Abstract
Pancreatic cancer is one of the leading causes of cancer-related deaths, for which serological biomarkers are urgently needed. Most discovery-phase studies focus on the use of one biological source for analysis. The present study details the combined mining of pancreatic cancer-related cell line conditioned media and pancreatic juice for identification of putative diagnostic leads. Using strong cation exchange chromatography, followed by LC-MS/MS on an LTQ-Orbitrap mass spectrometer, we extensively characterized the proteomes of conditioned media from six pancreatic cancer cell lines (BxPc3, MIA-PaCa2, PANC1, CAPAN1, CFPAC1, and SU.86.86), the normal human pancreatic ductal epithelial cell line HPDE, and two pools of six pancreatic juice samples from ductal adenocarcinoma patients. All samples were analyzed in triplicate. Between 1261 and 2171 proteins were identified with two or more peptides in each of the cell lines, and an average of 521 proteins were identified in the pancreatic juice pools. In total, 3479 nonredundant proteins were identified with high confidence, of which ∼ 40% were extracellular or cell membrane-bound based on Genome Ontology classifications. Three strategies were employed for identification of candidate biomarkers: (1) examination of differential protein expression between the cancer and normal cell lines using label-free protein quantification, (2) integrative analysis, focusing on the overlap of proteins among the multiple biological fluids, and (3) tissue specificity analysis through mining of publically available databases. Preliminary verification of anterior gradient homolog 2, syncollin, olfactomedin-4, polymeric immunoglobulin receptor, and collagen alpha-1(VI) chain in plasma samples from pancreatic cancer patients and healthy controls using ELISA, showed a significant increase (p < 0.01) of these proteins in plasma from pancreatic cancer patients. The combination of these five proteins showed an improved area under the receiver operating characteristic curve to CA19.9 alone. Further validation of these proteins is warranted, as is the investigation of the remaining group of candidates.
Highlights
EXPERIMENTAL PROCEDURESCell Lines—Six pancreatic cancer cell lines (MIA-PaCa2 (CRL1420), PANC1 (CRL-1469), BxPc3 (CRL-1687), CAPAN1 (HTB-79), CFPAC-1 (CRL-1918), and SU.86.86 (CRL-1837)) were obtained from the American Type Culture Collection (ATCC, Manassas, VA)
From the ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; §Department of Clinical Biochemistry, University Health Network, Toronto, ON, Canada; ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada; ʈThe Fred Hutchinson Cancer Research Center, Seattle, Washington; **Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technical University of Dresden, Germany; ‡‡Zane Cohen Familial Gastrointestinal Cancer Registry and Department of Surgery, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada
Increasing Protein Yield—Once the cell lines were grown and conditioned media (CM) collected, the samples were subjected to a twodimensional (2D)-LC-MS/MS analysis, which combined Strong Cation Exchange (SCX) liquid chromatography on an High Pressure Liquid Chromatography (HPLC) system, followed by LCMS/MS
Summary
Cell Lines—Six pancreatic cancer cell lines (MIA-PaCa2 (CRL1420), PANC1 (CRL-1469), BxPc3 (CRL-1687), CAPAN1 (HTB-79), CFPAC-1 (CRL-1918), and SU.86.86 (CRL-1837)) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The files generated from MASCOT (DAT files) and X!Tandem (XML files) for the three replicates of each cell line or pancreatic juice pool were integrated through Scaffold 2 software (version 2.06; Proteome Software Inc., Portland, Oregon) resulting in a nonredundant list of identified proteins per sample. Data Analysis—Scaffold prot-XML reports were generated and uploaded onto Protein Center Professional Edition (version 3.5.2.1; Proxeon Bioinformatics, Odense, Denmark) to facilitate comparisons between cell line CM and pancreatic juice proteomes, and to obtain Genome Ontology information. One Scaffold file containing all of the normalized spectral counts of each of the three replicates from the seven cell lines was generated for proteins identified with two or more peptides. The area under the curves (AUC) were calculated using ROCR software and the corresponding variances were calculated with a bootstrap method
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