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

多年来，人们进行了各种尝试，以开发能够从实体癌患者的血液和骨髓中检测和分离循环（CTC）或播散性肿瘤细胞（DTC）的技术（见综述：Pantel K et al., Nat Rev cancer 2008）。重要的是，我们小组最近的一项系统综述和荟萃分析证实了CTC在结直肠癌（CRC）中潜在的预后相关性（Rahbari， Aigner等人，手稿正在准备中）。然而，该分析揭示了主要的研究间变异性，这可能反映在这些细胞构成了一个异质细胞池，部分具有癌症干细胞（CSC）样，即致癌特性。csc是一种罕见的肿瘤细胞，能够自我更新、分化和增殖（Reya et aL, Nature 2001）；异种移植到免疫缺陷小鼠体内后，这些细胞重建了原始肿瘤形态（O Brien CA et al., Nature 2007; Ricci-Vitiani L et al.）。自然2007)。因此，本子项目的重点和目标是更好地定义CTC和DTC的表型特性。因此，我们首先计划从CRC患者中分离CTC和DTC，然后对这两种与CRC干性相关的标志物进行表型表征(例如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., clinicalcancer Res 2008])和（ii）上皮-间质转化(EMT，如E-cadherin， N-cadherin, vimentin, fibronectin] [Mani SA et al.， Cell 2008]和Twist [Ansieau S et al., Nature 2007]。癌症细胞2008；杨明辉等，《细胞生物学》2008；Valdes-Mora F et al. [j]。此外，将新鲜分离的DTC注射到免疫缺陷小鼠的肾包膜中，可以证明我们的假设，即CTC和DTC部分含有具有致癌能力的癌症干细胞。总之，我们的数据不仅揭示了转移形成的早期阶段的新见解，而且我们的结果与检查患者的临床随访的相关性（参见C-Project）也可能有助于评估循环癌起始细胞（CIC）的预后相关性。

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).