title: Secretome protein signature of human pancreatic cancer stem-like cells.
abstract: Emerging research has demonstrated that pancreatic ductal adenocarcinoma (PDAC) contains a sub-population of cancer stem cells (CSCs) characterized by self-renewal, anchorage-independent-growth, long-term proliferation and chemoresistance. The secretome analysis of pancreatic CSCs has not yet been performed, although it may provide insight into tumour/microenvironment interactions and intracellular processes, as well as to identify potential biomarkers. To characterize the secreted proteins of pancreatic CSCs, we performed an iTRAQ-based proteomic analysis to compare the secretomes of Panc1 cancer stem-like cells (Panc1 CSCs) and parental cell line. A total of 72 proteins were found up-/down-regulated in the conditioned medium of Panc1 CSCs. The pathway analysis revealed modulation of vital physiological pathways including glycolysis, gluconeogenesis and pentose phosphate. Through ELISA immunoassays we analysed the presence of the three proteins most highly secreted by Panc1 CSCs (ceruloplasmin, galectin-3, and MARCKS) in sera of PDAC patient. ROC curve analysis suggests ceruloplasmin as promising marker for patients negative for CA19-9. Overall, our study provides a systemic secretome analysis of pancreatic CSCs revealing a number of secreted proteins which participate in pathological conditions including cancer differentiation, invasion and metastasis. They may serve as a valuable pool of proteins from which biomarkers and therapeutic targets can be identified.
  
  
  CONCLUSIONS
  The secretome of CSCs is a rich reservoir of biomarkers of cancer progression and molecular therapeutic targets, and thus is a topic of great interest for cancer research. The secretome analysis of pancreatic CSCs has not yet been performed. Recently, our group has demonstrated that Panc-1 CSCs isolated from parental cell line by using the CSC selective medium, represent a model of great importance to deepen the understanding of the biology of pancreatic adenocarcinoma. To our knowledge, this is the first proteomic study of pancreatic CSC secretome. We performed an iTRAQ-based analysis to compare the secretomes of Panc1 CSCs and Panc1 parental cell line and identified a total of 43 proteins secreted at higher level by pancreatic cancer stem cells. We found modulation of different vital physiological pathways (such as glycolysis and gluconeogenesis, pentose phosphate pathway) and the involvement of CSC secreted proteins (for example 72kDa type IV collagenase, galectin-3, alpha-actinin-4, and MARCKS) in pathological conditions including cancer differentiation, invasion and metastasis. By ELISA verification we found that MARCKS and ceruloplasmin discriminate between controls and PDAC patients; in addition ROC curve analyses indicate that MARCKS does not have diagnostic accuracy, while ceruloplasmin could be a promising marker only for patients negative for CA19-9. We think that the findings reported in our manuscript advance the understanding of the pathways implicated in tumourigenesis, metastasis and chemoresistance of pancreatic cancer, and also identify a pool of proteins from which novel candidate diagnostic and therapeutic biomarkers could be discovered.
authors: University of Verona, Department of Biotechnology, Proteomics and Mass Spectrometry Laboratory, Verona 37134, Italy.
authors: Jessica
authors: J
authors: Brandi
authors: University of Verona, Department of Life and Reproduction Sciences, Verona 37134, Italy.
authors: Elisa
authors: E
authors: Dalla Pozza
authors: University of Verona, Department of Life and Reproduction Sciences, Verona 37134, Italy.
authors: Ilaria
authors: I
authors: Dando
authors: University of Verona, Department of Life and Reproduction Sciences, Verona 37134, Italy.
authors: Giulia
authors: G
authors: Biondani
authors: University of Piemonte Orientale, Department of Sciences and Technological Innovation, Alessandria 15121, Italy.
authors: Elisa
authors: E
authors: Robotti
authors: University of Liverpool, MRC Centre for Drug Safety Science, Department of Molecular & Clinical Pharmacology, Liverpool L69 3GE, United Kingdom.
authors: Rosalind
authors: R
authors: Jenkins
authors: NIHR Liverpool Pancreas Biomedical Research Unit, Department of Molecular and Therapeutic Cancer Medicine, Liverpool L69 3GA, United Kingdom.
authors: Victoria
authors: V
authors: Elliott
authors: University of Liverpool, MRC Centre for Drug Safety Science, Department of Molecular & Clinical Pharmacology, Liverpool L69 3GE, United Kingdom.
authors: Kevin
authors: K
authors: Park
authors: University of Piemonte Orientale, Department of Sciences and Technological Innovation, Alessandria 15121, Italy.
authors: Emilio
authors: E
authors: Marengo
authors: NIHR Liverpool Pancreas Biomedical Research Unit, Department of Molecular and Therapeutic Cancer Medicine, Liverpool L69 3GA, United Kingdom.
authors: Eithne
authors: E
authors: Costello
authors: University and Hospital Trust of Verona, Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, Verona 37134, Italy.
authors: Aldo
authors: A
authors: Scarpa
authors: University of Verona, Department of Life and Reproduction Sciences, Verona 37134, Italy. Electronic address: marta.palmieri@univr.it.
authors: Marta
authors: M
authors: Palmieri
authors: University of Verona, Department of Biotechnology, Proteomics and Mass Spectrometry Laboratory, Verona 37134, Italy.
authors: Daniela
authors: D
authors: Cecconi
mesh_terms: D002460:Cell Line; D006801:Humans; D010179:Pancreas / Q000166:cytology / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*
chemical_list: D020543:Proteome
keywords: Pancreatic cancer; Secretome; iTRAQ-based proteomic