Specific sample preparation in colorectal cancer

Abstract

Large tissue samples from ten patients operated for colorectal cancer were prepared in the operating room in iced phosphate buffered saline, containing ethylene diaminetetraacetic acid and protease inhibitors. After cutting the specimens into small fragments, the tissues were gently pressed through a steel mesh. Membranes were permeabilized in chilled ethanol 70% to allow cytosolic fluoresceine isothiocyanate labeling, performed with anti‐cytokeratin (CAM 5.2) antibodies. Samples were quantitatively sorted with a fluorescence activated cell sorter (FACS) and denatured before processing separation by two‐dimensional electrophoresis on polyacrylamide gels. This procedure made it possible to sample about 4 × 10⁷ viable normal and tumoral cells before fixation, and up to 4 × 10⁶ cells after FACS. The gels run before and after fixation showed no major differences. The rate of cytokeratin‐positive cells in the samples was the following (mean, Cl 5–95%): mucosa 29.5% (8.9–66.7%), tumor 44.3% (6.6–94.8%). The epithelial cell content in colorectal cancer and normal mucosa shows important intersample variations. This is important for any comparison of fresh samples, whether at DNA, RNA, or at the protein level. We propose a method allowing the preparation of pure epithelial cell samples from normal and tumoral colonic fresh mucosa.