Our goal is to discover plasma and tissue-based tumor biomarkers that work well enough together to identity colon cancer at early stages, lead to accurate diagnosis and could ultimately allow for individualized treatment.
Specific Aim 1. To evaluate the expression/activation of proteins important in proliferation, angiogenesis, and inflammation in plasma collected 0-24 months prior to diagnosis of colon cancer.
Approach: For discovery, we will utilize our high density array supplemented with antibodies targeting proteins such as cytokines, chemokines, growth factors, ubiquitination related proteins, insulin-related/resistance proteins, kinases, phosphatases and other regulators of cell signaling (IGF, IGFBP, STAT, TGF-β, AKT, MAPK). For biomarker candidates discovered during our previous research, we will perform “pre-validation” analyses utilizing smaller format antibody microarrays.
Specific Aim 2. To evaluate changes in the glycosylation status of proteins during colon cancer progression in plasma collected 0-24 months prior to diagnosis.
Approach: We will utilize our large format antibody arrays to fractionate proteins and then probe their glycosylation status through the use of 6 lectins (Aleuria aurentia lectin (AAL), Concanavalin A (ConA), Len culinaris agglutinin (LCA), Phaseolus vulgaris leucoagglutinin (L-PHA), Maackia amurensis lectin II (MAL II), and Sambucus nigra bark lectin (SNA). Differences will be followed up with immunopurification and glycan characterization (ELLA).
Specific Aim 3. To develop CRC-specific diagnostic biomarkers using pretreatment cancer tissue and plasma from colorectal cancer patients.
Approach: We will compare protein expression/activation in tumor vs. normal colon mucosa and in plasma vs. controls via antibody array to discover diagnostic biomarkers. The best markers will then be rescreened on a second array with independent samples. After triage, we will determine whether the surviving biomarkers are tumor or stromal derived by analyzing its expression in fixed tumor tissue.
Specific Aim 4. To validate colon cancer early detection and diagnostic biomarkers on independent sample sets.
Approach: We will utilize sandwich ELISA to judge the sensitivity and specificity of the plasma biomarkers that have survived either the early detection or diagnostic biomarker triage process or both (the latter will be particularly interesting). For the tumor derived diagnostic biomarkers, we will examine their expression in tissue microarrays (TMA) created from cores of tumors from people with known disease status and outcome. Thus, we will be able to determine if there is a correlation between biomarker expression and disease outcome.
High-density antibody microarrays
To discover potentially informative biomarkers for CRC early detection and diagnosis, we will utilize a high-density microarray antibody platform, developed by the Lampe laboratory, which has capacity for over 18,000 antibodies (we typically print up to ~4500 in triplicate). We will tailor this microarray specifically for application to the clinical management of CRC. Our current arrays contain ~3000 commercial antibodies from several vendors (~400 Hypromatrix, Inc, ~400 Genway Biotech, Inc., ~1200 cancer related antibodies from SDIX, ~400 from Abnova and ~800 from Aviva). The antibodies were produced to a wide variety of signaling proteins, cytokines, apoptotic, proteases and cancer related proteins. Where possible, we selected antibodies to secreted proteins. For the recombinant antibodies, sub-libraries specific to CRC plasma have already been selected from 2 master libraries (Tomlinson I and J libraries) and 310 CRC-specific scFv have shown excellent performance on arrays probed with prediagnostic CRC plasma.
A. Fabrication/printing of arrays
Buffers, protocols, slide surfaces and techniques were modified from DNA array-based technology and existing antibody array-based literature. Triplicate features of each antibody are printed. Previous array experiments confirmed that spatial separation resulted in no loss of reproducibility among the three common features. Coupling of the antibody primary amines on the array surface is essentially complete in 10 min. When required, antibodies will be diluted in PBS (50 mM potassium phosphate, pH 7.2, 150mM NaCl) to a final concentration of 0.20 to 0.25 mg/ml. This mixture is combined with 2x volume (10 µl) of 3x Protein Printing Buffer (3x PBS, 0.03% Tween, 3% glycerol). Arrays are contact printed onto HiSens Hydrogel H glass slides (Nexterion) using a Genetix Q-Array 2 microarray platform. Printed arrays have good shelf life (we saw no deterioration up to one month after printing when stored at 4°C and 1 year at -80°C). Arrays are blocked just prior to usage by placing in a slide rack and washing vigorously in phosphate buffered saline-0.01% Tween 20 (PBST) by dunking 30 times, washing twice in fresh water by dunking 30 times, then blocking with 0.3% ethanolamine in 0.05 M sodium borate pH 8.0 by dunking 30 times. After 2-3 hours incubation (orbital shaker, 80 rpm) in block solution, the slides are dunked 30 times in PBST, twice in fresh water, and then dried immediately by spinning 1000 x g for 5 min. Dried slides are immediately challenged with labeled plasma.
B. Fluorescent labeling of plasma and tissue lysate proteins
The detection of protein on the array requires direct incorporation of a tag or label. For plasma, we first remove the two most abundant proteins (Albumin and IgG) using a Sigma Prot I/A column. The protein solution is then reconcentrated to about half of its original volume. Protein (100 µg) from each sample is then labeled with the fluoropho
There are currently no biomarkers annotated for this protocol.
No datasets are currently associated with this protocol.
Thank you to everyone who helped make the 9th EDRN Scientific Workshop a success. We look forward to seeing everyone at the 28th EDRN Steering Committee Meeting from March 31-April 2, 2015, in Atlanta, GA.
Funding Opportunity Available
Both RFAs for Molecular and Cellular Characterization of Screen-Detected Lesions have been published.