Serum Derived Exosomes From Pancreatic Cancer Patients Promoted Metastasis: An iTRAQ-Based Proteomic Analysis

Abstract

Background: Pancreatic cancer (PC) is one of the most aggressive malignancies and has a poor prognosis despite being extensively researched. The role of serum-derived exosomes in tumorigenesis and the development of PC is still unclear.

Method: The present study employed iTRAQ-based proteomic analysis to search for differences between the serum exosomes of PC patients and those from control patients. Then, bioinformatics methods were used to analyze the functions of the identified proteins, and the possible functions were verified through cell culture experiments.

Results: A total of 611 proteins were identified from exosomes, and 141 proteins were differentially expressed, with 91 up- and 50 down regulated proteins in PC cancer compared to healthy controls. Further analysis indicated that APOE serves as an important hub in the network. In addition, CRP, VWF, APOA2, NIN, and GSK3B potentially interact with many other proteins. We then tested the effect of patient serum-derived exosomes on pancreatic cancer cells and found that patient serum-derived exosomes, but not those from healthy controls, induced cell proliferation, migration, and EMT, supporting the role of exosomes in metastasis.

Conclusion: Our data suggest that exosomes derived from PC patients may promote PC metastasis.

Keywords: metastasis; pancreatic cancer; proteomic analysis; serum exosome.

Figure 1

The relationship between identified proteins and Exocarta database.

Figure 2

GO classification of proteomic data for downregulated and upregulated proteins. Differently expressed proteins were submitted to the GO classification system.

Figure 3

GO analysis shows that blood microparticle, antigen binding and receptor-mediated endocytosis were the most enriched term incellular component, molecular function and biological process.

Figure 4

Network of proteins identified in overall differentially expressed proteins. Up- or downregulation is indicated by the color of nodes (upregulated in red and downregulated in green).

Figure 5

CCK8 test. PC cells (MiaPaCa-2, AsPC-1, 4×10⁶) were treated with serum exosomes derived from PC patients or healthy controls after 12 h of seeding (equal PDAC cells seeded first, and the OD value showed a significantly difference at 24h and 48h after treated with the exosomes, P<0.05). Compared with the control group, exosomes derived from PC patients can promote the proliferation of PC cells.

Figure 6

Wound-healing assay in vitro of PC cells (MiaPaCa-2, AsPC-1) added serum exosomes derived from PC patients. As can be seen from the figure, exosomes derived from PC patients can promote the migration of PDAC cells (***P < 0.001).

Figure 7

Transwell invasion experiment of Miapaca-2 and Aspc-1 treated with or without exosomes derived from PC patients. As can be seen from the figure, exosomes derived from PC patients can increased the trans magration of PDAC cell lines significantly (***P < 0.001).

Figure 8

The laser confocal imaging of EMT related proteins. Vimentin was up expressed, while the E-cadherin was down expressed when treated with exosomes derived from PC patients. In order to make each group comparable, the fluorescence brightness of the nucleus was set at the same level in the laser confocal imaging of the three groups (***P < 0.001).