A human colonic neoplastic cell line, LS174T, established and characterized in our laboratory, develop organoid structures reminiscent of the patient's tumor pathology when grown on synthetic hollow fiber capillaries, which simulate a vascular system through which nutrient medium and dissolved gases are continuously perfused. Cloned LS174T cells will be characterized and cell masses attaining solid tissue-like densities will be compared on the basis of morphologic, biochemical, and biological properties to the parental cell line cultured by hollow fiber or standard monolayer techniques. Cell masses growing on bundles of hollow fibers will be fixed in situ, prepared for light, scan, or transmission electron microscopy and examined for cell social behavior, organoid structures, and organization as well as the presence of brush border-like features. Carcinoembryonic antigen (CEA) content of the extracapillary fluid and total cell mass glycocalyx, using ruthenium red staining, will be compared to the parental cell line. If some or all cloned cell cultures fail to produce glandular structures in hollow fiber cultivation, the presence of soluble differentiation-associated signals will be assessed by the addition of cell-free, extracapillary fluids, harvested from parental population hollow fiber cultures, to cloned matrix and monolayer cultures. The activity of these fluids will also be assessed upon a LS174T cell strain adapted to serial cultivation in serum-free media and known to be incapable of organoid expression in hollow fiber culture and on other human colon adenocarcinoma cell lines displaying varying degrees of differentiation. The presence of mesenchymal elements, within the parental LS174T population, and their interaction with epithelial cells for organoid expression will be evaluated on cloned cells. These studies may afford not only information on organotypic expression in vitro, which may be harnessed to effect phenotypic modulations of in vivo colonic neoplasms, but also insights into cellular characteristics and molecular triggers of differentiation of mammalian epithelial tumor cells.