NEW YORK (GenomeWeb) – A group led by University of Illinois at Chicago (UIC) researchers has developed a circulating tumor cell (CTC) isolation method using gravity and microfluidics.
The team plans to commercialize the microfluidic chip for cell separation and isolation for downstream applications such as sequencing and gene expression analysis.
In a study published earlier this month in Microsystems and Nanoengineering, senior author and UIC bioengineering professor Ian Papautsky and his team extracted and isolated CTCs from 2 milliliters of blood from eight patients with NSCLC and five control patients using a multi-flow, label-free device.
Papautsky explained that his group built the CTC collection device around the concept of size-dependent inertial migration. They found that larger cells, specifically CTCs, will move to targeted positions faster than smaller cells like red blood cells (RBCs) and white blood cells in a liquid sample, which his team decided to manipulate when building the microfluidic chip.
The researchers therefore designed the microfluidic channel to contain multiple flows to initially contain cells near channel sidewalls. After lysing the blood to remove RBCs, the team passes the samples through the microchip. Three flow streams develop in the main channel as a clean buffer flows in the middle to collect larger target cells (CTCs), which Papautsky said are then subjected to size-dependent inertial forces and pushed into groups based on geometry.
"We found that if we could create a lane of clear fluid, the larger cells migrating within blood will have an HOV lane that, where they could migrate from the main traffic of cells," Papautsky said. "So we created this flow system where the center buffer is a place where CTCs migrate into and we're able to isolate them."
To prevent contamination in the center channel, the team chose a threshold size of 14 micrometers to differentiate CTCs from WBCs, which are normally 8 to 13 micrometers in diameter.
In the study, the team pumped the blood samples through the microfluidic channels. They then inspected the collected cells from the middle channel and identified them as CTCs after they met certain criteria, including the presence of cytokeratin-8,18,19, intact nuclei, a high nucleus-to-cytoplasmic ratio, a lack of CD45 antigen, and a morphologically larger size than background WBCs.
According to Papautsky, the CTC device would typically use a standard patient blood draw of about 7.5 milliliters. While the device could potentially run on a smaller sample, such as the 2 milliliters of blood in the study, Papautsky noted that smaller samples make it harder for researchers to establish flow parameters and can lead to a lower recovery rate of CTCs.
In the study, Papautsky and his team achieved a recovery rate of greater than 93 percent using spiked cancer cells at clinically relevant concentrations. Papautsky noted that the overall time needed to collect and detect targeted cells was about two hours. The researchers also successfully detected CTCs from six of the eight NSCLC patients, with a recovery rate of greater than 93 percent and a purity of greater than 87 percent using spiked cancer cells.
Papautsky also noted that prior research studies needed to dilute the blood samples when attempting to extract CTCs, as RBCs and WBCs dislodged CTCs from collection points.
"In earlier work, we would take a blood sample and dilute it initially 100 times, but now we only need to dilute it 5 or 10 times to minimize cell collisions," Papautsky said. "If we dilute the samples, we minimize cell-to-cell interactions, allowing larger cells to migrate to the right position and be isolated."
The researchers have previously used the microfluidic device to capture CTCs from other tumors. In a study published last month in Cancers, Papautsky and his group used the microfluidic chip to successfully focus and identify CTCs in 10 of a cohort of 21 head and neck cancer patients.
While Papautsky's team does not have any current plans to use other liquid sample types on the CTC isolation platform, he noted that the team is happy to collaborate with groups interested in using the tool. He believes that urine samples may pose a challenge because CTCs are even rarer in urine than in blood. Saliva also poses a similar problem because the sample size is very small, which means researchers would need to perform a significant amount of dilutions, Papautsky said.
Papautsky also acknowledged that his team dealt with several limitations in the study when attempting to collect targeted CTCs. One issue that he noted included selecting an accurate cutoff for the center channel to minimize WBC contamination.
The study authors also highlighted that the device's sensitivity and recovery performances when the size of target and non-target cells overlaps significantly.
In addition, Papautsky and his team noted that using the size-based separation method requires additional steps to identify targeted cells. In the study, the group noted that downstream immunofluorescence and molecular characterization steps are needed in order to potentially apply the collection method in clinical situations.
Another issue that the researchers encountered was the relatively slow flow rate when extracting the targeted cells. They noted that while the "device shows much better performance in terms of efficiency, recovery rate, and sensitivity, the effective throughput is 1.2 milliliters of whole blood per hour, which necessitates further improvement."
Overall, Papautsky and his team believe the approach is a promising alternative for reliable CTC capture in order to extract information from the cell to personalize treatment strategies for solid tumor patients.
In addition, Papautsky and his team believe researchers could potentially use the device to separate circulating tumor microemboli (CTM). Because of the stronger inertial migration for a larger target, they believe that the system could easily isolate CTMs from different cancers for downstream analysis.
"Recent studies are suggesting that it is the clusters/CTMs that lead to metastasis and maybe a better predictor of survival than CTCs alone," Papautsky said. "I think there is a lot of interesting work [that] remains on the makeup of these CTM/WBC aggregates, their response to therapy, and even perhaps genomic analysis that may differentiate them from healthy cells or even from other CTCs."
Papautsky and his team therefore plan to pursue two major goals in future studies. After isolating the cancer cells, the researchers aim to identify genetic biomarkers that can act as targets for drugs. In addition, the team will perform studies on the potential efficacy of treatment with immunotherapy, as Papautsky highlighted that oncologists need to find better methods for specific types of patients.
The team has a portfolio of patents related to the CTC isolation technology, of which some have been issued and others are currently pending. The UIC researchers plan to establish a joint venture with their partners at the University of Queensland of Technology to form a startup based on the CTC isolation platform. While in the early stages of discussion, Papautsky believes that UQT will help provide support for clinical trials and to validate the technology.
Many other academic groups and companies are developing or currently offer their own methods of CTC isolation using microfluidics, such as Menarini-Silicon Biosystems with its Cellsearch platform and Akadeum, which has developed a microbubble technology platform.
In addition, Angle offers its Parsortix microfluidic cassette system, which the firm said collects and enriches rare cells like CTCs based on their less-deformable nature and larger sizes.
Papautsky argued that his team's platform stands out from current methods because of its sized-based approach and ability to produce viable cells for downstream usage. Highlighting the difference between the group's CTC tool and Menarini-Silicon Biosystem's Cellsearch platform — which uses surface markers and antigens, Papautsky argued that size-based separation frees the user from worrying if a cancer cell expresses a specific surface biomarker.
In addition, Papautsky touted the CTC's gentle flow system, which he said minimizes tear and damage on the CTCs for downstream applications.
"We are taking a lot of steps to optimize the system so that the shear forces acting on these cells are gentle and [allow] us to culture [the CTCs] after capturing them," Papautsky explained. "We [currently] have follow-up studies where we are tracking the cells from one to two weeks on the chip."
Papautsky envisions the assay as a supplementary tool that clinicians could use to detect CTCs if the standard of care fails to identify cancer cells in a patient's bloodstream. In addition, he believes the CTC platform could be used as a research tool to develop downstream immunotherapies and improved diagnostic tools.
"In Chicago, we have large underrepresented minority groups (African American and Hispanic) that come to our clinic, which is challenging for them to travel across the city to get blood tested," Papautsky said. "If there was a test that could be done on a blood sample, that could be collected in a tube of blood, it could be done quickly and be beneficial in terms of cost savings to the patient."