Figure 7

Functional analysis of the colon CTC lines. (A) Representative images of the EPISPOT results. Secretion, release and shedding of CK19 (upper), OPG (middle) and VEGF (lower panels) by viable CTCs were assessed using the fluoro-EPISPOT assay (C.T.L. Elispot reader). The breast cancer cell lines SKBR3 and BC-M1 were used as positive controls for CK19, and for OPG and VEGF, respectively. The negative control well was without cells (magnification X5). Each fluorescent immunospot corresponds to the protein fingerprint of one secreting cell. (B) Endothelial cell tube formation assay. Endothelial cell tube formation was observed after 6 h (representative images; magnification X5) of cultures with endothelial cell medium (positive control), basal medium (RPMI 1640 alone; negative control), and supernatant from the nine CTC lines cultured in basal medium for 48 h. (C) ALDH activity assay by flow cytometry. Gates for positive signal were designed for each CTC line based on the negative control performed with the DEAB reagent. The lung cancer cell line A549 was used as positive control. The percentage of ALDH-positive cells is indicated for each condition.