Antigen Profiling of Colorectal Cancer Uses Fluorescence Multiplexing

Surface antigen profiling of colorectal cancer (CRC) tissue has been achieved using antibody microarrays with fluorescence multiplexing.

The profiling method disaggregates CRC and normal intestinal mucosal tissues to produce suspensions of viable single cells, which are then captured on customized antibody microarrays recognizing 122 different surface antigens.

A study was carried out at the University of Sydney, (Sydney, NSW, Australia) to investigate the surface antigens of surgical samples from 40 CRC patients. Cell binding patterns are recorded by optical scanning of microarrays provide a surface profile of antigens on the cells. The DotScan Microarrays were custom-made at Medsaic Pty Ltd., (Eveleigh, NSW, Australia). Subpopulations of cells bound on the microarray were profiled by fluorescence multiplexing using monoclonal antibodies tagged with Quantum Dots or other fluorescent dyes.

Statistical analysis revealed significant differences between profiles for CRC samples and mucosal controls. Hierarchical clustering of CRC data identified several disease clusters that showed some correlation with clinicopathological stage as determined by conventional histopathological analysis. Fluorescence multiplexing using Phycoerythrin- or Alexa Fluor 647-conjugated antibodies was more effective than multiplexing with antibodies labeled with Quantum Dots. This relatively simple method yields a large amount of information for each patient sample and, with further application, should provide disease signatures, and enable the identification of patients with good or poor prognosis.

While the small size of CRC samples precluded separation of mixed CRC populations into subsets of interest prior to profiling, fluorescence multiplexing was used to profile T-cells (CD3+) and epithelial cell adhesion molecule (EpCAM)-expressing cells of epithelial origin in CRC and corresponding normal intestinal mucosa. The study was published in April 2010 issue of the Journal of Immunological Methods.

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