NEW YORK (GenomeWeb) – A team led by researchers at the University of California, Berkeley, has developed a single-cell western blotting system for multiplexed analysis of proteins in circulating tumor cells.
Described in a paper published last week in Nature Communications, the system is an extension of the single-cell western blotting technology developed in the lab of Amy Herr, a professor of bioengineering at UC Berkeley and senior author on the study.
Using the system, Herr and her colleagues measured an eight-protein panel in individual CTCs collected from the blood of estrogen receptor-positive breast cancer patients, identifying two distinct patient subpopulations and demonstrating the general feasibility of the approach for measuring protein panels in such cells.
CTCs are cells shed from tumors into patient blood where they can then be collected and analyzed for genomic, proteomic, and other information that could be informative as to the status of a patient's cancer. Their utility is still under investigation, but researchers and clinicians believe they could help, for instance, with monitoring for cancer recurrence or the development of drug resistance.
Sequencing-based methods for analyzing CTCs are relatively advanced, but approaches for multiplexed protein analysis have lagged behind. While a variety of technologies are capable of multiplexed protein analysis at the single-cell level, these methods typical involve some degree of sample loss, which makes them untenable for analyzing a cell type as rare as CTCs, which, according to the Nature Communications paper, are typically present in cancer patient blood at levels of between one and 500 cells per 7.5 ml.
Conventional immunoassay techniques like ELISA and immunohistochemistry can be used, but these methods are limited by the availability of highly specific antibodies, which makes multiplexing difficult and also hampers the analysis of protein post-translational modifications.
Western blotting, on the other hand, increases assay specificity by first separating proteins by size and then probing them with antibodies, allowing researchers to multiplex panels of proteins.
In 2014, Herr's lab presented a microfluidic device capable of single-cell western blotting, using the system, which they named scWestern, for a study published in Nature Methods.
However, like other single-cell proteomics systems, the device was poorly suited to CTC analysis due to sample loss. According to the Nature Communications paper, the method required on the order of thousands of cells to account for loss of cells as they settled into the device's microwells.
This sample loss stems from Herr and her colleagues' desire to make the scWestern a high-throughput method capable of performing single-cell analyses of thousands of cells. However, she noted, in the case of CTCs, the assays they wanted to do were inherently low throughput, and low-throughput methods for transferring single cells without sample loss already existed.
"So for us that meant that, well, maybe we didn't have to reinvent the wheel," she said. "We could use something like microtransfer to be able to pipette selected cells into [the scWestern system]. And that actually worked quite well."
The researchers used Vortex Biosciences' HT chip CTC enrichment system which selects cells based on size and deformability (several Vortex researchers were co-authors on the study) and then introduced them into the scWestern system where they measured levels of eight proteins. Among their findings were differences in EpCAM expression and two patient sub-populations with statistically different GAPDH expression levels.
Having demonstrated the feasibility of the method for analyzing CTCs, Herr said she and her colleagues are now using the system to further investigate these cells in prostate and breast cancer.
She noted that she is particularly interested in developing the technique to measure different post-translationally modified proteins, something that is challenging for existing immunoassays due to the high specificity needed to distinguish between modified forms of the same protein.
Looking at, for instance, protein phosphorylation, could help researchers "understand what signaling pathways are active," she said, citing the case of the breast cancer patients she has been working with. "Those patients are undergoing treatment, so we're really trying to do things like detect the development of resistance to a cancer drug early."
Herr said that her group is also working to incorporate isoelectric focusing up front of western blot analysis, which would add an additional form of separation that could further increase the method's ability to distinguish between different proteoforms.
They are also working to expand the technique's multiplexing capabilities. The researchers looked at eight different proteins in the Nature Communications paper, but Herr said that they hope to push the number of proteins they can analyze in a given cell to the 50 to 100 range.
She added that the assay format is well suited to long-term storage, meaning that samples can be analyzed and then stored for future use. She suggested that this was particularly significant for CTC work given the relatively newness of the field.
"Not so much is known, and there is some debate as to what markers or what proteins are specific to the [CTC] biology," she said. "It is an evolving field, for sure. So being able to hold on to those single-cell materials in a way that we can come back later and start probing for new stem cell markers that come out or other markers that other people have interest in is pretty exciting."
In September 2014, Herr co-founded a company, Zephyrus Biosciences, to commercialize the scWestern technology. The firm launched with $1.86 million in funding, including $1.5 million in seed funding and a $350,000 Phase I SBIR grant. In 2016, life sciences firm Bio-Techne purchased the company for an undisclosed sum and integrated it into its protein products division, which includes its ProteinSimple western blot line.
Herr said this week that she did not know if the intellectual property underpinning the CTC analysis method described in the Nature Communications study was owned by Bio-Techne through its purchase of Zephyrus and the scWestern system.
Inquiries placed with Bio-Techne were not returned as of press time.