Researchers from the University of California, San Francisco and the California Institute for Quantitative Biosciences have developed a method dubbed PCR-activated cell sorting (PACS) to sort and analyze single cells from large populations of heterogeneous cells based on gene expression.
According to UCSF researcher and Mission Bio co-founder Adam Abate, the principal investigator on the project, the method could prove useful in clinical applications such as analyzing individual cancer cells from genetically diverse tumor samples and monitoring latent infections in HIV patients.
In addition, a startup company called Mission Bio — which was founded last year by Abate and collaborators — is tasked with moving the technology to market.
Abate shared details of the new method in a presentation last week at Cambridge Healthtech Institute's Molecular Medicine Tri-Conference in San Francisco. Abate first introduced the method at Stanford University's Frontiers of Single-Cell Analysis conference in September, and also discussed it earlier this month at the American Society of Mechanical Engineering's conference on Nanoengineering for Medicine and Biology in San Francisco.
The technique builds on the concept of fluorescence-activated cell sorting, a time-tested and oft-used method for rapidly selecting cells of interest based on phenotype — usually through the use of a fluorescent tag for a particular surface protein of interest.
However, "until now FACS has been limited to sorting cells based on what's on the outside," Abate said at Tri-Con. "We haven't been able to sort cells based on gene expression."
PACS aims to do just that by isolating up to 1 million individual cells into discrete aqueous picoliter- or even sub-picoliter-volume droplets suspended in oil — "basically using small droplets of water as test tubes for cells," Abate said — then performing PCR reactions on the cell lysates.
Using multiplexed TaqMan PCR assays, the system can then interrogate each cell for the expression of specific combinations of transcripts, mutations, or non-coding RNAs. In addition, similar to FACS, PACS can sort the cell lysate into different containers, allowing the lysates of cells with a unique transcriptional signature to be recovered for additional downstream analysis using any number of molecular biology techniques.
According to Abate, his lab recently made an important technical breakthrough with the PACS technique by incorporating a novel microfluidic workflow that overcomes lysate-mediated inhibition of reverse transcription PCR in the droplets. Essentially, he noted at Tri-Con, it is now feasible to "analyze millions of cells at a time using PCR," adding that the group has specifically demonstrated a throughput of 108 individual cells per day, or about 100-fold greater than can be achieved with existing techniques.
Abate and collaborators also published a study last summer demonstrating how a technique called picoinjection can be used to precisely and efficiently add reagents to individual reaction volumes without compromising assay quality or detection rates.
Although the group demonstrated the picoinjection technique on individual digital PCR reaction volumes, it noted at the time that it could be used in a workflow where "populations of cells are first encapsulated in drops, lysed, and subsequently picoinjected with PCR reagents" to enable rare cell detection from heterogeneous populations of cells.
In fact, such rare cell detection is one of the primary applications that Abate and colleagues are pursuing along with their startup company, which they founded last year as Torrent Bio but subsequently changed the name to Mission Bio.
The ability to examine different cell types independently of one another is extremely important when examining tumors, the heterogeneity of which is an oft-cited problem for uniform and homogenous analysis techniques that analyze many cells at once and provide an average measurement of gene expression levels.
Abate said at Tri-Con that his lab and Mission Bio are currently collaborating with various laboratories at the University of California, San Diego, to use PACS to examine how stem cell markers such as LGR5 and Myc affect cancer development.
Another potential application area is microbiology, Abate said. Many important bacteria and viruses are not able to be cultured, which makes it difficult to generate antibodies against them for traditional FACS analysis. Thus, most FACS methods for enriching bacteria or viruses from a diverse population are based on non-specific surface markers.
The UCSF, QB3, and Mission Bio researchers are collaborating with the University of California, Berkeley on this application, developing a PACS assay for bacteria that eat perchlorate for environmental applications, Abate said.
Finally, Abate and colleagues are exploring the use of PACS to investigate latent HIV infection in blood cells, which can also be quite heterogeneous. This application is also emerging as a sweet spot for droplet digital PCR, although false positive data continue to be a hurdle for that technology.
In general, Abate noted, the group continues to develop the PACS method and is currently investigating ways to further increase throughput and to more consistently and robustly recover cell lysates from individual droplets, the latter of which has also been cited as a downside to droplet digital PCR.
Abate and colleagues are hoping that the PACS method can eventually serve to compliment FACS, enabling the differentiation and sorting of cells based on both genomic and phenotypic markers.