Circulating tumor cells : approaches to isolation and characterization

Correspondence to Daniel A. Haber: dhaber@partners.org Abbreviations used in this paper: CAM, cell adhesion molecule; CTC, circulating tumor cell; DTC, disseminated tumor cell, EGFR, EGF receptor; EMT, epithelial– mesenchymal transition; EpCAM, epithelial CAM; HB, herringbone; PSA, prostatespecific antigen. Introduction Blood-borne metastasis is initiated by cancer cells that are transported through the circulation from the primary tumor to vital distant organs, and it is directly responsible for most cancer-related deaths. Addressing this challenge, however, is confounded by our limited understanding of the process by which tumor cells exit from their primary site, intravasate into the circulation, and then establish distant lesions in the lung, brain, liver, or bone. Tumor cells that are identified in transit within the blood stream are referred to as circulating tumor cells (CTCs). Although their exact composition is unknown, a fraction of these are thought to be viable metastatic precursors capable of initiating a clonal metastatic lesion. However, CTCs are extraordinarily rare (estimated at one CTC per billion normal blood cells in the circulation of patients with advanced cancer); our understanding of their biological properties has thus been limited by the availability of technologies capable of isolating them in sufficient numbers and under conditions that are compatible with detailed molecular and functional experiments. Despite the limitations of current CTC-isolating methods, circulating cancer cells have been detected in a majority of epithelial cancers, including those from breast, prostate, lung, and colon. Patients with metastatic lesions are more likely to have CTCs detected in their blood; however, these have also been reported in some localized cancers. A better understanding of the identity of CTCs and the factors underlying their shedding into the vasculature is critical to identifying the key drivers of human cancer metastasis and devising rational therapeutic approaches. Much of our current understanding of processes involved in cancer metastasis has been derived from mouse models of metastasis. Recent studies in these models have raised interesting mechanistic insights. For example, CTCs captured in xenograft prostate cancer models have highlighted the importance of pathways conferring resistance to apoptosis in these cells (Berezovskaya et al., 2005; Howard et al., 2008; Helzer et al., 2009). In a mouse model of breast cancer, disseminated tumor cells (DTCs) in the bone marrow can be detected in the premalignant phases of breast cancer, suggesting an early spread to distant organs (Hüsemann et al., 2008). Studies of the effects of epithelial– mesenchymal transition (EMT) in the generation of CTCs and distal metastases have suggested that this mesenchymal transformation may enhance the ability of cells to intravasate but may reduce their competence to initiate overt metastases (Tsuji et al., 2008, 2009). Mouse studies have also identified bone marrow– derived hematopoietic progenitor cells that express VEGF receptor 1 (VEGFR1) and may form a premetastatic niche that precedes the arrival of tumor cells (Kaplan et al., 2005). Moreover, Kim et al., (2009) have recently proposed a new concept of tumor selfseeding, in which injected tagged human cancer cell lines may colonize an existing tumor deposit, with the newly recruited tumor cells conferring increased aggressiveness to the existing tumor. Finally, the possibility of intravascular proliferation of CTCs adherent to vascular endothelium has been proposed based on in vivo imaging of tagged cells (Al-Mehdi et al., 2000). Although these mouse studies offer fascinating insights into potential mechanisms of metastasis, certain limitations apply: xenograft models using established human cancer cell lines do not recapitulate the complex evolving vasculature and microenvironment of endogenous cancers, nor, of course, does direct intravascular inoculation of cancer cell lines into the tail vein. On the other hand, most endogenous mouse tumor models metastasize late, if at Circulating tumor cells (CTCs) shed from primary and metastatic cancers are admixed with blood components and are thus rare, making their isolation and characterization a major technological challenge. CTCs hold the key to understanding the biology of metastasis and provide a biomarker to noninvasively measure the evolution of tumor genotypes during treatment and disease progression. Improvements in technologies to yield purer CTC populations amenable to better cellular and molecular characterization will enable a broad range of clinical applications, including early detection of disease and the discovery of biomarkers to predict treatment responses and disease progression. Circulating tumor cells: approaches to isolation and characterization