CellSearch gives a count of EpCAM+, DAPI+, cytokeratin+, CD-45- cells, and Veridex sells research use only kits allowing the magnetic/fluorescence-based CellSearch system to identify which of these is, for example, Her-2/neu+. Researchers are also developing a host of other assays to be used with this or the more than 50 (by Dr. Liu’s count) various technologies to collect CTCs.
Some are based on staining with antibodies to intracellular and cell-surface targets before the cells are sorted. For example, Dr. Wang looks for the effect of third-generation topoisomerase I inhibitors by staining cells for phosphorylated H2AX (γH2AX), a marker of DNA double-strand breaks.
Some, like Dr. Kirby, stain the cells right on the chip after they have been captured. To demonstrate that the device had captured CTCs, for example, cells from patients with metastatic prostate cancer were fixed and stained for prostate-specific antigen, CD45, EpCAM, and DAPI and examined by confocal microscopy.
Researchers have extracted nucleic acids from CTCs. Some lyse all the cells right on the chip, using the lysate for pooled analysis expression patterns. Others, like Dr. Jeffrey’s group, for example, used a microfluidic approach to measure the expression of 87 cancer-associated and reference genes in over 100 individual CTCs isolated from breast cancer patients. Their profiles differed from one another, as well as from those of single cells taken from breast cancer cell lines—leading them to conclude that individual CTCs may be the way forward in drug discovery.
Groups may use antibodies other than an anti-EpCAM to capture different subsets of CTCs. Dr. Kirby, for example, coats his GEDI device with a monoclonal antibody recognizing the extracellular domain of prostate-specific membrane antigen (PSMA). And On-Q-ity has “developed some markers that we feel confident will recognize cells undergoing EMT,” the epithelial–mesenchymal transition thought to be involved in metastasis, said Dr. Sprott. “It may be picking up more cells in each patient, or potentially picking up patients that don’t have epithelial cells in their blood but have cells undergoing EMT.”
And various techniques can also be used to look at other cells and cell fragments that are irregular in shape, that may not be counted by CellSearch-based methods.
In addition to examining fixed or lysed cells, considerable effort is being put into vital assays and growing CTCs as well.
Dr. Kirby’s group has performed functional assays right on-chip, incubating GEDI-captured cells with taxanes for 24 hours prior to fixing and staining. They were able to demonstrate microtubule bundling in the drug-treated, compared to control, cells.
Dr. Jeffrey is trying to grow cells in vitro for drug sensitivity testing and also developing a chip to test chemoresponsiveness. “The question is whether we will be able to obtain enough CTCs, because to do drug-sensitivity testing you need to start with a certain number and measure dose response curves,” she noted. “Since the CTCs are obtained fresh, hopefully the cells will still retain features that reflect microenvironment interactions they’ve had that predispose to further cancer spread.”