Found in the blood of some cancer patients, circulating tumor cells (CTCs) are thought to be released into the bloodstream, in some manner, by malignant tumors. Further, it is likely that CTCs are involved in the metastatic process of secondary tumor formation.
The biology of CTCs is exceedingly complex. CTCs are often cytokeratin-positive (CK+) and CD45-negative (CD45-), but not always. The epithelial cell adhesion molecule EpCAM is present on the surface of CTCs associated with some cancers, but not others. CTCs are larger than and morphologically distinct from lymphocytes, except in the case of the “small CTC” subtype, when they are neither. CTCs can be genetically homogenous or heterogeneous relative to their apparent tumors of origin, and genetic heterogeneity can occur even between CTCs taken from the same patient, in the same blood draw sample.
The clinicians who care for cancer patients hope that one day a “liquid biopsy” analysis of CTCs isolated from a routine blood draw sample will take the place of many surgically invasive solid tumor biopsies. Clearly, there is much to learn about these complicated cells.
Though not as valuable as the hoped-for liquid biopsy, the mere enumeration of CTCs in a blood sample can be of prognostic significance. CTCs are bad actors in cancer, and their presence is associated with poorer outcomes, roughly in proportion with their abundance.
CTCs are exceedingly difficult to study, though, because even when they are clinically “abundant,” they are, by any other index, exceptionally rare. Among the white blood cells it may resemble, a CTC can be a one-in-a-million event; even in a sick patient, there might be fewer than 10 CTCs found in a typical blood sample. Therefore, in CTCs we have an object of great clinical research interest that is both highly complex and very rare.
Go with the flow
It’s no wonder, then, that researchers have been investigating flow cytometry as a potentially productive method of CTC analysis. “Flow cytometry is unique in its ability to look at rare events from a heterogeneous sample,” says Trent Colville, marketing manager at BD Biosciences, “and fluorescent-dye-labeled antibodies to surface markers allows identification of CTCs,” he adds.
With ever-higher rates of cellular throughput and relatively low operating and sample processing costs, the BD Biosciences FACSCanto II cell-analysis system and other commercial flow cytometry systems answer the needle-in-a-haystack challenge of finding CTCs for enumeration and analysis. Increasingly capable and diverse flow cytometry detection technologies can help researchers unfold the mysteries of these rare and complex cells.
Adapting the method
Making note of the potential clinical utility of routine CTC enumeration and tracking in breast-cancer medicine, researchers Alison Allan, et al., explored the use of multiparametric flow cytometry for this application by using a mouse model of human breast cancer to simulate the challenges of CTC analysis in a clinically relevant manner [1]. The authors found, using the forward-scattering measurement capability of a Beckman Coulter XL-MCL flow cytometer to measure cell size and three of the instrument’s fluorophore signal-detection channels to measure HLA, CD45 and aneuploid DNA content, that they could identify human breast-cancer cells against a background of mouse leukocytes. Foreshadowing many studies to follow, the authors found that a preliminary step of immunomagnetic enrichment for CTCs, by removing CD-45-positive lymphocytes from the sample, boosted the information yield from their experiments.
For best results, sample preparation and analysis of CTCs should be gentle and quick. “CTCs are rare, fragile and time-sensitive,” notes Mike Ward, senior staff engineer at Thermo Fisher Scientific. “Any time you can eliminate a sample-preparation step, your opportunity to analyze a CTC improves. A lot of enrichment strategies are dilutive, so the high volumetric throughput of the Attune NxT flow cytometer is an advantage when you don’t have to concentrate your cells by centrifugation or some other method where cell loss or damage can occur.”
Enrichment
Indeed, any CTC sample-enrichment strategy involves separating cells with CTC-like properties from those without, and introduces the risk that one or more rare CTCs will be lost prior to detection. Seeking to reduce this risk, Tsvetana Hristozova, et al., developed a new flow cytometry protocol for the detection of epithelial cancer CTCs [2]. The approach they described replaces immunomagnetic depletion of CD45+ lymphocytes with an electronic thresholding protocol that biases flow cytometry data acquisition in favor of CTC-like EpCAM+ CK+ cells. The investigators configured a BD Biosciences FACSCanto II flow cytometer to detect UT-SCC-23 (squamous cell carcinoma) cells spiked into blood samples, while rejecting low forward-scattering signals from cellular debris, as well as events producing non-CTC-like fluorescence profiles from the experiments’ cell-surface-marker staining schema. Results indicated that an electronic threshold applied during data collection can indeed eliminate the need for tumor-cell pre-enrichment prior to CTC analysis. When applied to actual blood samples from patients with squamous cell carcinoma of the head and neck (SCCHN), the thresholding protocol detected CTCs at the same frequencies and numbers as previously found using the conventional immunodepletion-based enrichment approach. The authors concluded that both methods are equally useful for the analysis of CTCs in SCCHN, but that their thresholding approach is more cost-effective and less laborious.
Introducing a technically differentiated method of CTC analysis at the 2014 Annual Meeting of the American Association for Cancer Research, scientists from Amnis Corporation (part of EMD Millipore) presented the poster “Detection and enumeration of circulating tumor cells using Imaging Flow Cytometry.” The approach described uses of multispectral imaging and Her-2 and EpCAM SmartFlare probes, which target intracellular RNA sequences rather than cell-surface proteins. The poster authors used the Amnis ImageStream MK II imaging flow cytometer to detect live SKBR-3 human breast-cancer cells emitting EpCAM and Her-2 SmartFlare fluorescence signals. Further discrimination of the cancer cells, which were spiked into a 100,000-fold excess of leukocytes, was provided by cell size and circularity analysis of images acquired during flow cytometry. The poster data indicate the ability of this imaging flow cytometry method to detect tumor cells at the experimental ratio of one per 100,000 leukocytes; the authors point out that in a clinical setting the additional analysis of microscopic cellular images acquired during flow cytometry would prevent the production of false-positive artifacts.
Considering flow cytometry
The advantages of flow cytometry for CTC analysis are several. High rates of cell flow enable researchers to overcome the one-in-a-million prevalence of CTCs in leukocyte-rich blood samples. Samples can be run at relatively low cost, and researchers have an expansive canvas for experimental design thanks to a variety of commercially available fluorescent stains and probes and instrumentation featuring multiple excitation and detection wavelengths, forward- and side-scattering measurement and even microscopic imaging. Whether flow cytometry will become the dominant platform for liquid biopsy is difficult to predict. The principal shortcoming of flow cytometry as a CTC-analysis platform is the sample flow-through aspect itself. Until the capability exists to sequence the genomes of rapidly moving cells, there will continue to be a need to separate, isolate and recover individual CTCs for detailed molecular characterization. Systems that approximate, or actually provide, this functionality exist but either lack the specificity required for individual cell selection and analysis or abandon the high-throughput advantages of flow in favor of potentially flawed upstream CTC-enrichment strategies. In the meantime, the advantages of flow cytometry for the analysis of CTCs will continue to accumulate, as increasingly sophisticated reagents and instrumentation for rapid, detailed interrogation of cells in flow are developed and commercialized.
References
[1] Allan, AL, et al., “Detection and Quantification of Circulating Tumor Cells in Mouse Models of Human Breast Cancer Using Immunomagnetic Enrichment and Multiparameter Flow Cytometry,” Cytometry, 65A.1:4-14, 2005. [PubMedID: 15810015]
[2] Hristozova, T, et al., “A Simple Multicolor Flow Cytometry Protocol for Detection and Molecular Characterization of Circulating Tumor Cells in Epithelial Cancers,” Cytometry, 81A.6:489-495, 2012. [PubMedID: 22438318]