Enrichment and molecular characterization of single disseminated tumor cells (DTCs) and proof of circulating tumor cell (CTC)- and DTC-mediated tumorigenicity in vitro and in vivo

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For many years, various attempts have been made to develop techniques enabling to detect and isolate circulating (CTC) or disseminated tumor cells (DTC) from the blood and bone marrow of patients with solid cancer (see review: Pantel K et al., Nat Rev Cancer 2008). Importantly, a recent systematic review and meta-analysis from our group corroborate the potential prognostic relevance of CTC in colorectal cancer (CRC) (Rahbari, Aigner et al., manuscript in preparation). The analysis, however, reveals major inter-study variability, which may be reflected by the fact that these cells constitute a heterogeneous cell pool with partly cancer stem cell (CSC)-like, i. e. cancer-initiating, properties. CSCs constitute a rare tumor fraction which are able to self-renew, to differentiate and to proliferate (Reya T et aL, Nature 2001); following xenotransplantation into immunodeficient mice, these cells re-establish the original tumor s morphology (O Brien CA et al., Nature 2007; Ricci-Vitiani L et al.. Nature 2007). Thus, the focus and aim of this subproject is to better define the phenotypic properties of CTC and DTC. Therefore, we first plan to isolate and then to phenotypically characterize CTC and DTC from patients with CRC with regard to both (i) markers associated with CRC stemness (e. g. GD133 [O Brien CA et al., Nature 2007; Ricci-Vitiani L et al., Nature 2007], CD166 [Dalerba P et al., PNAS 2007], CD44 [Du L et al., Clin Cancer Res 2008]) and (ii) markers involved in epithelial-mesenchymal transition (EMT, e. g. E-cadherin, N-cadherin, vimentin, fibronectin [Mani SA et al., Cell 2008] and Twist [Ansieau S et al.. Cancer Cell 2008; Yang MH et al., Nat Cell Biol 2008; Valdes-Mora F et al., Ann Surg Oncol 2008]). Furthermore, the outgrowth of freshly isolated DTC injected into the renal capsule of immunodeficient mice should serve as a proof for our hypothesis that the CTC and DTC fraction contains cancer stem cells with cancer-initiating capacities. In conclusion, our data should not only reveal new insights into the early steps of metastasis formation, but correlation of our results with the clinical follow-up of the examined patients (see also C-Project) may also help to assess the prognostic relevance of circulating cancer-initiating cells (CIC).