CTC - in situ RNA hybridization. (Source: Wikimedia/Ryan Jeffs)When cancers spread into the bloodstream, they often take on different characteristics, requiring different therapies. But it is hard to find these rare blood-borne cells. So relapsed patients often do not get personalized care.

Massachusetts General Hospital (MGH) has come up with a solution that is exciting many oncologists. In ScienceTranslational Medicine, MGH Cancer Center Director Daniel Haber recently reported on a new technology that zeros in on elusive circulating tumor cells (CTCs).

 “CTCs are promising biomarkers for the diagnosis and therapy of systemic cancer,” Dario Marchetti told Bioscience Technology via email. Director of the CTC Core Facility at Baylor College of Medicine, he was not involved in this study, but has cultured CTCs in a hailed approach himself. “[Haber’s] work is of value for both CTC biology and clinical areas.”

“I did see the paper, and loved it. Very cool. Great potential,” emailed Steffi Oesterreich, director of education at the University of Pittsburgh Cancer Center.  (Oesterreich, too, was uninvolved in the study.)

Stanford Medical School Chief of Surgical Oncology Research Stefanie Jeffrey called it “a critically important proof-of-concept study” providing a path for “improved tumor-specific biological studies” and “facilitating patient-specific and, importantly, real-time drug selection for patients with cancer.”

If all goes well, Jeffrey said by email: “I expect this work to lead to translation into clinical trials within the next five years.”

Negative depletion

Right now, the one commercial CTC technology is CellSearch, “the girl we all brought to the party,” Michigan University Breast Oncology Director Daniel Hayes told Bioscience in an interview. Noting he gets research funding from the owners of CellSearch, and has a patent for a novel use of the assay, he said CellSearch expertly fishes out breast cancer cells expressing the epithelial cell marker EpCam. But, he said, cancer cells can down-regulate that marker when switching from epithelial in nature, to mesenchymal. “Cancer cells can make themselves look like mesenchymal white cells to get where they are not supposed to be…It lets them transfer out of blood and squeeze in tight spaces.  Epithelial cells are very rigid--have to be, they make glands... Mesenchymal cells are fluid and plastic. That is why when you cut yourself, white blood cells rush over, leave the blood, and start fighting bacteria.”

He continued: “CellSearch is a great assay, highly reproducible, very accurate for what it is supposed to detect.” But it may miss the above transitioning cells, among others. By contrast, the MGH approach detects cells independent of EpCam, so while it “hasn’t been proven superior yet…it is a very exciting new technology, with much promise.”

Said Haber, in an emailed interview with Bioscience Technology: “The strength of our approach is that it doesn’t rely on looking for [tumor] cells with particular markers (hence bias)...Instead, it removes known white blood cells (WBCs) and leaves behind all potential tumor cells. We have ongoing studies aimed specifically at defining the entire transcriptome of single CTCs, and searching for markers.”

The approach enables the tracking of tumor cells’ changing genetic mutations and drug susceptibility. Changeability is a huge problem. Often, attempts to lasso and study floating tumor cells involves using antibodies to glom to particular tumor antigen on cell surfaces.

But if tumor cells are mutating— along with surface antigen— how can researchers know which antibodies to use?

MGH went in the opposite direction. Up front, the team removed the blood cells— not cancer cells— from samples using antibodies. Normal blood cell antigen targets are less changeable, so they are easy to find.

What is left behind? CTCs. A negative depletion approach.

Trolling for breast cancer CTCs

For the study, the crew fished CTCs from the blood of 36 patients with metastatic, estrogen-receptor (ER) positive breast cancer. Long-lived cell lines were grown in culture from six patients. Samples from three were used to create additional cell lines for monitoring changes in tumors during treatment.

MGH biopsies almost all solid tumors, looking for mutations to match therapies, but it currently only screens for a select group of known mutations. CTC-derived cell lines, via the new approach, were screened for mutations in 1,000 genes.

The team found many new mutations. One CTC line showed evolution of a new PI3 kinase mutation that a known drug can target. A rare ER mutation forming in patients given aromatase inhibitors (AIs) was found in CTC cell lines from three patients on AIs. Drugs blocked CTC growth in vitro and in mice.

The approach— to remove blood cells, leaving CTCs behind—has been tried in the past. But it has involved bulk depletion, said Haber, which yields few cells and inadequate purity. MGH’s version, devised by Harvard University biomedical engineer Mehmet Toner, is more “efficient,” said Haber.

The way it works: “We first add antibody against WBCs much more reliable and well-defined than antibodies against tumor cells. The antibodies are conjugated with magnetic beads." Next, the blood is passed through a microfluidic device. “The proteins, platelets, red blood cells, and unbound antibody/beads are removed based on size. Remaining CTCs and WBCs are passed through a series of curved channels which line them ‘single file’ in the channel." Then: "CTCs and WBCs pass through a magnet that diverts the magnetically tagged WBCs into the waste chamber, leaving behind untagged CTCs."

Haber continued: "The essence is the curved channels, which work through a physical principle (inertial focusing) to line up the cells in single file, when it’s easy for a minimal number of magnetic particles to deflect them into a different path with extraordinary efficiency.” This was first described in a 2013 Science Translational Medicine paper.

The CTC-iChip

The technology stars the CTC-iChip, which is “exceptional in that it allows us to remove all normal blood cells, leaving behind untagged and un-manipulated tumor cells,” Haber said. It lets “negative depletion…actually work.” Un-manipulated, viable cells “grow in vitro,” he said.

Marchetti believes the iChip “is not more efficient than multi-parametric flow cytometry and/or other methods for blood depletion. Further, some of these CTC lines are tumorigenic, but their metastatic propensities are unknown. The iChip has promise but the strength of the study comes from two other points.”

First, Marchetti said, “CTCs are promising biomarkers for diagnosis and therapy of systemic cancer.” But hurdles impeding widespread use of CTCs as non-invasive “liquid biopsy” for diagnosis and therapy include the fact that “the circulating phase of cancer progression is not as simple as it was previously thought. CTCs include a heterogeneous mixture of epithelial-derived cells with a wide spectrum of markers, tumorigenic properties, and prognostic potential.”

Haber’s study “has relevance” in establishing CTC cultures from ER-positive breast cancers with distinct tumorigenicity in xenografts, and pre-existing, or newly acquired, mutations, Marchetti continued. “Although CTCs with metastatic competence have been reported with the initial characterization of their properties, [the Haber study] sheds light on varying expression of mutations/genetic aberrations even within the same breast cancer subtype, thus trying to address one of the fundamental questions in the field. That is: Do CTCs have properties that make them capable of surviving in blood distinct from cells in the neoplasm?”

Aberrant CTCs, said Marchetti, are “informative,” offering a window into the “evolutionary stage of cancer” and enabling assessment of the extent to which CTCs drive relapse. He added that his lab consistently grows “mammosphere cultures out of CTCs isolated by flow cytometry [unpublished data].”

Ohio State University engineering professor Jeffrey Chalmers agreed by email that “developing CTC cell line(s) is very important (for studying the evolution of cancer cells; providing targets to test drugs; establishing more accurate standards.).” But he added he believed “insufficient data exist to compare the MGH negative depletion technique to our and other negative approaches, so I do not agree the MGH system provides a higher recovery and purity. Further work is needed. Our paper in Breast Cancer Research demonstrates our approach can find CTCs that would not normally be positively selected, that can be correlated to patient overall survival, while traditional positively selected CTCs do not have this correlation. I believe this demonstrates the importance of negative depletion no matter which technology is used.”

Marchetti agreed more studies are needed. These will “relate to the molecular and genetic characterization of CTC subsets unique to a tumor." Success will enable cancer monitoring “in real-time, predicting therapy response.”

Jeffrey said future work should involve “intense molecular scrutiny of cultured cells…to better determine how representative the cultures are of a patient's disease at any given time; experiments to demonstrate whether replicate cultures of CTCs from a patient's blood sample are molecularly similar even if obtained on different days within a short time-frame; and most importantly, whether these replicate CTC cultures show the same drug sensitivities for that short time period.”

Haber said his team is working on “further optimization of culture conditions, and extension to other cancers in which genetic characterization may identify novel mutations that arise during the course of therapy.”

(This story was updated on August 4, 2014.)