Fig. 1.

Generation and characterization of HCA-7–derived CC and SC. (A) A human CRC cell line, HCA-7, exhibited two distinct morphologies when grown in 3D in type-1 collagen: hollow unilamellar cystic colonies (CC) and spiky colonies (SC) that formed solid masses with protrusions into the collagen matrix. Individual CC and SC colonies were manually isolated and expanded on plastic. CC and SC maintained their characteristic appearance upon repeated passage in 3D. Individual colonies from CC and SC 3D cultures were scored for cystic or spiky morphology. Results are displayed as the mean fraction of total colonies (%) ± SEM. (B) DIC images of representative CC (Left) and SC (Right) colonies. (Scale bars: 50 µm.) (C) Confocal images through the equatorial plane of representative colonies from CC and SC clones stained for DAPI (blue) and phalloidin (red). (Scale bars: 50 µm.) (D) Confocal images through the equatorial plane of representative CC and SC colonies stained for EGFR (blue) and ezrin (red). (Scale bars: 50 µm.) (E) Representative CC and SC colonies were fixed and processed for transmission electron microscopy as described in Materials and Methods. (Top, Left and Right) A section of colonies with CC- and SC-like morphology, respectively. Lu, the luminal (apical) side; Cg, the collagen (basal) side of cysts. Both colonies are displayed in identical orientation. (Scale bars: 2 µm.) (Lower panels) Higher magnifications from each morphology. AJ, adherens junction; Mv, microvilli; tight junction, TJ. Note the lack of TJ in the magnified region corresponding to the protrusions in SC colonies. (Scale bars: 500 nm.) (F) Two thousand cells were cultured in type-1 collagen for 17 d. Fresh medium was added with or without cetuximab (CTX, 3 μg/mL) every 2–3 d. Colony count was determined using a GelCount plate reader. Results are plotted as mean counts ± SEM. P values from a two-tailed unpaired t test are 0.0003 for CC and 0.0049 for SC.