NEW YORK (GenomeWeb) – Researchers at the University of British Columbia have developed a new methodology for isolating and analyzing single circulating tumor cells (CTCs) that is compatible with downstream sequencing. They believe that the technique overcomes limitations of currently available approaches by integrating biophysical enrichment and laser capture microdissection (LCM) to minimize cell loss.
Most current methods to isolate and sequence single CTCs use micromanipulation, which involves suctioning individual cells into a micropipette, depositing them into reservoirs, and performing whole-genome amplification on each cell. While the technique works for collecting a small number of cells, the UBC researchers highlighted that micromanipulation can be time consuming and burdensome for routine clinical settings.
The UBC team demonstrated that its methodology enabled isolation and next-generation sequencing of single CTCs from a patient with metastatic prostate cancer, providing a platform for somatic mutation discovery and examining the genetic heterogeneity of CTCs. Hongshen Ma, senior author and associate professor at UBC, explained that his team had previously developed similar methods to isolate circulating tumor cells.
"There's a lot of interest in single-cell sequencing, but there aren't a lot of good ways to select cells you want to sequence individually," Ma noted. "[However], we found how to alter [LCM] to isolate rare cells" in liquid biopsies.
In the study, published earlier this month in The Royal Society of Chemistry, Ma and his colleagues collected blood samples from healthy human donors, spiking them with UM-UC13 cells and tracking the cells through hydrogel encapsulation and laser capture microdissection.
In order to enrich the CTCs, the researchers first lysed the sample's red blood cells and used a microfluidic ratchet-based cell sorting method to separate white blood cells and circulating tumor cells.
To extract single CTCs for LCM, the team identified the tumor cells by fixation-free staining and embedded the cell samples in a hydrogel matrix, which encapsulated and immobilized the cells. Ma explained that the team developed a poly-ethylene glycol diacrylate (PEGDA) hydrogel matrix in order to mimic solid tissue samples. After centrifugation, the researchers cured the hydrogel using ultraviolet light to limit DNA damage to the cells. They saw that the cells embedded in hydrogel retained a strong fluorescence signal, and that a region of interest could be excised with high precision.
The team then extracted individual PEGDA hydrogel-embedded cells on the slide by laser pressure catapulting. After identifying a target cell, the team used a short defocused UV laser pulse to launch the embedded cells onto an adhesive cap in a collection tube.
"We [were able to] cut out the hydrogel portion [we] wanted, with the cell embedded in the middle," Ma explained. After the LCM step, the team added reagents "for a whole-genome amplification step."
After microdissection, the team amplified the cells' DNA and performed targeted next-generation sequencing on the genetic material to generate a library of genes relevant to prostate cancer.
Ma explained that while researchers enriched the sampe in one to two hours, the overall process required about two days to produce results due time needed for immunostaining and LCM.
To demonstrate proof of principle for clinical use of the method, Ma and his team isolated single CTCs and cell-free DNA (cfDNA) from a patient with castration-resistant prostate cancer. Collecting 8 milliliters of whole blood, the researchers performed the single-cell sequencing workflow on the sample. Identifying 191 CTCs with LCM, the team then evaluated eight individual cells for for single-cell analysis by qPCR.
After selecting five cells for next-generation sequencing, the team found identical mutations in prostate cancer driver genes —TP53, PTEN, FOXA1 — in both single CTCs and cfDNA.
However, certain CTCs showed somatic mutations that the researchers did not identify in cfDNA. The team therefore believes that the mutations may be relevant because they were not detected in all isolated CTCs and may represent CTC subpopulations that differ in their metastatic potential.
In terms of potential limitations, Ma noted that the workflow's overall sample to result time is a major challenge for CTC detection. While Ma noted that LCM is a quicker method than micropipetting, the technique does not produce results "fast and fully automated" enough for larger patient populations.
"Because we only sequenced a few cells, we wanted to identify mutations in more than one cell, [since] whole-genome applications tend to make mistakes," Ma said. "If you only see the mutation one time, you're unsure and don't know if it's a mistake in the whole genome, or something real. We therefore wanted to point out things that appeared in more than one CTC"
According to Ma, the team has filed for a provisional patent on the PEGDA hydrogel matrix, which they created in house for the workflow. The group is currently working with UBC's industry liaison office to potentially commercialize the CTC enrichment and hydrogel technology.
Ma and his team will be entering a very crowded field for CTC capture technology, as many firms and research groups have developed their own methods for cell collection and extraction, some of which feature approaches that are parallel, or similar aspects of the UBC team's. Vortex Biosciences, for example, has released a platform for DNA extraction that uses a microfluidic chip to capture single CTCs via "micro-scale vortices."
Researchers at the University of Toronto have also recently released a study on a technology called "single-cell mRNA cytometry," that used magnetic particles to separate single CTCs from a small cohort of patients with metastatic castration-resistant prostate cancer, and asses the cells' RNA without PCR amplification.
In addition, startup CytoLumina Technologies has been developing its "NanoVelcro" approach in a system it calls CytoTrapNano to isolate CTCs in patients with prostate cancer. The firm's microchip technology captures microvilli protruding from CTCs and uses LCM to remove the cells for isolation, purification, and single-cell sequencing.
Ma believes that his team's workflow distinguishes itself from current CTC collection technologies because of its combination of enrichment and single cell isolation abilities. He noted that a key advantage of the team's workflow is that it can capture a higher amount of cells and has access to those cells in a fluid suspension after enrichment.
In addition, Ma argued that his team's workflow, which takes the suspended cells and place them in hydrogel, allows the use of LCM to isolate the cells separately for downstream single-cell sequencing in ways other platforms do not.
In the study, the researchers noted that the mutations they found may be missed by bulk sequencing libraries, whereas single-cell sequencing could potentially allow characterization of key CTC subpopulations that appear during metastasis.
Ma said that the workflow can be applied to a variety of different cancers and other diseases.
"You're selecting cells based on what you see, and therefore [the workflow] could be used for isolating diseased cells, like parasites or bacterial cells," he explained.
In the future, Ma and his team believe a extensive analysis of patient CTCs could provide a crucial insight into metastatic behavior. Ma noted that his main objective is to improve the sample to result speed, reducing the time needed to perform LCM. In addition, Ma wants to scale the technology for a larger patient population in a later study.
"We can isolate hundreds of CTCs, but it would take too long with our current methodology," Ma noted. "We need to find ways to improve detection and sequencing of hundreds of CTCs at a time in a patient setting."