the 152 proteins found in common between Fraction 3 samples and the common cancer proteins (red dashed box). Enriched Reactome pathways were identified via GO enrichment analysis.