Epithelial immunospot (EPISPOT) is an antibody-based method for quantification of live CTCs by detection of CTCs which are capable of secreting proteins such as cathepsin D, MUC1 and CK19.[115,116] Briefly, CTCs in blood samples are enriched by negative selection using anti-CD45 immunomagnetic beads. The isolated CTCs are then cultured in tissue culture plates pre-coated with antibodies which capture the secreted protein of interest. The secreted proteins are therefore immobilized around the CTC which produce them and forms a 'spot' around it. After the incubation period, the cells are removed and secreted protein 'spots' are detected by immunological techniques and counted, where each spot corresponds to 1 live CTC (Figure 4).[115–117] In cancer cell line spike-in experiments, this method was capable of detecting one cancer cell per 5 ml blood. Also, cathepsin D or MUC1 secreting cells were successfully detected in breast cancer patients but not in healthy controls. Moreover, CK19 releasing cells were found to be detectable in up to 65 and 70% of CRC and breast cancer patients, respectively, and correlated with presence of metastasis and poor survival in breast cancer.[116,118] However, a recent report found that CK19 releasing cells could also be detected in patients with benign colon diseases such as diverticulosis and Crohn's disease. It was concluded that circulating epithelial cells may be present in patients with benign inflammatory colon which were detectable by CTC assays. Further studies are warranted to better characterize circulating cells identified by this technique.
EPISPOT assay procedure.
CTCs in the blood sample are enriched by negative selection using anti-CD45 immunomagnetic beads. The isolated CTCs are then cultured in tissue culture plates pre-coated with antibodies which capture the released protein of interest (e.g., CK19). After the incubation period, cells are washed out and the released protein spots are detected by the incubation with a fluorochrome-conjugated antibody and counted. Each spot corresponds to one viable CTC.
Expert Rev Proteomics. 2013;10(6):579-589. © 2013 Expert Reviews Ltd.