Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However, CTC investigation is hampered by their low number, making the establishment of permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy, and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype, stem-cell like characteristics, angiogenesis potential, an osteomimetic signature and the capacity to escape from the immune system. Moreover, they showed changes in mRNA and protein expression (e.g., DEFA6, ABCB1 and GAL), whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits, treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time.

Identification and characterization of circulating tumor cells (CTCs) in peripheral blood can provide information on the direction and the efficacy of treatments. Current techniques such as CellSearch are limited in differentiating between apoptotic and viable CTCs. In contrast, the fluorescent EPISPOT assay allows for the identification of viable cells by detecting proteins secreted/released/shed by functional single epithelial cancer cells. In addition, as CTCs are rare events, it is required to combine the EPISPOT assay with an enrichment step. In this article, the EPISPOT assay, as well as two technologies for enrichment of viable CTCs, RosetteSep™ and Parsortix™ techniques, will be presented and discussed in detail.