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Cancer Studies Demonstrate Potential of CalTech Immunoassay Barcode Chip in Clinical Research


In a pair of recent studies, research teams led by the laboratory of California Institute of Technology professor James Heath have offered demonstrations of potential applications of the lab's single-cell barcode chip technology to clinical research.

Introduced in a May 2011 paper in Nature Medicine, the single-cell barcode chip – SCBC – platform is a microfluidic immunoassay-based device that enables researchers to perform highly multiplexed protein detection at the single-cell level.

As detailed in the Nature Medicine study, the device consists of 1,040 3-nl volume microchambers, each of which can be loaded with single cells or small defined numbers of cells. Each microchamber contains a series of immunoassays on antibody barcodes, with duplicate barcodes per microchamber allowing for the collection of single-cell protein measurements.

In the original paper, Heath and his team used the device to measure levels of 12 secreted proteins in tumor antigen-specific cytotoxic T lymphocytes, finding significant heterogeneity in these CTLs.

Last month, the researchers further demonstrated the platform's potential, publishing two papers that employed it: one in Cancer Discovery profiling T cells used in adoptive cell transfer immunotherapy, the other in Proceedings of the National Academy of Sciences investigating the effect of hypoxia on mTOR signaling in glioblastoma multiforme cancer.

According to Heath, he and his colleagues have filed patents on the technology and are looking to commercialize it in the near future, although he didn't provide details on these commercialization plans. Heath has considerable start-up experience, having co-founded several biotech firms including Seattle, Wash.-based protein diagnostics outfit Integrated Diagnostics.

In an email to ProteoMonitor, he suggested that the platform's ability to measure proteins at the level of copy numbers per cell was an important aspect of its clinical research potential, noting that the researchers have published findings showing that the platform can detect proteins at a level of "a couple hundred copy numbers per cell" and that with amplification, it could detect proteins at levels as low as 50 to 100 copies per cell.

Heath cited the PNAS study as an example of the sort of analysis such quantitative data could enable. In that work, the researchers used the SCBC platform to measure in GBM cancer cells levels of seven proteins involved in HIF-1α and mTORC1 signaling, studying how these pathways are affected by hypoxia.

Using the device they found that at low oxygen levels mTORC1 becomes deregulated in a manner that causes it to be unresponsive to mTOR kinase inhibitors, suggesting a potential mechanism of drug resistance.

"The platform is extremely useful for looking at cell signaling pathways within cancer cells and for understanding how those pathways respond to perturbations," Heath said. "We're working towards moving those types of measurements [in the PNAS study] into the clinic as a means of identifying combination therapies that can be used to treat patients with highly heterogeneous tumors."

In the Cancer Discovery study, the researchers used the SCBC platform to investigate another form of cancer therapy – adoptive cell transfer, in which genetically engineered T-cells expressing cancer-specific T-cell receptors are generated ex vivo and then introduced into patients, where they then attack cancer cells.

While ACT has proven effective in some cancer patients, the modified T cells typically lose their antitumor activity. To investigate why, Heath and his colleagues performed a time-course study of engineered T-cells taken from three patients receiving ACT therapy, using the SCBC technology to measure changes over time in levels of 19 proteins secreted by these cells.

The researchers identified time-dependent changes in levels of these proteins that they were able to coordinate with the patients' clinical outcomes.

As they noted in the paper, all three patients showed "an initial reduction in tumor volume, followed by tumor regrowth, but with different times to tumor relapse." Cellular phenotyping using cell-surface markers showed relatively uniform changes among the three patients' T cells. On the other hand, the patients' T cells exhibited "clear differences" in their functional changes, as represented by the SCBC-based cytokine measurements.

In particular, the CalTech team identified a population of "polyfunctional" T cells that they hypothesized are the main drivers of the antitumor immune response. These cells – which the researchers defined as those producing five or more cytokines upon stimulation – represented on average only 10 percent of a given cell type but secreted roughly 100 times the amount of protein than the other 90 percent of the cell population.

This polyfunctional population specifically, the authors wrote, showed significant functional changes over time and differences between patients that tracked with their response to ACT treatment.

The SCBC platform and its multiplexing ability were key to identifying these polyfunctional cells, Heath said, calling it one of the key findings of the study.

"Those cells, while only 10 percent of the total for a given phenotype, secreted around 100-fold more copies of any given protein than did the other, less functional 90 percent of cells," he said. "I'm not sure how one could identify those polyfunctional T cells without assaying for a large number of proteins."

Given the platform's multiplexing, antibody cross-reactivity is a significant challenge, Heath said, noting that "the entire panel of proteins and antibody pairs involved in our assays must be validated to ensure that there isn't significant cross-reactivity between the different protein assays."

He estimated that the device's upper limits of multiplexing probably lie in the range of 75 to 100 proteins.

"Much more than that would likely require a fundamentally different platform and would require replacing antibodies with something more stable and less subject to batch-to-batch variations," he said.

Heath added that in addition to the platform's high sensitivity and multiplexing abilities, its relative simplicity and cheapness could prove advantageous.

"The platform is just a little glass and plastic microchip, albeit still requiring a significant skill set to operate," he said.