NEW YORK – Researchers from the University of Georgia are commercializing a method called integrated ferrohydrodynamic cell separation (iFCS) as part of a device to isolate circulating tumor cells (CTCs) while minimizing white blood cell (WBC) contamination.
Through a startup called FCS Technology, the researchers are hoping to market the platform for cancer research and, eventually, diagnostic applications.
Leidong Mao, FCS Technology's founder and an electrical and computer engineering professor at UGA, noted that his team initially explored the use of ferrofluids for diamagnetic particle separation. He explained that ferrofluids are nanometer-sized superparamagnetic particles covered by a surfactant, which prevents them from coming too close to each other. Ferrohydrodynamics then incorporates ferrofluids and magnetic fields to separate particles based on the contrast of magnetization.
Mao said that the microfluidic research community has shown interest in applying particle separation for diagnostic purposes, and that his group initially tested the technology by collecting cervical cancer cells in a 2015 study published in Advanced Functional Materials.
"When you take a cervical sample, only 10 percent [of cells] are abnormal and mutated," Mao said. "After enriching [cells] for cervical cancer screening, we [then] decided to apply the technology to CTC enrichment and separation."
Initially relying on size-based differences to separate CTCs and WBCs in their microfluidic system, Mao's team saw that over 1,000 WBCs still "contaminated every ml of whole blood" when they tried to purify CTCs. Concluding there was "no perfect threshold to separate CTCs and WBCs," Mao modified the system by incorporating magnetic microbeads to minimize WBC contamination.
Before a liquid sample is processed through the iFCS device, the WBCs in a patient's blood sample are labeled with magnetic microbeads via leukocyte surface biomarkers. Mao said that this step ensures that the magnetization of WBCs is larger than that of surrounding ferrofluid and unlabeled CTCs.
After the researchers perform red blood cell lysis, a magnetic field guides unlabeled CTCs to the upper boundary of a microchannel, while attracting magnetic beads and highly magnetized WBCs towards a waste outlet at the lower boundary of the microchannel.
Then, a symmetric magnetic field with its maximum at the middle of a new channel is used to attract remaining weakly magnetic WBC-bead conjugates towards the channel's center for rapid depletion, while unlabeled CTCs flowing along the upper and lower channel boundaries pass through separate outlets for imaging and analysis.
In a May 2019 feasibility study published in Lab on a Chip, Mao and his team tested the technology's ability to enrich for CTCs in several different patient cohorts. The team included four performance goals for the iFCS devices in the different cohorts: a complete CTC recovery of greater than 99 percent regardless of surface antigens and sizes; a minimal WBC contamination of about 500 cells for every 1 ml of blood processed; a blood processing throughput of more than 10 ml per hour; and unaffected cell integrity after enrichment (including viability and proliferation).
For each cohort, Mao's team processed blood samples through the iFCS device, immobilizing remaining cells onto a glass slide with a customized cell collection chamber. The group then immunostained the cells with primary antibodies, later washing and storing them for detection via bright-field microscopy.
In order to validate the iFCS system, the team first studied cell enrichment using eight cultured cancer cell lines with different average cell sizes, including four breast cancer, two non-small-cell lung cancer, and two small-cell lung cancer cell lines.
On average, the researchers found that the recovery rate across the eight cancer cell lines was about 99 percent, indicating a near-complete recovery of spiked cancer cells, regardless of their size profiles. Enriched cells were also suitable for immunofluorescent and cytopathological staining.
Mao's team then collected samples from late-stage cancer patients — three late-stage breast and three late-stage lung cancer samples — to determine if heterogeneous populations of CTC cell types could be enriched using iFCS.
Both sample cohorts indicated that CTCs were highly variable in cell diameters and often overlapped with the size of contaminating WBCs. Surface antigen expressions also varied across lung and breast cancer CTCs. However, the authors concluded that the IFCS device is "insensitive to both size and antigen variations, [and] can enrich CTCs and preserve these variations."
In a third cohort, Mao's team used the iFCS device to process blood samples from six early-stage breast cancer patients. Using similar approaches to the prior two cohorts, the researchers found that they could detect clinically significant amounts of CTCs in the early-stage cancer patients' blood samples.
Based on the study's results, Mao's team is now licensing the iFCS platform from UGA through Athens, Georgia-based FCS Technology, which Mao launched in 2015 at the university. The team initially received a patent for the technology in 2015, in addition to a few follow-on patents describing the ferrohydrodynamic process and iFCS platform.
Mao said his team is also developing a second device to be used in conjunction with the initial device and further deplete WBCs while enriching for additional CTCs. The group is collaborating with UGA and undisclosed clinical partners to collect a larger cohort of lung and breast cancer samples in order to validate the updated platform.
Mao declined to elaborate on the specific methods used in the second device, but explained that a patient's blood sample will pass through it following the initial instrument as part of a potential clinical workflow to curb WBC impurity.
While the system can collect CTCs from a patient's bloodstream, Mao acknowledged that the system lacks the ability to enrich for CTCS in lymphatic cancers.
"Rather than [developing] a fully commercialized [510(k)] instrument, we want to develop the technology for people performing fundamental cancer research," Mao said. "We hope to apply for more funding in Georgia and from the NIH as part of an [eventual] Phase II grant within the next two years."
Mao envisions offering the dual-instrument platform as part of a benchtop point-of-care clinical assay system to help doctors collect a purified population of metastatic CTCs for diagnostic or prognostic purposes. He believes that clinicians could use the platform with downstream approaches such as PCR and next-generation sequencing to capture the majority of CTCs and how they vary among cancer patients of different stages.
"We hope that clinicians would be able to inject a sample from a patient and extract information from CTCs, allowing them to help with treatment or, in the long run, do a better job of diagnosing cancer patients," Mao said.
FCS Technology is optimizing the CTC enrichment technology to eventually deploy it in universities, research centers, and hospitals.
Mao's team was awarded a $225,000 Phase I Small Business Technology Transfer grant in August from the National Institutes of Health to further develop the iFCS method for tumor antigen-independent and cell-size inclusive CTC enrichment. The team also plans to validate iFCS with 36 breast cancer patient samples as part of the firm's goal to enrich "the entire repertoire of intact CTCs with minimal WBC contamination."
Acknowledging that multiple startups in the liquid biopsy space are applying size-based and biomarker-specific methods to collect CTCs, Mao argued that FCS' technology allows users to perform antigen-independent enrichment and is unbiased toward biophysical size varieties of CTCs. He also believes that the two-instrument platform will eventually remove more than 99 percent of WBCs in a 'sample.
In addition to applying iFCS for CTC enrichment, Mao's team is also using the technology to enrich for tumor-derived extracellular vesicles. Mao noted that his team has submitted a manuscript for the EV isolation method, and expects to publish it by the end of the year.