The good, the bad and the savage — Cytometry of the rare

Circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) are thought to be responsible for metastasis, so the detection of CTCs may serve as an individual prognostic factor for patients suffering from various cancer types for therapy decision making and during therapy follow-up. Several clinical studies have clearly demonstrated the prognostic value of CTC enumeration. The up-to-date standard assays used rely on immunomagnetic enrichment of these extremely rare cells followed by their identification either by polymerase chain reaction or by phenotyping with tumor cell specific antibodies (1). However, these assays suffer from inaccuracies regarding precise enumeration of CTCs, as demonstrated in several studies.

Possibly, the most promising ways to quantitate CTCs would be the recently proposed approaches of in vivo Flow Cytometry, IVFC (2). These barrier-breaking innovative approaches could also revolutionize therapy of metastasizing cancer. Currently, IVFC works only in model systems of small vertebrates but for humans it remains science fiction for the near future. Therefore, improved and better standardized assays for CTCs are of imminent relevance. Recently, Takao and Takeda (3) suggested a cross-contamination free assay, using disposable microfluidic chips. Normally with flow cytometry, verification of the CTCs based on morphology is only feasible with image flow cytometers. An example for such an approach is presented by Bourton and colleagues from Middlesex, London and Camberley in the United Kingdom (this issue: page 130) for the enumeration of gamma-H2AX foci induced by gamma irradiation in a DNA repair impaired cell line.

For CTCs, Scholtens and colleagues from the University of Twente in The Netherlands (this issue: page 138) propose an image cytometry-based multicolor method for their automated identification. CTC measurement was performed following their magnetic enrichment from whole blood of cancer patients on the authors' recently introduced image cytometer: Cell-Tracks TDI (4). The novelty with the presented assay is that the captured of three-color plus light scatter images automatically analyze and identify CTCs using their newly developed classifier system. This automated system yields comparable data to the standard analyzer system but in addition enables the identification of CTC debris and CTC apoptosis. These new parameters could harbor additional value for therapy monitoring in the future.

Liu and colleagues from Palo Alto, California and Memphis, Tennessee, USA (this issue: page 169) push the limits even further and propose an image cytometry approach based on a very high-speed analytical setting that works without a priori magnetic enrichment of CTCs or other extremely rare cells. Although the way the instrument detects cells is comparable to that of traditional laser scanning and image cytometers, it has an incredibly high speed by which 64 cm² slides with up to 25 million nucleated cells can be scanned in only one minute. CTC detection and discrimination are based on a six color assay with the labeling of four different antigens in combination with cellular and nuclear labeling. Both the abovementioned approaches are with respect to the instrumentation and optimized preanalytics, highly promising for future implementation into improved clinical diagnosis and improvement of therapy development and follow-up.

As already mentioned, IVFC is a promising innovative approach to monitor rare circulating cells in living organisms in a sampling free manner (5). Zhang and colleagues, an international group from Boston, Massachusetts, USA and Shanghai, China (this issue: page 176) developed a sophisticated integrated microscopic system that enables simultaneous multicolor IVFC and laser scanning confocal microscopy on the same spot in live zebrafish of the casper line. These animals have the invaluable property of being transparent to visible light and are therefore ideal models for cell proliferation analysis in developmental biology (6), as well as the investigation of tumorogenesis. The authors demonstrate in this work the feasibility of their instrument, wherein the confocal microscopy part of the instrument enables precise positioning of the IVFC measurement in blood vessels. The advantage of such a complex analytical device is evident and convincingly demonstrated in the study: because time-series measurements are done in the identical animal, intraindividual variations during time-series become less relevant and monitoring of drug effects, for example, more precise. Furthermore, because sacrificing the animal is not needed, the number of experimental animals used is substantially reduced.

In summary, innovations in microscopic cytometry in vitro and in vivo are on their way to increase sensitivity and precision in the detection of rare, harmful cells and to foster novel insights into tumor biology and therapy efficacy.