SPECIFIC SAMPLE PREPARATION IN COLORECTAL-CANCER

Large tissue samples from ten patients operated for colorectal cancer were prepared in the operating roam in iced phosphate buffered saline, containing ethylene diaminetetraacetic acid and protease inhibitors. After cutting the specimens into small fragments, the tissues were gen tly pressed through a steel mesh. Membranes were permeabilized in chil led ethanol 70% to allow cytosolic fluoresceine isothiocyanate labelin g, pet-formed with anti-cytokeratin (CAM 5.2) antibodies. Samples were quantitatively sorted with a fluorescence activated cell sorter (FAGS ) and denatured before processing separation by two-dimensional electr ophoresis on polyacrylamide gels, This Procedure made it possible to s ample about 4 x 10(7) viable normal and tumoral cells before fixation, and up to 4 x 10(6) cells after FAGS, The gels run before and after f ixation 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 c olorectal cancer and normal mucosa shows important intersample variati ons. This is important for any comparison of fresh samples, whether at DNA, RNA, or at the protein level, We propose a method allowing the p reparation of pure epithelial cell samples from normal and tumoral col onic fresh mucosa.