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.
On January 10, next-generation DNA sequencing powerhouse Illumina announced it was branching out into the field of medical diagnostics.
According to published reports, the company said it had formed a spin-off company called Grail, which would pursue the idea of using Illumina technology to detect tumors using only fragments of genetic material, such growths shed into the circulatory system.
Such tests are sometimes called “liquid biopsies,” and they come in two basic flavors. Grail is pursuing tests based on naked, circulating tumor DNA (ctDNA). Others, though, hope to diagnose disease and assess prognosis and treatment efficacy via intact cells called circulating tumor cells (CTCs).
Blood-borne cells that either passively slough off or actively emigrate from intact tumors, CTCs are like canaries in the oncologic coal mine, trumpeting the presence, health and molecular characteristics of tumors that may otherwise be asymptomatic. Their abundance is closely tied to patient prognosis, says Ron Mazumder, head of R&D and operations at Janssen Diagnostics. “In metastatic breast, colon and prostate cancer, you can segment [patients] into those with better or worse prognosis based on CTC counts.”
Yet finding them is easier said than done, as CTCs are exceptionally rare. “In 1 ml of blood, there’s about 7 million leukocytes and about 10 CTCs,” says Brian Hall, a senior scientist at MilliporeSigma. “So you’re really looking for a needle in a haystack.” (The precise CTC burden, of course, varies over a relatively wide range from patient to patient and over the course of disease and treatment.)
To sift through that haystack, researchers and tools developers have devised several options (reviewed in [1]).
Positive and negative
Because CTCs are so rare, most CTC analysis tools adopt one of two fundamental strategies, says Olaf Hardt, R&D oncology manager at Miltenyi Biotec. In positive selection approaches, CTCs are concentrated using bead-coupled antibodies targeting known CTC antigens. Janssen Diagnostics’ CellSearch assay, for instance, the only CTC diagnostic to be approved by the U.S. Food and Drug Administration, exploits the fact that many epithelial tumors express the EpCAM surface ligand to enrich CTCs. Those isolated cells are then stained with DAPI and cytokeratin to ensure their status as CTCs (and counterstained for the leukocyte marker CD45 to ensure they aren’t white blood cells).
Negative selection strategies use antibodies to remove cells that aren’t CTCs, for instance, those expressing CD45; these strategies may be superior for several tumor entities, as they don’t rely on the knowledge of an expressed CTC antigen.
Neither strategy yields pure CTCs with 100% recovery, notes Hardt. Cells can aggregate, so precious CTCs may get thrown out with the bathwater during negative selection, for instance. By the same token, positive selection strategies inevitably retain non-CTCs for the same reason. As a result, researchers often combine positive and negative enrichment, or pair them with downstream analyses, such as microscopy or flow cytometry.
Of course, these strategies depend on identifying a marker or markers that will define the CTC of interest. Not every CTC is EpCAM-positive, for instance—nonepithelial tumors, such as melanomas, aren’t. And neither are tumors that have undergone a so-called epithelial-mesenchymal transition to a more “stem-like” state, says Jason Johnson, senior director of oncology product management at Thermo Fisher Scientific.
Whatever the enrichment method, once purified, the CTCs may be cultured in isolation, stained for microscopy or flow cytometry analysis, or sequenced. In one recent study, for instance, Massachusetts General Hospital’s Daniel Haber (director at Massachusetts General Hospital Cancer Center) and Shyamala Maheswaran (associate preofessor of surgery at Harvard Medical School)and colleagues used a custom positive selection-based microfluidic device called a CTC-iChip to study the RNA-seq gene-expression profiles of 77 single CTCs isolated from 13 patients [2]. (A 2014 article in Nature Protocols details the design and use of the CTC-iChip [3].)
CTC enrichment tools
With a wide array of antibody-conjugated magnetic MACS MicroBeads, Miltenyi Biotech supports both positive and negative selection strategies, says Hardt. Researchers can combine their cell mixtures with the appropriate beads and load them on the company’s MultiMACS (for parallelization) or AutoMACSpro (automated) processing systems.
Miltenyi Biotec plans to launch a new series of MicroBeads for direct enrichment of CTCs from whole blood later in 2016, Hardt says. At the moment, researchers must remove red blood cells prior to selection using either cell lysis or density-gradient centrifugation; otherwise, there are simply too many cells for the beads to find their targets, he says. The new beads are optimized to work without erythrocyte clearance, providing a significant time savings. “And the risk of losing your CTCs is lower,” he adds.
Another CTC-enrichment option is Thermo Fisher Scientific’s LiquidBiopsy workflow. Launched in February 2015, the LiquidBiopsy workflow comprises sample collection tubes capable of retaining CTCs for up to 96 hours; a sample-processing workstation and microfluidic chips for separating ctDNA, CTCs and leukocytes; and an Ion Torrent sequencer (such as Thermo Scientific’s new S5 sequencer) to read out the results.
Researchers can use any antibody they desire to effect CTC isolation, Johnson says (EpCAM and EMT-specific markers are default selections), and the different libraries enable researchers to distinguish cancer mutations from germline mutations found in all of a patient’s cells. “You can see three CTCs in a 7.5-ml blood draw.”
Visualizing CTCS
Isolated CTCs can be analyzed by microscopy, flow cytometry or sequencing. Yet microscopy is a relatively low-throughput approach, and neither flow cytometry nor sequencing enable researchers to actually visualize the presumptive CTCs. But MilliporeSigma’s ImageStream technology (originally from Amnis) does.
ImageStream, explains Hall, is “an imaging system that combines flow cytometry with microscopy.” As cells flow through the device at up to 10,000 cells/second, they are individually imaged at 60x magnification to measure up to 12 parameters each: side scatter, two brightfield images and up to nine fluorescent channels. As a result, Hall says, researchers can compile comprehensive data on millions of cells to identify those rare CTCs, whether based on morphology, protein expression or even RNA abundance, using fluorescence in situ hybridization (FISH) or MilliporeSigma’s SmartFlare technology.
One particular advantage of the system, Hall says, is its flexibility in accommodating new markers of interest. In one recent study, for instance, researchers stained hepatic CTCs for tumor-suppressor antigens (p53, p16 and smad4) that had previously been associated with poor prognosis [4]. “Once the scientific community discovers other markers and other ways to identify CTCs, we can simply add that as a probe,” Hall says.
References
[1] Ho, KF, et al., “Quantification techniques for circulating tumor cells,” Trends in Analytical Chemistry, 64:173-82, 2015.
[2] Miyamoto, DT, et al., “RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance,” Science, 349:1351-6, 2015. [PMID: 26383955]
[3] Karabacak, NM, et al., “Microfluidic, marker-free isolation of circulating tumor cells from blood samples,” Nature Protocols, 9:694-710, 2014. [PMID: 24577360]
[4] Catenacci, DVT, et al., “Acquisition of Portal Venous Circulating Tumor Cells From Patients With Pancreaticobiliary Cancers by Endoscopic Ultrasound,” Gastroenterology, 149:1794-1803, 2015. [PMID: 26341722]