(1058-C) Novel Platform for Full-Scale Single-Cell Biology through Laser Particle Barcoding of Individual Cells

Abstract: To address complex diseases such as cancer, neurodegenerative disorders and auto-immunity, researchers are turning towards new single-cell analysis tools that can resolve the cellular heterogeneity responsible for disease pathogenesis and treatment resistance. These tools include flow cytometry and single-cell sequencing, which obtain high-dimensional molecular profiles for each cell. However, these tools provide only a snapshot of cellular properties within each modality. The inability to measure single cells over time, across modalities, and at scale is a major bottleneck for single-cell analysis.

Here, we describe a breakthrough platform that overcomes this bottleneck through laser particle (LP) barcoding of individual cells. LPs are micron-sized semiconductor particles that emit narrowband laser light ( < 0.5nm) in the infrared wavelengths (1100-1600nm). Multiple LPs with randomly selected wavelengths tagged to a cell via antibody binding links a high-fidelity optical barcode to the cell that can be read-out quickly and non-destructively, allowing it to be repeatedly identified among a sample of millions of cells across multiple separate measurements. This represents orders-of-magnitude improvement in encoding capacity compared with earlier optical barcoding technologies.

As a first application, we show integration of this barcoding platform to flow cytometry for time-resolved, high-dimensional analysis of immune cells. We developed a new optical system employing a 1064 nm pump laser and high-throughput infrared spectrometer that identifies each LP-barcoded cell within a few microseconds and records the barcode along with its fluorescence and scattering parameters. The flow cytometer also incorporates a specially designed fluidic system to enable reliable and gentle collection of measured cells without excessive dilution by sheath fluid. When the collected sample runs through the cytometer again, additional measured parameters are added to those from the previous run on a single-cell level according to matching LP barcodes, thereby multiplying the dimensionality and depth of the full dataset.

We applied this system in an in vitro model of T cell activation and exhaustion relevant to immunotherapy development. One of the major challenges facing immunotherapies is the variability in patient response and inability to predict treatment efficacy. Using our method, we identified novel subsets of cells with distinct kinetic patterns in expression of PD1, LAG3 and CD69 that correlate to functional phenotypes (cytokine secretion). We expect this novel platform will give access to new biological insights, especially in the study of heterogeneous processes in immunology and immune-oncology.