Abstract
Abstract e14591 Background: The important role played by activated pancreatic stellate cells (aPSCs) in PDAC has only been recently appreciated. By shaping the tumor microenvironment, APSCs contribute to tumor growth, metastasis and drug resistance. Innovative stable isotope labeling with amino acids in cell culture (SILAC) and multiple reaction monitoring (MRM)-MS provide tools for interrogating human serum samples for aPSC derived biomarkers of PDAC. 600 proteins secreted by aPSCs were previously characterized (Wehr, 2010). Selected proteins were shown to be overexpressed in pancreatic tumors. An immortalized PSC cell line was SILAC labeled; secreted proteins were collected from activated cells and used as labeled internal standards. An MRM-MS method was developed to quantitate the 40 most abundant secreted aPSC proteins in serum. Internal standard proteins were added to human serum samples prior to immunoaffinity (IA) removal of abundant serum proteins, trypsin digested and analyzed by liquid chromatography (LC)/MRM-MS. Powered primary endpoint: characterize the sensitivity and specificity of biomarker panel in a case control study of 10 patients with PDAC and 10 healthy controls. Secondary endpoint: biological pathway analysis of discriminating biomarkers. 183 unique peptides were identified for the selected 40 aPSC proteins. 69 peptides, representing 36 proteins, were reproducibly detected by MS. During method development, a prototypical biomarker candidate, MMP2, was monitored; Western blot and MS analysis showed that MMP2 was not lost by IA processing, and removal of abundant proteins was required for detection of MMP2 by MS. The method was found to be linear over a range of 4-100 fmol/µL media, and met precision criteria of
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