Search

Exosomes are small vesicles found in extracellular environments including blood, urine, and cell culture medium. Their contents are cell‑type specific, and molecules embedded in exosomes can be useful fluid‑based clinical biomarkers. To identify proteins with metastatic marker potential, we conducted a comparative exosomal proteome analysis using human pancreatic cancer cell lines derived from metastasis, ascites, and primary tumors. Metastatic potential of cell lines was assessed by migratory and invasive activities. A pancreatic cancer cell line from metastasis (SU.86.86) revealed 23‑fold and 20‑fold increases in cell migratory and invasive activities, respectively, compared to the MIA PaCa‑2 cell line derived from primary tumor cells. Liquid chromatography‑mass spectrometry‑based proteome analysis and subsequent validation by immunoblot analysis revealed that epidermal growth factor receptor pathway substrate 8 (Eps8) was highly abundant in exosomes from metastasis‑derived SU.86.86 cells. Comparison of 12 pancreatic cancer cell lines derived from different stages of malignancy revealed a strong relationship between exosomal Eps8 protein levels and cell motile activities (migration: r=0.85, P=4.2x10‑4; invasion: r=0.60, P=3.2x10‑2). Conversely, relationships between intracellular Eps8 protein levels and cell motile activities were moderate (migration: r=0.65, P=2.0x10‑2; invasion: r=0.51, P=9.2x10‑2). It was therefore concluded that exosomal Eps8 protein levels were correlated with the migratory cell potential of human pancreatic cancer cells, indicating that exosomal Eps8 has the potential to be a metastatic biomarker for human pancreatic cancer.

Exosomes are small vesicles secreted from cells that transport their embedded molecules through bidirectional exocytosis- and endocytosis-like pathways. Expression patterns of exosomal molecules such as proteins and RNAs can be indicative of cell type since their signature is thought to be unique among cells. Using human primary (AZ-521) and metastatic (AZ-P7a) duodenal cancer cell lines, we conducted a comparative exosomal proteome analysis to identify proteins with metastatic marker potential. As determined by LC-MS/MS and Western blot analyses, polyadenylate-binding protein 1 (PABP1) was found to be predominantly abundant in AZ-P7a exosomes. The amount of exosomal PABP1 in AZ-P7a cells increased by treating the cells with inhibitors for the classical ER/Golgi secretory pathway (brefeldin A and monensin) and the ubiquitin-proteasome pathway (MG-132 and PYR-41). Treatment of AZ-P7a cells with the neutral sphingomyelinase inhibitor GW4869, which suppresses exosome release, not only reduced the amount of exosomal PABP1 but also produced PABP1-immunoreactive products cleaved via a proteolysis-like process. Taken together, these results suggest that AZ-P7a cells do not tolerate intracellular PABP1 accumulation and are thus exported into the extracellular milieu by the exosome-mediated pathway. In addition, PABP1 has a potential use as a biomarker for metastatic duodenal cancer.

BackgroundExosomes play a major role in cell-to-cell communication, targeting cells to transfer exosomal molecules including proteins, mRNAs, and microRNAs (miRNAs) by an endocytosis-like pathway. miRNAs are small noncoding RNA molecules on average 22 nucleotides in length that regulate numerous biological processes including cancer pathogenesis and mediate gene down-regulation by targeting mRNAs to induce RNA degradation and/or interfering with translation. Recent reports imply that miRNAs can be stably detected in circulating plasma and serum since miRNAs are packaged by exosomes to be protected from RNA degradation. Thus, profiling exosomal miRNAs are in need to clarify intercellular signaling and discover a novel disease marker as well.Methodology/principal findingsExosomes were isolated from cultured cancer cell lines and their quality was validated by analyses of transmission electron microscopy and western blotting. One of the cell lines tested, a metastatic gastric cancer cell line, AZ-P7a, showed the highest RNA yield in the released exosomes and distinctive shape in morphology. In addition, RNAs were isolated from cells and culture media, and profiles of these three miRNA fractions were obtained using microarray analysis. By comparing signal intensities of microarray data and the following validation using RT-PCR analysis, we found that let-7 miRNA family was abundant in both the intracellular and extracellular fractions from AZ-P7a cells, while low metastatic AZ-521, the parental cell line of AZ-P7a, as well as other cancer cell lines showed no such propensity.Conclusions/significanceThe enrichment of let-7 miRNA family in the extracellular fractions, particularly, in the exosomes from AZ-P7a cells may reflect their oncogenic characteristics including tumorigenesis and metastasis. Since let-7 miRNAs generally play a tumor-suppressive role as targeting oncogenes such as RAS and HMGA2, our results suggest that AZ-P7a cells release let-7 miRNAs via exosomes into the extracellular environment to maintain their oncogenesis.