Research Interests
All cells secrete molecules that are used to send signals and perform functions in the local and distant spaces of the body. The molecular secretions of cancer cells often are significantly different from those of their normal counterparts. Our lab studies particular proteins and carbohydrates secreted by cancer cells in order to understand their roles in cancer progression, as well as to develop novel clinical tests for the detection and diagnosis of cancer.
Glycoprotein biomarkers for pancreatic cancer
A great need exists for better tools to detect and diagnose incipient pancreatic cancer. Our laboratory is addressing this problem by taking advantage of a frequently observed molecular feature of pancreatic cancer: alterations to the carbohydrate side chains of cell-surface and secreted proteins. Most secreted proteins have carbohydrates known as glycans attached to them, and some of the secreted proteins with altered carbohydrates are released into the blood of cancer patients. The measurement of certain secreted glycoproteins, along with their attached glycans, could form the basis of effective diagnostic markers.
A particularly valuable platform for probing glycan variants on specific proteins is the antibody-lectin sandwich array (ALSA), developed earlier in our laboratory. The method starts with a microarray of antibodies that target various glycoproteins of interest. A complex biological sample is incubated on the array, resulting in the capture of glycoproteins by the antibodies. Then the array is probed with a labeling lectin (a protein with carbohydrate-binding activity), which binds to the captured glycoproteins that bear the glycan targeted by the lectin. The amount of lectin binding at each antibody indicates the amount of glycan on the proteins captured by that antibody. Diverse lectins can be used to probe a variety of glycans on a given sample. In addition, the captured proteins can be probed with antibodies targeting the core proteins, as in a “sandwich” immunoassay, to obtain the levels of the proteins in parallel assays.
Relative to other technologies, the platform offers a unique combination of capabilities such as reproducible glycan measurements on specific proteins, high-throughput sample processing, and high-sensitivity detection directly from biological samples. These features make the platform ideal for glycoprotein-based biomarker studies. A product based on this technology is now available from GenTel Biosciences (Madison, Wisconsin).
Using this tool, we can now explore the hypotheses that particular glycan structures on specific proteins are found uniquely in certain disease states and that their measurement yields effective detection of cancer. We have characterized the prevalence in pancreatic cancer patients of a variety of glycan structures on several types of mucin proteins. Some glycan alterations were found in a high percentage of the cancer patients but not at all in healthy subjects. Furthermore, the glycan levels were altered independently of changes to the protein level, so that measuring both the glycan and protein level gives improved biomarker performance relative to measuring only protein levels as in standard immunoassays. The performance of these initial studies already suggests improvement upon the current best biomarkers for pancreatic cancer. Now we are working to characterize and develop detection methods for both the protein forms that carry cancer-associated glycans and the glycans themselves.
We work with clinical collaborators at several institutions to address various clinical needs. One such need is to help doctors make a more accurate diagnosis of patients with suspected pancreatic cancer. Since pancreatic cancer can be difficult to distinguish from benign conditions of the gastrointestinal tract, highly accurate biomarkers are needed to match patients to the appropriate procedures at the earliest possible time. We also are developing biomarkers to screen for clinically undetectable pancreatic cancer. Among populations at an increased risk for developing pancreatic cancer—including those suffering from chronic pancreatitis or with a family history of pancreatic cancer—an accurate screening test could detect new cancers early enough to allow more effective treatment. We also are testing our novel biomarkers for use in drug trials. Biomarkers that give early indications of the effectiveness of a candidate drug could accelerate drug trials and better match patients with the drugs that benefit them most.
Another novel class of biomarker we are developing is for the diagnosis of patients with pancreatic cysts. Cystic lesions of the pancreas are increasingly being recognized due to the widespread use of high-resolution abdominal imaging. Since certain cyst types are precursors of invasive cancer, this situation presents an opportunity to intervene prior to malignant progression. Effective implementation of that strategy has been hampered by difficulties in clearly distinguishing cystic lesions based on differences in their malignant potential. In collaboration with Dr. Diane Simeone at the University of Michigan, we have identified glycan variants of secreted mucins that distinguish benign from pre-cancerous cysts with an 87% accuracy—better than the best current markers. Ongoing work is aimed at validating and building upon these results. Ultimately, we hope to implement a test that could be used to determine which pancreatic cysts should be surgically removed in order to prevent progression to cancer.
Origin and function of secreted glycan alterations in pancreatic cancer
Our laboratory also studies the origins and functions of cancer-cell secretions bearing altered glycans. The carbohydrate alterations observed in pancreatic tumors are strongly associated with accelerated disease progression, but it is not known whether these alterations functionally contribute to that progression. We have shown that certain glycoprotein alterations are likely the product of subpopulations of tumor cells that are more likely to be aggressive. Using ALSA in a study of cultured pancreatic cancer cells, we have shown that cells bearing markers of high tumor-forming capability (termed “cancer stem cell markers”) display distinct glycan characteristics. The glycans of such cancer cells are distinctly altered in response to inflammatory signaling from the environment, showing the link between secreted glycan structures and the cellular state. Additional studies have shown distinct glycan alterations produced when cells transition from a stationary to a migratory state. This transition initiates metastasis and results in tumors at new sites. This work clearly links the origin of particular cancer-associated glycans with aggressive cancer cells.
We are pursuing the hypothesis that the distinct glycans and glycoproteins secreted by aggressive or tumor-initiation cancer cells contribute to cancer progression through interactions with cells and proteins of the tumor environment. Evidence from our laboratory suggests that these secretions produce a higher state of inflammation and weaker immune recognition of the cancer cells. Our goals are to characterize the glycan alterations and their protein carriers that are unique to aggressive subsets of cancer cells and to understand the mechanisms by which these molecules affect host cells and promote tumor progression. In addition, we are investigating new strategies for treating cancer based on these observations. Targeting the functions of the aggressive subpopulations of cancer cells could be highly effective.
Figure 1
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Figure 1. Protein and glycan detection using antibody arrays. a) Array-based sandwich assays for protein detection. Multiple antibodies are immobilized on a planar support, and the captured proteins are probed using biotinylated detection antibodies, followed by fluorescence detection using phycoerythrin-labeled streptavidin. b) Antibody-lectin sandwich arrays (ALSA). This format is similar to above, but the detection reagents target the glycans on the capture proteins rather than the core proteins. The glycans on the immobilized antibodies are chemically derivatized to prevent lectin binding to those glycans. c) Example antibody array results for core protein detection (left) and glycan measurement (right). SA-PE, streptavidin-phycoerythrin. |
Figure 2
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Figure 2. Distinct changes to glycan levels associated with cell type. Cell lines were treated with various pro-inflammatory signals, including the cytokines IFNg, TNFa, IL-a1, and oxidative stress (H2O2). The cell lines and their treatments are indicated by the column labels. Six cell lines were treated: two bearing cell-surface markers characteristic of tumorigenicity (labeled in red); two not bearing the markers (labeled in black); and two partially bearing the markers (labeled in green). Using the ALSA assay, the levels of various glycans on the mucins MUC1, MUC5AC, and MUC16 in the secretions of the cells were measured before and after treatment. The row labels indicate the lectin used for detection (which determines the glycan detected) and the capture antibody. The color of each square represents the fold-change of the signal after treatment divided by the signal before treatment. The cells bearing markers of tumorigenicity uniquely increased particular glycans, showing a difference from the other cells in their glycan characteristics. See Wu et al., J. Proteome Research, 2009. |
