Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Cancer is unpredictable. Tumors can shrink upon initial treatment only to rebound stronger than ever. The disease can spread, and it can mutate, with genetic alterations arising to make the cancer either more susceptible to therapeutics or less so.
Oncologists traditionally rely on invasive biopsies and non-invasive imaging modalities to track tumor size, spread and therapeutic response. But circulating tumor cells (CTCs) offer an alternative, and potentially promising approach. Here, we review some of the methods for isolating and studying these oncogenic bellwethers.
Defining CTCs
CTCs are cancer cells that have sloughed off the main tumor and entered the circulation, where they can be detected via blood tests. These cells can serve as cancer biomarkers, indicating with a simple blood test whether distant tumors are present as well as their genetic status and drug susceptibility. Unlike surgical interventions, they are compatible with longitudinal studies, acting effectively as “liquid biopsies.” Yet making sense of what they reveal, isn’t necessarily straightforward.
CTCs, like tumors themselves, are highly heterogeneous, says says Zena Werb, professor of anatomy at the University of California, San Francisco, who studies CTC biology. Some have the ability to seed new tumors, but not all do. Some CTCs appear to have the properties of cancer stem cells, but some do not. At the moment, the only U.S. Food and Drug Administration-approved clinical assay for CTCs is Janssen Diagnostics’ CellSearch®, which defines CTCs effectively as cells that are epithelial in origin—i.e., positive for EpCAM, DAPI and cytokeratin and negative for CD45 (a lymphocyte marker). But of course, not every tumor is epithelial, and even epithelial tumors may lose epithelial markers over time (CellSearch is approved for metastatic breast, colorectal and prostate cancers). And there are indications other than cancer, such as inflammation, that can lead to the presence of epithelial cells in the blood. “The story is much more complex than we would have imagined,” says Werb, whose own research effectively sidesteps the question of molecular phenotype by capturing human-derived CTCs in mouse models using anti-human antibodies.
According to Ron Mazumder, head of research and development and operations at Janssen Diagnostics, the fraction of patients with detectable CTCs in the blood varies with tumor type, from 50% to 70% in colorectal, ovarian and breast tumors, for instance, to as low as 30% in non-small-cell lung cancer. The CTC counts in those patients can vary wildly, too, from as few as a single cell to several thousand. “The median value is typically two to three dozen cells,” Mazumder says, but the company has shown that metastatic breast and prostate cancer patients with more than five CTCs per 7.5 ml of blood (or three, in the case of metastatic colorectal cancer) “have a much worse prognosis than those who have fewer [cells]. So five was set as a cutoff—there is a statistically significant impact on survival if you have more or less CTCs, and that was generally true across multiple cancer types.”
Microfluidic alternatives
Originally, CellSearch was based entirely on cell counting. Today, users also can obtain genetic information on those cells: In mid-2014, Janssen announced a partnership with Asuragen to perform next-generation DNA sequencing of isolated CTCs, to identify mutations in a couple hundred cancer-associated genes.
CellSearch is based on positive selection, using antibody-coated magnetic beads to capture, image and count EpCAM-positive cells. As such, it relies upon knowing a priori the characteristics of the cells you’re looking for, says Shannon Stott, assistant professor of medicine at the Massachusetts General Hospital (MGH) Center for Cancer Research. Yet not all CTCs have the same physical and biological properties.
Stott has a background in mechanical engineering, and working with Mehmet Toner, Daniel Haber and Shyamala Maheswaran at MGH/Harvard Medical School, she has developed novel methods for CTC isolation. In 2010, as a post-doc with Toner, she developed a “herringbone CTC-chip,” a microfluidic device “with patterned chevrons or herringbones on the upper surface,” that uses anti-EpCAM antibodies to capture CTCs [1]. The design, she says, provides “reasonably fast processing of blood [~2 ml per hour], good sensitivity and selectivity.” The flip-side is that it cannot account for different antigenic profiles, for instance as tumor cells undergo an epithelial to mesenchymal transition. Also, the captured cells are, well, captured, or stuck to the chip surface, making follow-up culture problematic.
In 2013, the Mass General team developed a “third-generation” technology called the CTC-iChip, to alleviate those concerns [2]. The “i” in iChip stands for inertial focusing, Stott says, a process in which cells are manipulated by fluidic forces into single file. The chip uses hydrodynamic sorting to remove the smallest blood constituents. It then uses positive selection—i.e., antibody-coated magnetic beads targeting white blood cells—to direct lymphocytes to waste, retaining everything else. The CTCs that emerge are thus defined by what they are not—the components of blood that are neither platelets, plasma proteins nor red or white blood cells.
According to Stott, the iChip is far faster than the herringbone chip, capable of handling 20 ml per hour. It also produces unlabeled cells that can be readily cultured and analyzed. In one example, Haber’s team established CTC cell lines from six breast cancer patients [3]. Sequencing of those lines identified newly acquired mutations and drug susceptibilities, information that could be used to develop better treatment strategies, Stott says. In another study, Haber’s team performed single-cell RNA-seq on isolated CTCs and compared them to CTC aggregates from the same patients to understand the nature of these clustered CTC structures, which have greater metastatic potential than stand-alone CTCs [4]. The analysis identified a cell-junction-associated gene called plakoglobin in the formation of these clusters.
According to Mazumder, Janssen Diagnostics is now working with the Mass General team to transform the iChip into a “next-generation CTC-analysis platform.”
Imaging CTCs
The principles of CTC analysis and characterization were reviewed recently in Nature Reviews Clinical Oncology [5]. The article includes a table of selected detection technologies, most of which operate based either on CTCs' antgenic profiles or physical characteristics. One alternative strategy the table mentions is imaging cytometry, a method commercialized by EMD Millipore.
According to David Basiji, the company's senior director of cellular analysis, enrichment-based strategies sacrifice yield for specificity. “If you have an average of only one or two CTCs in a tube of blood, you’re already fighting statistics,” he says. “There’s very, very high statistical variance with so rare an event, so you cannot afford to lose any CTCs in the enrichment.”
ImageStream and FlowSight are imaging cytometers—essentially flow cytometers with a camera. As each cell passes, it is imaged in brightfield and darkfield modes, as well as in as many as 10 fluorescent channels, producing up to 12 images per cell at 2,000 cells per second. The difference between the two platforms is that the ImageStream can magnify up to 60x, whereas FlowSight offers a fixed 20x magnification.
“By being able to image at these speeds, you can greatly reduce the enrichment burden you need to apply before analysis,” Basiji explains –as many as three to four orders of magnitude lower.
Although the resulting cells cannot be cultured—they currently are lost to waste—users can perform extensive analyses as the cells pass through the system, Basiji says. For instance, they can stain for both extracellular and intracellular markers to identify a cell’s provenance or stain with RNA-FISH probes to count transcripts.
Many of these parameters can be obtained using traditional flow cytometry, of course. What imaging cytometry provides is confidence that the few positive cells are, in fact, CTCs. “Seeing is believing,” Basiji says. Now the company is working to enable isolation of the analyzed cells for downstream studies. “At that point,” he says, “we’ll be talking about all sorts of amazing things.”
References
[1] Stott, SL, et al., “Isolation of circulating tumor cells using a microvortex-generating herringbone-chip,” Proc Natl Acad Sci USA, 107:18392–7, 2010. [PubMed: 20930119]
[2] Ozkumur, E, et al., “Inertial focusing for tumor antigen-dependent and –independent sorting of rare circulating tumor cells,” Sci Transl Med, 5:179ra47, 2013. [PubMed: 23552373]
[3] Yu, M, et al., “Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility,” Science, 345:216–220, 2014. [PubMed: 25013076]
[4] Aceto, N, et al., “Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis,” Cell, 158:1110–22, 2014. [PubMed: 25171411]
[5] Krebs, MG, et al., “Molecular analysis of circulating tumour cells—biology and biomarkers,” Nat Rev Clin Oncol, 11:129–44, 2014. [PubMed: 24445517]
Image: iStockPhoto