CTC plus whole transcriptome amplification, made it possible to hybridise cDNA from the CTC population onto TWS119 GSK-3 inhibitor geneexpression microarrays. By applying this procedure to a group of mCRC patients compared to the background of unspecific isolated hematopoietic cells, the population of immunoisolated CTC was profiled specifically. In addition to the molecular characterization of CTC for the understanding of the biology of a main source of metastasis in CRC, these data provided potential therapeutic targets and diagnostic/prognostic biomarkers. Molecular profiling is widely employed as a powerful strategy to characterize specific types of tumors, specific subtypes of carcinomas or specific events associated with carcinogenesis. In addition to contribute to the understanding of molecular events associated with the genesis and progression of cancer, geneexpression profiling has led to the identification of therapeutic targets and biomarkers in an effort to improve the management of cancer patients at the clinical setting. In this work, we aimed to tackle with a very specific and attractive population of tumor cells that are at the origin of metastasis, the circulating tumor cells. Whole-genome amplification and massive gene-expression profiling for the characterization and interpretation of the biology of CTC in metastatic colorectal cancer is presented here. To technically validate this strategy, the profiling of CTC from mCRC patients was approached by subtracting the background of non-specific isolation from a group of healthy controls. The next challenge is to compare the gene-expression profile of the CTC population with the primary CRC lesions and with the overt metastases, in order to better understand the mechanisms of adaption of tumor cells during the process of metastasis and the crosstalk with the environment. The subtraction of the background from a group of controls was also considered to be more relevant than the background from the same mCRC patients as it would require two rounds of CTC isolation within the same samples, representing a significant source of technical artefacts. Differences in the background could not be excluded due to the systemic metastatic disease, although the analysis of the remaining fraction after CTC isolation did not render any differences within the selected genes. Regarding immunoisolation, and although CTC enrichment with EpCAM-coupled antibodies has demonstrated to be superior to other cytometric methods and a reliable method for CTC detection in mCRC patients, this CTC capture procedure has raised some debate due to the reliance of this technique on the expression of EpCAM. Initially described 30 years ago as a dominant antigen in human colon carcinoma tissue, it is assumed that a decreased expression of epithelial markers occurs during the epithelial to mesenchymal transition associated with tumor invasion. Importantly, EpCAM is apparently needed to maintain distinct cancer cell attributes and, potentially, the cancer stem cell phenotype. CD133+ cells, currently one of the best markers for the characterization of colon cancer stem cells and an independent prognostic.