Circulating tumor cells (CTCs) are malignant cells shed into the bloodstream from a tumor that have the potential to establish metastases in different anatomical sites. The separation and subsequent characterization of these cells is emerging as an important tool for both biomarker discovery and the elucidation of mechanisms of metastasis. Established methods for separating CTCs rely on biochemical markers of epithelial cells that are known to be unreliable because of epithelial-to-mesenchymal transition, which reduces expression for epithelial markers. Emerging label-free separation methods based on the biophysical and biomechanical properties of CTCs have the potential to address this key shortcoming and present greater flexibility in the subsequent characterization of these cells. In this review we first present what is known about the biophysical and biomechanical properties of CTCs from historical studies and recent research. We then review biophysical label-free technologies that have been developed for CTC separation, including techniques based on filtration, hydrodynamic chromatography, and dielectrophoresis. Finally, we evaluate these separation methods and discuss requirements for subsequent characterization of CTCs.

Circulating tumor cell (CTC) enumeration promises to be an important predictor of clinical outcome for a range of cancers. Established CTC enumeration methods primarily rely on affinity capture of cell surface antigens, and have been criticized for underestimation of CTC numbers due to antigenic bias. Emerging CTC capture strategies typically distinguish these cells based on their assumed biomechanical characteristics, which are often validated using cultured cancer cells. In this study, we developed a software tool to investigate the morphological properties of CTCs from patients with castrate resistant prostate cancer and cultured prostate cancer cells in order to establish whether the latter is an appropriate model for the former. We isolated both CTCs and cultured cancer cells from whole blood using the CellSearch® system and examined various cytomorphological characteristics. In contrast with cultured cancer cells, CTCs enriched by CellSearch® system were found to have significantly smaller size, larger nuclear-cytoplasmic ratio, and more elongated shape. These CTCs were also found to exhibit significantly more variability than cultured cancer cells in nuclear-cytoplasmic ratio and shape profile.

Mutations in FLVCR2, a cell surface protein related by homology and membrane topology to the heme exporter/retroviral receptor FLVCR1, have recently been associated with Fowler syndrome, a vascular disorder of the brain. We previously identified FLVCR2 to function as a receptor for FY981 feline leukemia virus (FeLV). However, the cellular function of FLVCR2 remains unresolved. Here, we report the cellular function of FLVCR2 as an importer of heme, based on the following observations. First, FLVCR2 binds to heminconjugated agarose, and binding is competed by free hemin. Second, mammalian cells and Xenopus laevis oocytes expressing FLVCR2 display enhanced heme uptake. Third, heme import is reduced after the expression of FLVCR2-specific small interfering RNA (siRNA) or after the binding of the FY981 FeLV envelope protein to the FLVCR2 receptor. Finally, cells overexpressing FLVCR2 are more sensitive to heme toxicity, a finding most likely attributable to enhanced heme uptake. Tissue expression analysis indicates that FLVCR2 is expressed in a broad range of human tissues, including liver, placenta, brain, and kidney. The identification of a cellular function for FLVCR2 will have important implications in elucidating the pathogenic mechanisms of Fowler syndrome and of phenotypically associated disorders.Membrane transporters play essential roles in cellular homeostasis by importing substrates critical for cell growth and differentiation or by exporting substrates that cause toxicity. There are five major categories of membrane transporters consisting of over 550 transporter superfamilies (41). The major facilitator superfamily (MFS) is the largest and most diverse superfamily, consisting of over 10,000 members (31, 41). Transporters in this superfamily consist of 12 to 14 transmembrane (TM)-spanning segments and transport substrates as diverse as sugars, polyols, drugs, neurotransmitters, amino acids, organic/inorganic ions, and peptides (31). Recently, a disruption of MFS transporters that is associated with human diseases has been described, further confirming their role in the maintenance of normal cell homeostasis. The DIRC2 MFS transporter (substrate transported unknown) is disrupted in renal cell carcinoma cosegregating with a t(2;3)(q35;q21) chromosomal translocation (4). Mutations in the thiamine transporter THTR1 have been shown to be responsible for Rogers syndrome (14, 21), a thiamine-responsive megaloblastic anemia. We have recently reported that a disruption in the heme exporter FLVCR1 (MFSD7B) plays a role in Diamond Blackfan anemia (DBA) (40), a fatal infant anemia characterized by a block in erythroid progenitor cell development (3, 12, 13). The abrogation of FLVCR1 function in primary human hematopoietic stem cells (40) or in a human erythroid cell line (37) specifically disrupts erythropoiesis, mimicking the hematological features observed for patients with DBA. We have reported previously that FLVCR1 is disrupted not as a consequence of mutations in the FLVCR1 coding region but due to the aberrant...