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Single-Cell, 42-plexed Protein Analysis Achieved with a New Microchip Technology

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The laboratories of Drs. Rong Fan and Kathryn Miller-Jensen in Department of Biomedical Engineering at Yale University have invented a novel microdevice capable of detecting 42 unique immune effector proteins, a record number for a single-cell protein secretion assay; using the device, the team was also able to demonstrate that a phenotypically identical cell population still exhibits a large degree of intrinsic heterogeneity at the functional and cell behavior level.

“Our device complements existing technologies for cell sequencing and profiling by making such analyses available even with very small sample sizes,” said, Associate Professor of Biomedical Engineering Rong Fan, who led the development of this innovative technology and published this research on February 2, 2015 in the Proceedings of the National Academy of Sciences of the United States of America (PNAS). “For this reason, our device could provide a low-cost avenue for clinical immunomonitoring.”

Developed and tested in collaboration with Assistant Professor of Biomedical Engineering, and molecular, cellular & developmental biology Kathryn Miller-Jensen, the device consists of a glass slide attached to a microchamber array; the glass slide is striped with 15 different bands of antibodies — three per band for fourteen of the stripes, with an additional control band. The bands change colors in the presence of various immune effector function proteins, including pro-inflammatory cytokines, chemokines, cytolytic enzymes, and growth factors.

The researchers also used their device to generate novel insights into how immune cells respond to pathogens. In particular, distinct subpopulations of genetically identical immune cells exhibited varied reactions to pathogen stimulation — reactions that though diverse appeared dynamically structured instead of randomized. For example, the researchers identified one subpopulation that secreted a macrophage migration inhibitory factor that potentiates the production of a range of inflammatory cytokines.

“This research opens the door for deep functional phenotyping and comprehensive dissection of immune functional states of single cells,” said Fan. “We hope that this might soon be a common tool in the clinic, where these capabilities could be key to, say, measuring the effectiveness and toxic effect of cancer immunotherapy on an individual patient. In this way, medicines might be tailored to the individual’s cellular activation for optimal results and minimal side effects.

Additional authors of the research include Yao Lu, Qiong Xue, Markus R. Eisele, Endah S. Sulistijo, and Lin Han of Yale; Kara Brower of IsoPlexis; and El-ad David Amir and Dana Pe’er of Columbia University.

This research resulted from a project funded through the NIH Program “Library of Integrated Network-based Cellular Signatures” for which Fan and Miller-Jensen were principal investigators. It was also supported by the DFCI Physical Oncology Cancer Center where Fan has been directing its Single Cell Analysis core.