11 months ago

2006 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Research Interests Cancer induces a variety of changes in the protein composition of blood. Increased cell breakdown in the tumor environment, oxidative stress, inflammation, new blood vessel formation, secretions from tumors, and immune responses against tumors all may contribute to such changes. Measurements of these various alterations may be useful in understanding the roles of secreted and circulating proteins during cancer progression and in developing improved blood tests for cancer diagnosis and management. We are characterizing protein changes that are prevalent in the blood of cancer patients, developing and testing hypotheses relating to the origin and functional consequences of the changes, and exploring the use of these measurements for cancer diagnostics. We have developed several array-based protein analysis methods to look at various aspects of proteins, including their abundance, glycosylation, immunoreactivity, and protein-protein interactions. Array-based methods allow the analysis of multiple proteins simultaneously in very small sample volumes, which is useful for efficiently gathering information from small clinical samples or for testing hypotheses that may not be practical to test using conventional technologies. We use high-precision robotics to deposit tiny droplets of protein or antibody solutions onto the surfaces of coated microscope slides, which are then incubated with biological samples. Interactions between species in the samples and the spotted proteins are observed in a variety of ways. Conventional protein analysis methods are used to complement the information obtained from our newly developed platforms. 28 Glycosylation (the attachment of carbohydrate structures to proteins) is an important determinant of protein function, and changes in glycosylation are thought to play roles in certain disease processes. Glycosylation changes occur, for example, on cancer-cell adhesion molecules, on secreted proteins that regulate the environment of the cancer cells, and on stress-response proteins. Those changes may have roles in modulating the adhesion or migratory capabilities of cancer cells or in regulating protein-protein interactions and signaling. We have developed a novel array-based strategy to probe the levels of specific glycan structures on multiple different proteins. Serum samples are incubated on antibody microarrays and, after washing away unbound serum proteins, the glycan levels on the captured proteins are detected with a biotin-labeled lectin (Fig. 1A). (Lectins are plant and animal proteins with natural carbohydrate-binding functionality.) A variant of the method allows the measurement of both the protein level and the glycan level in a single experiment (Fig. 1B). In that method, serum proteins are labeled with digoxigenin prior to application to an array, and the biotinylated lectin and digoxigenin-labeled proteins are detected with streptavidin-linked phycoerythrin (green fluorescence) and Cy5-labeled anti-digoxigenin antibody (red fluorescence), respectively. The detection of glycans on proteins captured by immobilized antibodies was confirmed using both methods (Fig. 1C). This method for profiling variation in specific glycans on multiple proteins should be useful in diverse areas of glycobiology research. Figure 1. Figure 1. Detection of glycans on antibody arrays. a) One-color glycan detection. b) Two-color detection of glycans and proteins, using digoxigeninlabeled proteins. c) Antibody arrays were incubated with no serum, unlabeled serum, or digoxigenin-labeled serum. The arrays were then incubated with biotinylated SNA lectin, followed by detection with Cy5-labeled anti-digoxigenin (red, 633 nm) and Cy3-labeled anti-biotin (green, 543 nm). The arrays were scanned at both 543 nm and 633 nm, and fluorescence from the spots is shown in both color channels from the indicated antibodies.

VARI | 2006 Pancreatic cancer The glycan-detection arrays were used to find glycosylation changes on specific proteins in the sera of pancreatic cancer patients. The proteins CEA and MUC1 are thought to be involved in controlling the tumor cell environment, and glycan alterations on those proteins may be involved in altering cancer cell migratory or adhesive properties. A blood-group-related carbohydrate structure—the sialyl-Lewis structure, targeted by the CA19-9 monoclonal antibody—is elevated on both CEA and MUC1 in cancer patients (Fig. 2). That structure is the ligand for the endothelial cell-surface receptor E-selectin, which controls the homing of white blood cells to sites of inflammation and has been implicated in cancer-cell invasion. We are currently investigating that glycan on secreted MUC1 and CEA molecules in terms of its possible role in modulating cell migration or in signaling to cell surfaces. We also are investigating the roles of glycan structures in mediating interactions between inflammatory cytokines and MUC1, CEA, and other proteins. Our screens have turned up other cancer-associated glycan changes that we are pursuing, and additional glycan structures continue to be probed. 29 Figure 2. Figure 2. Comparison of protein and glycan levels using parallel sandwich and glycan-detection assays. A) Representative array images. A pool of 30 cancer serum samples was incubated on the left pair of arrays, and a healthy-patient serum sample and cancer-patient serum sample were incubated on the middle and right pairs of arrays (same two samples in each pair). The arrays were detected using the indicated antibodies. The spots appearing at the lower right of each array were biotinylated control proteins. The other spots that showed signals were anti-haptoglobin in the array detected with anti-MUC1 (second image from left), and anti-CA19-9, anti-pan CEACAM, and the WGA lectin in the array detected with anti-CA19-9 (right-most image). B) Scatter-plot comparison of protein and glycan levels. Forty-six serum samples (23 from cancer patients and 23 from controls) were incubated on replicate sets of antibody arrays. One set of 46 arrays was detected with antibodies against CEA and MUC1 to obtain the variation in levels of these two proteins, and the other set of arrays was detected with the glycan-binding antibody anti-CA19-9. The graphs depict the levels detected at the CEA- (left graph) or MUC1- (right graph) capture antibodies by either the anti-protein antibodies (y-axis) or the anti-CA19-9 antibody (x-axis) for each control-patient serum sample (dark triangles) and each cancer-patient serum sample (open circles). The protein alterations that we are discovering in the sera of cancer patients could be used to develop improved diagnostic tests. Blood tests could be an important improvement in diagnostics because they can be routinely and inexpensively administered for early detection of disease, to aid in diagnosis or staging, or to follow the progress of treatment. Early detection tests for pancreatic cancer could greatly improve outcomes for many patients, since that cancer is usually not detected until it has advanced past a treatable stage. Our previous studies have shown that a number of proteins have altered abundances in the sera of pancreatic cancer patients and that the measurements of multiple proteins simultaneously, as is possible with array-based tools, can be used in combination to form diagnostic tests having improved accuracy over tests based on single protein measurements. In collaboration with Randall Brand (Evanston Northwestern Healthcare), Diane Simeone (University of Michigan), and Michael Hollingsworth (University of Nebraska), as well as with the Early Detection Research Network (EDRN), we are combining our results with other discoveries of protein alterations in pancreatic cancer to further develop, refine, test, and validate these new approaches.

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