Circulating tumor cells in metastatic colorectal cancerfrom basic understanding to clinical practice

Abstract

Colorectal cancer (CRC) represents the third most diagnosed cancer type worldwide, accounting for more than 600.000 deaths in 2012, which approximately represents 8,5% of all cancer-related deaths. It arises from the inner intestinal layer, usually from preexisting polyps that, after a series of molecular and genetic alterations, finally generate colorectal tumors. It is very important to consider that in CRC patients, the appearance of cancer metastasis, tumor masses that originate from primary tumors at distant sites, strongly determine patient prognosis. Only 8-10% of patients diagnosed with CRC at advanced stages survive after five years of follow up, which reinforces the importance of metastasis research to improve patient management and survival rates in CRC. Circulating tumor cells (CTCs) are cells that escape the primary tumor and reach the bloodstream. CTCs are proposed as one of the major players in tumor dissemination, and represent main intermediates between primary tumors and metastases. During the last years it has been shown that the presence of CTCs in the bloodstream of CRC patients determined their prognosis. The idea that CTCs could represent a ¿liquid biopsy¿ of tumors, as they are easily accessible and may provide information about disease status, have made that CTCrelated research incredibly increased in the last years. Moreover, the biological information obtainable from CTCs opens the possibility of a better understanding of the generation metastasis, process that remains largely unknown. This thesis encompasses a global approach that, focusing on the study of CTCs in the process of cancer metastasis, aims to improve the current clinical tools available for a better patient management, providing at the same time clues about the biology of CTCs specifically related to the process of extravasation and subsequent invasion and implantation. One of the main limitations in CTC research is given by the scarcity of CTCs in the bloodstream of cancer patients, and the elevated quantity of contaminating leucocytes on which they are present event after CTC enrichment. To overcome these shortcomings, we have developed an improved methodology for CTC detection in terms of sensitivity, by combining magnetic CTC isolation from blood, with qPCR quantification. The addition of a preamplification step, combined with the selection of optimal detection markers finally generated a highly sensitive and reproducible detection method. Despite the importance that CTC population may have in the process of cancer metastasis, little is known about their global gene expression profile. The availability of these type of gene sets, is of a key importance, in order to provide new CTC detection markers, that could serve as prognostic and predictive clinical tools, as the same time that could open a door for targeted therapies discovery. Here, and applying the methodology previously described, we have carried out a global molecular characterization of CTCs in metastatic CRC patients, in terms of their gene expression profile. We finally obtained a set of 410 CTC-specific genes that characterized this population with migratory and adhesive attributes. Moreover, validated genes showed a good prognosis value, which reinforces the validity of our strategy. In fact, markers identified in this part, were found to have a strong value for therapy response prediction, when analyzed in CTCs at different time points during treatment. In the last part of this thesis, and go deepen in the functional biology of CTCs, we have identified Talin1, as a CTC-specific marker probably involved in the process of CTC extravasation from the circulation, by mediating interactions between cancer and endothelial cells of blood vessels. Facts like Talin1 depleted cells failed to form metastasis, or that patient with high Talin1 levels in enriched CTCs from metastatic CRC patients, propose this molecule as a novel an interesting target against cancer dissemination. As a whole, the establishment of new prognosis and predictive biomarkers, as well as potential therapeutic targets such as Talin1, widely evidence the clear translational commitment of this thesis. It represents an effort to transfer basic CTC-derived knowledge into a clinical application, with the ultimate intention of improving patient¿s welfare