EPISPOT assay: detection of viable DTCs/CTCs in solid tumor patients.

The enumeration and characterization of circulating tumor cells (CTCs) in the peripheral blood and disseminated tumor cells (DTCs) in bone marrow may provide important prognostic information and might help to monitor efficacy of therapy. Since current assays cannot distinguish between apoptotic and viable DTCs/CTCs, it is now possible to apply a novel ELISPOT assay (designated 'EPISPOT') that detects proteins secreted/released/shed from single epithelial cancer cells. Cells are cultured for a short time on a membrane coated with antibodies that capture the secreted/released/shed proteins which are subsequently detected by secondary antibodies labeled with fluorochromes. In breast cancer, we measured the release of cytokeratin-19 (CK19) and mucin-1 (MUC1) and demonstrated that many patients harbored viable DTCs, even in patients with apparently localized tumors (stage M(0): 54%). Preliminary clinical data showed that patients with DTC-releasing CK19 have an unfavorable outcome. We also studied CTCs or CK19-secreting cells in the peripheral blood of M1 breast cancer patients and showed that patients with CK19-SC had a worse clinical outcome. In prostate cancer, we used prostate-specific antigen (PSA) secretion as marker and found that a significant fraction of CTCs secreted fibroblast growth factor-2 (FGF2), a known stem cell growth factor. In conclusion, the EPISPOT assay offers a new opportunity to detect and characterize viable DTCs/CTCs in cancer patients and it can be extended to a multi-parameter analysis revealing a CTC/DTC protein fingerprint.