Cloning and Characterizing a Human Colon Carcinoma Cell Line With 3-D Tumor Models

Mutations in phosphatidylinositol-3-kinase (PI3K), an enzyme critical for growth signaling, occur in 1/3 of colorectal cancers and have been shown to alter metastatic behavior. Due to the difficulty of studying the effects of PI3K mutation in vivo, we are using multicellular spheroid 3-D tumor models in vitro to investigate mechanisms linking PI3K mutations to metastasis. In initial studies with a human colon carcinoma cell line, DLD1, two distinct morphologies were observed and two tumor sublines were selected. In monolayer culture, the “brainy” morphology showed flattened, pseudopodia-like attachments while the “smooth” cells were spherical. Spheroids of the brainy subline developed tortuous tubular structures, while smooth cell spheroids had a compact spherical geometry. Smooth and brainy clones were isolated to ensure that each cell line used for future experiments is a distinct subline derived from a single cell. On monolayer, the smooth cell lines, “S9”, “BM6”, and “B9” had doubling times of 15.7, 12.2, and 17.2 hours, respectively. The brainy cell line, “BM2” had a doubling time of 13.7 hours. As spheroids, S9, BM6, B9 and BM2 had diameter growth rates of 94.3, 21.6, and 70.3 and 55.5 µm/day, respectively. Western blot analysis of PI3K mutational status and downstream regulators is underway for each cloned cell line. The spheroid cultures of these morphologically distinct cell lines will be used to further investigate the mechanisms by which PI3K mutations alter metastatic spread.