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2007 Scientific Report

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VARI | 2007 Research Interests Members of the Haab laboratory identify protein and carbohydrate abnormalities in the blood of cancer patients and investigate the significance and potential clinical usefulness of those abnormalities. We develop novel experimental methods to facilitate this work, and we collaborate with both clinicians and basic scientists to pursue research on pancreatic and prostate cancers. Low-volume, high-throughput antibody and protein arrays We have developed the ability to probe multiple proteins or carbohydrate structures using low sample volumes, which provides a powerful tool for identifying and measuring protein and carbohydrate abnormalities in cancer. Antibody and protein arrays immobilized on the surface of a microscope slide are the key to such a capability. A biological sample such as blood serum can be incubated on an array to investigate interactions between the immobilized molecules and the proteins or antibodies in the sample. Those interactions can be probed to obtain information such as protein abundance, glycosylation level, or protein-protein interaction level. The routine use of these tools was made possible by the development of a practical method for processing multiple arrays on a microscope slide (Fig. 1). A stamp imprints a wax pattern onto the surface of a slide, creating hydrophobic partitions that segregate various samples. Distinct stamp designs can be used to form differing sizes and numbers of partitions. A design that imprints 48 arrays on one slide requires only 6 μl of sample per array, with each array composed of 144 distinct spots of immobilized molecules. Such a design enables the efficient processing of many samples or testing of many conditions in parallel, as demonstrated in the projects described below. The device for creating these slides is commercially available from The Gel Company, San Francisco. 27 Figure 1A. Figure 1B. Figure 1C. Figure 1. High-throughput sample processing using a novel slide partitioning method. A) Wax is imprinted onto a microscope slide to form borders around multiple arrays. Wax is melted by the hotplate under the bath, and a slide is inserted upside-down into the holder. Bringing the lever forward raises a stamp out of the wax bath to touch the slide, imprinting the design onto the slide. Two stamps are shown in front of the machine. B) Loading samples onto a slide containing 48 arrays. The arrays are spaced by 4.5 mm, which is compatible with the 9 mm spacing of standard multichannel pipettes. C) Samples loaded onto slides containing 12 (top), 48 (middle), and 192 (bottom) arrays (96 samples loaded).

Van Andel Research Institute | Scientific Report Glycans in pancreatic cancer One of the major interests of the lab is characterizing and studying the changes in carbohydrate structures (glycans) on particular proteins from pancreatic cancer patients. A novel technique developed in our laboratory enables the measurement of specific glycans on multiple proteins in biological samples (Fig. 2A, B). We use lectins—proteins that bind specific glycan structures—as well as glycan-binding antibodies to probe the levels of particular glycans on the proteins captured on the antibody arrays. Several types of lectins, each with its own carbohydrate binding specificity, can be used to identify the carbohydrate structures associated with each protein. We can analyze many different patient samples or cell culture conditions, looking at associations between glycan levels and disease states or at the effects of certain perturbations on glycan structures. This method is in development for commercial use by GenTel Biosciences (Madison, WI). Mucins are long-chain, heavily glycosylated proteins on epithelial cell surfaces that have roles in cell protection, interaction with the extracellular space, and regulation of extracellular signaling. Screening studies in collaboration with Randall Brand and Diane Simeone have revealed a variety of glycan alterations on mucin molecules from pancreatic cancer patients (a representative example is shown in Fig. 2C). Altered carbohydrates on mucins can affect critical processes in cancer such as cell migration or extracellular signaling to the immune system. We are characterizing the glycan structural variation on mucins secreted from cancer cells and other cells, and we are using cell culture systems to study the origins and effects of those variations. We are pursuing hypotheses about the effects of extracellular stress from an inflammatory tumor environment on mucin carbohydrate structures and the resulting interactions of those structures with inflammatory proteins and host cells. Figure 2A. 28 Figure 2B. Figure 2C. Figure 2. Complementary antibody array formats for protein and glycan detection. A) Sandwich assay with fluorescence detection to measure protein abundance. B) Antibody-lectin assay. The biotinylated lectin binds to glycans on the proteins captured by the immobilized antibodies. The antibodies are first chemically derivatized to prevent lectin binding to the glycans of the immobilized capture antibodies. C) Detecting protein and glycan variation in cancer and control sera. Sandwich detection of the MUC1 and CEA proteins showed similar levels in serum samples from a cancer patient and a control subject (left images). The anti-CA19-9 antibody, which targets a glycan structure, detected a significant glycan increase on MUC1 and CEA in the cancer serum (right images).

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