Catch me if you can: isolating circulating tumor cells from flowing blood.

Since the discovery that the concentration of circulating tumor cells (CTCs) is a strong predictor of survival rate in metastatic breast cancer (1 ), there has been great enthusiasm for the potential of these cells as a marker for cancer progression. The technical challenge is to isolate these rare cells (1–100 cells/mL) from whole blood with sufficient sensitivity and specificity, while maintaining cell viability for postcapture analysis. The most widespread methods of CTC isolation are based on immunocapture of antigens highly expressed on CTCs. Epithelial cell adhesion molecule and prostate-specific membrane antigen are the most extensively characterized antigens used for immunologic detection of carcinomas (2 ). These antigens are often used in conjunction with cytokeratin, an epithelial marker, to discriminate CTCs from leukocytes. Immunocapture methods can be divided into bead-based approaches and microfluidics approaches. In bead-based approaches, antibody-coated magnetic nanoparticles bind to CTCs in a suspension of blood cells, and the CTCs are then separated with a magnetic field. This is the basis for Veridex’s CellSearch®, the only CTC-enumeration assay cleared by the US Food and Drug Administration (3 ). The disadvantage of this assay is that it requires multiple processing steps— centrifugation, dilution, capture, separation—and leaves the captured cells nonviable. In microfluidics approaches, whole blood is perfused through channels within which cells collide with antibodies immobilized on the channel walls or obstacles within the channel (4 – 6 ). Microfluidics methods are compatible with whole blood, thus minimizing sample preparation, and the captured CTCs remain viable. Throughput is limited, however, to about 1 mL of whole blood per hour. The reason for this low throughput is that the rate of formation of the antigen–antibody bond is relatively slow. Consequently, if blood is perfused at a faster rate, cells will not adhere to immobilized antibodies. In this issue of Clinical Chemistry, Hughes and colleagues describe a CTC-isolation device for overcoming the inherent limitation of immunocapture by mimicking the endogenous cell adhesion process (7 ). In the vasculature, CTCs escape the fast-moving bloodstream and adhere to the vessel wall by forming fast but weak bonds that allow them to roll and slow down before firmly adhering and transmigrating the endothelium (8 ). Cell rolling on endothelial cells is mediated by E-selectin, a cell adhesion molecule that is also responsible for recruiting leukocytes as part of the inflammatory response. The authors’ approach is to coat a microtube with a mixture of E-selectin and an antibody. In experiments with diluted whole blood spiked with a known concentration of hematopoietic cancer cells, the mixture of selectins and antibodies yielded significantly higher recoveries of CTCs than with antibodies alone. Another notable feature is the use of a simple off-the-shelf tube that is the size (300 m) and shape of postcapillary venules where endothelial cells are stimulated by inflammatory stimuli. A capture efficiency of 50% was achieved at a flow rate of 4.8 mL/h, which is 5-fold higher than most flow-based immunocapture methods and comparable to the highest throughput reported for a microfluidics device (5 ). A drawback of this approach is that the purity is not particularly high, because leukocytes also adhere to selectins. The authors show, however, that altering the topography of the tube wall with adsorbing nanoparticles inhibits leukocyte adhesion and spreading compared with smooth surfaces. This surface modification pushes the captured-cell purity from 37% to 66%. The mechanism for this phenomenon is unclear, but there is some evidence that rough surfaces are less inflammatory than smooth ones (9 ). The microtube method compared favorably with the CellSearch assay with a limited data set (n 12) of patients with breast, prostate, lung, and ovarian cancer. CTCs were captured with the microtube method from all 12 cancer patients, whereas the CellSearch assay identified CTCs in only 7 of the samples. In addition, the number of cells captured by the microtube was higher than with the CellSearch assay for every sample. Three of the samples from 8 healthy donors were positively identified for CTCs with the microtube method. This result seems like a high false-positive rate; however, the number of cells captured from these samples Colorado School of Mines, Golden, CO. * Address correspondence to the author at: Colorado School of Mines, 429 Alderson Hall, Golden, CO 80401. Fax 303-273-3730; e-mail kneeves@ mines.edu. Received February 6, 2012; accepted February 9, 2012. Previously published online at DOI: 10.1373/clinchem.2012.182600 Clinical Chemistry 58:5 000 – 000 (2012) Editorials