title: Identification and validation of novel protein biomarkers in pancreatic ductal adenocarcinoma with the ability to distinguish molecular subtypes
creator: Kuhlmann, Laura 
description: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer, with a medium overall survival of only six months. The standard of care treatment, consisting of surgical resection followed by adjuvant chemotherapy, which incidentally can be offered to as little as 20% of the newly diagnosed patients, shows only marginal benefit for patients and no significant improvements to PDAC therapy have been made over the past two decades. PDAC is currently treated as a single, homogenous disease. Recently, three molecular subtypes termed classical, exocrine-like and quasimesenchymal (QM) have been defined, based on transcriptomic profiling of micro-dissected cancer cells from primary tumors and commercially available cell lines. The newly defined molecular subtypes were associated with differences in patient overall survival and are suggested to predict response to different chemotherapeutical agents. However, no clinical markers are available for the stratification of patients according to these molecular subtypes.  The aim of this study was the identification and validation of novel pan-PDAC as well as subtype-specific protein biomarker candidates, which can be employed in the development of novel clinical applications, such as diagnostics and/ or targeted PDAC therapies. Using a panel of twelve patient derived primary pancreatic cancer cell lines, grown in a chemically defined serum free medium, we employed LC-MALDI-MS to identify novel cell surface and secreted PDAC protein biomarker candidates. Two commercially available healthy pancreatic cell lines served as controls. The cell surface proteome was analyzed following in vitro biotinylation and subsequent streptavidin pull-down of the covalently biotin-labeled proteins, while the secreted proteome was analyzed by shotgun proteomics. In addition, we investigated in vivo vascular accessible PDAC biomarkers, by carrying out whole body perfusions of mice bearing orthotopic pancreatic tumors using a reactive biotin ester solution. Biomarker candidates of interest were selected after the proteomics discovery experiment and underwent thorough validations, using complementary antibody based (immunofluorescence, Western Blot) and antibody independent (single reaction monitoring, RT-qPCR) techniques. The validation experiments were performed both in vitro – using the PDAC cell culture model and in vivo – using tumor xenografts developed in immunodeficient mice.  More than 2500 proteins were identified after completing the cell surface proteome analysis, and over 1700 proteins could be reported in the conditioned cell culture medium. We selected the two pan-PDAC biomarker candidates, Pan-marker-1 and Pan-marker-2, as well as the two exocrine-like biomarker candidates, Exo-1 and Exo-2, for further validations.   In vitro immunofluorescence confirmed the exclusive presence of Pan-marker-1 and Pan-marker-2 on the surface of primary patient-matched PDAC cell lines, which correlated with elevated levels of the corresponding mRNAs. The expression of the two exocrine-like protein biomarker candidates was restricted to the predicted subtype, as proven by immunofluorescence and single reaction monitoring. The secretion of the two exocrine-like proteins in the cell culture medium was additionally confirmed by single reaction monitoring. The regulation of the two exocrine-like PDAC biomarkers was at least partially due to the upregulation of the corresponding mRNAs. Additionally, we could report that Exo-1 and Exo-2 colocalize on the surface of cultured exocrine-like PDAC cells. In vivo immunofluorescence analyses corroborated the presence of Pan-marker-1 and Pan-marker-2 on the surface of pancreatic cancer cells and in the tumor environment, while being completely absent in healthy human pancreata. Exocrine-like tumor xenografts expressed Exo-1 and Exo-2 at high levels, as evaluated by immunofluorescence. However, a limited activation of the two putative exocrine-like biomarkers could be detected in vivo in some of the classical pancreatic tumors. Protein expression patterns correlated with reported levels of the respective mRNAs for all the investigated biomarker candidates, evaluated in both EpCam+ epithelial cells isolated from orthotopic xenografts and whole tumor tissue.  Moreover, Pan-marker-2 and Exo-2 could be detected in the course of a proof-of-principle in vivo perfusion experiment using mice harboring orthotopic tumors. Therefore, we could conclude that the two proteins are accessible from the blood stream and could represent candidates for novel PDAC targeted therapies.    Taken together, our study identifies novel pan-PDAC as well as subtype-specific protein biomarkers candidates, which can be used in clinical applications for patient stratification. Additionally, the proteins were secreted by pancreatic cancer cells, and could therefore represent the basis for developing novel non-invasive diagnostic tools. Importantly, the validated protein biomarker candidates were also accessible from the vascular system, making them eligible targets for novel antibody based therapies of PDAC.   
date: 2015
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/17467/1/20140709_PhD%20thesis%20Laura%20Kuhlmann.pdf
identifier: DOI:10.11588/heidok.00017467
identifier: urn:nbn:de:bsz:16-heidok-174674
identifier:   Kuhlmann, Laura   (2015) Identification and validation of novel protein biomarkers in pancreatic ductal adenocarcinoma with the ability to distinguish molecular subtypes.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/17467/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng