Antibody microarray profiling reveals individual and combined serum proteins associated with pancreatic cancer.

We used antibody microarrays to probe the associations of multiple serum proteins with pancreatic cancer and to explore the use of combined measurements for sample classification. Serum samples from pancreatic cancer patients (n = 61), patients with benign pancreatic disease (n = 31), and healthy control subjects (n = 50) were probed in replicate experiment sets by two-color, rolling circle amplification on microarrays containing 92 antibodies and control proteins. The antibodies that had reproducibly different binding levels between the patient classes revealed different types of alterations, reflecting inflammation (high C-reactive protein, alpha-1-antitrypsin, and serum amyloid A), immune response (high IgA), leakage of cell breakdown products (low plasma gelsolin), and possibly altered vitamin K usage or glucose regulation (high protein-induced vitamin K antagonist-II). The accuracy of the most significant antibody microarray measurements was confirmed through immunoblot and antigen dilution experiments. A logistic-regression algorithm distinguished the cancer samples from the healthy control samples with a 90% and 93% sensitivity and a 90% and 94% specificity in duplicate experiment sets. The cancer samples were distinguished from the benign disease samples with a 95% and 92% sensitivity and an 88% and 74% specificity in duplicate experiment sets. The classification accuracies were significantly improved over those achieved using individual antibodies. This study furthered the development of antibody microarrays for molecular profiling, provided insights into the nature of serum-protein alterations in pancreatic cancer patients, and showed the potential of combined measurements to improve sample classification accuracy.

Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis.

BACKGROUND: Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. METHODS: Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance. RESULTS: Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and alpha-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified. CONCLUSION: Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer.

Modulation of cell adhesion molecules in various epithelial cell lines after treatment with PP2.

Regulation and expression of E-cadherin and other adhesion molecules were evaluated after exposure to a selective inhibitor of the Src family of tyrosine kinases and inducer of E-cadherin, PP2. E-cadherin is located within the intercellular junction, and it is involved in the management of paracellular permeability of various epithelial barriers in the body. Epithelial cell lines HCT-116, HT29, Caco-2, LS174T, and ARPE-19 were examined for morphological, functional, protein, and mRNA changes following 20 microM PP2 treatment. PP2 treatment caused cell clustering in Caco-2, HT29, and HCT-116 cells. E-cadherin also redistributed to the points of cell contact in Caco-2 cells. These changes suggest increased E-cadherin-dependent cell adhesion. Studies evaluating transepithelial electrical resistance, an established measurement of paracellular permeability, displayed increases in resistance for the Caco-2 cells following PP2 treatment, which correlates with our microscopy data. In addition, E-cadherin protein levels increased for all cells except HCT-116. ARPE-19 cells did not express E-cadherin at the protein or mRNA level. Expression of adhesion molecules varied for the cell lines, and only Claudin 3 mRNA expression was significantly increased in the three intestinal cell lines treated with PP2. Overall, our data suggest that E-cadherin is positively regulated by inhibition of Src tyrosine kinases at the functional and protein expression levels within these epithelial cell lines.