New insights in the composition of extracellular vesicles from pancreatic cancer cells: implications for biomarkers and functions.

BACKGROUND: Pancreatic cancer development is associated with characteristic alterations like desmoplastic reaction and immune escape which are mediated by the cell-cell communication mechanism and by the microenvironment of the cells. The whole of released components are important determinants in these processes. Especially the extracellular vesicles released by pancreatic cancer cells play a role in cell communication and modulate cell growth and immune responses. RESULTS: Here, we present the proteomic description of affinity purified extracellular vesicles from pancreatic tumour cells, compared to the secretome, defined as the whole of the proteins released by pancreatic cancer cells. The proteomic data provide comprehensive catalogues of hundreds of proteins, and the comparison reveals a special proteomic composition of pancreatic cancer cell derived extracellular vesicles. The functional analysis of the protein composition displayed that membrane proteins, glycoproteins, small GTP binding proteins and a further, heterogeneous group of proteins are enriched in vesicles, whereas proteins derived from proteasomes and ribosomes, as well as metabolic enzymes, are not components of the vesicles. Furthermore proteins playing a role in carcinogenesis and modulators of the extracellular matrix (ECM) or cell-cell interactions are components of affinity purified extracellular vesicles. CONCLUSION: The data deepen the knowledge of extracellular vesicle composition by hundreds of proteins that have not been previously described as vesicle components released by pancreatic cancer cells. Extracellular vesicles derived from pancreatic cancer cells show common proteins shared with other vesicles as well as cell type specific proteins indicating biomarker candidates and suggesting functional roles in cancer cell stroma interactions.

A soluble form of the giant cadherin Fat1 is released from pancreatic cancer cells by ADAM10 mediated ectodomain shedding.

In pancreatic cancer, there is a clear unmet need to identify new serum markers for either early diagnosis, therapeutic stratification or patient monitoring. Proteomic analysis of tumor cell secretomes is a promising approach to indicate proteins released from tumor cells in vitro. Ectodomain shedding of transmembrane proteins has previously been shown to contribute significant fractions the tumor cell secretomes and to generate valuable serum biomarkers. Here we introduce a soluble form of the giant cadherin Fat1 as a novel biomarker candidate. Fat1 expression and proteolytic processing was analyzed by mass spectrometry and Western blotting using pancreatic cancer cell lines as compared to human pancreatic ductal epithelial cells. RNA expression in cancer tissues was assessed by in silico analysis of publically available microarray data. Involvement of ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in Fat1 ectodomain shedding was analyzed by chemical inhibition and knockdown experiments. A sandwich ELISA was developed to determine levels of soluble Fat1 in serum samples. In the present report we describe the release of high levels of the ectodomain of Fat1 cadherin into the secretomes of human pancreatic cancer cells in vitro, a process that is mediated by ADAM10. We confirm the full-length and processed heterodimeric form of Fat1 expressed on the plasma membrane and also show the p60 C-terminal transmembrane remnant fragment corresponding to the shed ectodomain. Fat1 and its sheddase ADAM10 are overexpressed in pancreatic adenocarcinomas and ectodomain shedding is also recapitulated in vivo leading to increased Fat1 serum levels in some pancreatic cancer patients. We suggest that soluble Fat1 may find an application as a marker for patient monitoring complementing carbohydrate antigen 19-9 (CA19-9). In addition, detailed analysis of the diverse processed protein isoforms of the candidate tumor suppressor Fat1 can also contribute to our understanding of cell biology and tumor behavior.

Characterization of soluble and exosomal forms of the EGFR released from pancreatic cancer cells.

AIMS: Members of the epidermal growth factor receptor (EGFR) family represent validated targets for anti-cancer therapy and EGFR inhibitors have also shown efficacy in pancreatic carcinoma. We here described in detail molecular forms of the EGF receptor released by pancreatic cancer cells. We found peptides specific for the EGFR in the secretomes of five pancreatic cancer cell lines. Secretomes from cultured cancer cells are widely used as sources for serum biomarker discovery. MAIN METHODS: The detailed analysis of EGFR forms in secretomes of human pancreatic cancer cells is a compilation of results from mass spectrometry (MS) and Western blotting with intracellular and extracellular domain specific antibodies. KEY FINDINGS: Pancreatic cancer cells secrete a 110 kDa soluble form of the EGFR (sEGFR) representing the ligand binding extracellular EGFR domains and presumably released by ectodomain shedding. At the same time, as constituents of exosomes, the EGFR is released as full-length intact receptor (170 kDa) and as a 65 kDa processed form, the C-terminal remnant fragment that corresponds to the intracellular kinase domain. SIGNIFICANCE: The detailed characterization of diverse EGFR forms released by pancreatic cancer cells in vitro and presumably in vivo bears important implications for functional studies, for the validation of soluble EGFR as a serum biomarker and for the design of targeted therapies.