Project summary A major goal of cancer research is to define the composition of the tumor micro-environment (TME) across individuals. Once measured, differences in TME composition can be correlated with prognosis, targeted by therapy, and used to test or generate novel therapeutic hypotheses. Our appreciation of TME complexity was significantly advanced with the development of single cell RNA-Sequencing (scRNA-Seq). But scRNA-Seq remains expensive, noisy at the level of single cells, and has a slow turn-around time (typically weeks). scRNA- Seq also typically analyzes only 1000s of cells per sample. As a result, scRNA-Seq is not practical for deep profiling of large patient or animal cohorts, or for routine hypothesis-testing in cancer research. Faster and more scalable alternatives to scRNA-Seq are flow cytometry (FC) and Cytometry by Time of Flight (CyToF) but these methods do not resolve the complexity seen in the TME by scRNA-Seq. Thus, there is an unmet need for rapid, sensitive, highly-multiplexed TME profiling. The focus of this grant is to address this unmet need by advancing a versatile and novel `micro-capsule' technology. Capsules represent an evolution of droplet microfluidics, which is a mature technology for carrying out single cell genomic assays in nanoliter-scale compartments, isolated by oil. Capsules overcome severe technical limitations of water-in-oil droplets: their fragility to handling, and their complete isolation by immiscible oil. By contrast, capsules are resilient, semi-permeable compartments that can be dispersed and processed in any aqueous biological buffer. Prior to this proposal, we optimized capsules to retain cellular mRNA and DNA, while simultaneously enabling rapid exchange of salts, enzymes, primers and probes with the surrounding medium. We have now shown that capsules enable multi-step reactions and serial analyses on single cells and specifically on surface proteins and mRNA molecules. This in turn enables rapid, versatile, highly-multiplexed cytometry. In this R33 we will benchmark and optimize two related capsule-derived methods: the first, “CapFlow”, implements robust multiplexed mRNA flow cytometry with rapid capsule-based signal amplification. The second, “CapCycle”, extends CapFlow to quantifying the abundance of ≥50 gene transcripts and cell surface proteins, by replacing flow cytometry with cyclic imaging of immobilized capsules. With these methods, capsules will enable sensitive, versatile, rapid, low-cost, highly-multiplexed phenotyping of tumor heterogeneity. Thus, this proposal fills an important analytical gap, and develops a versatile microfluidic technology with long-term potential to improve biological assays on single biomolecules and cells.

Project Narrative This proposal develops a novel method for carrying out multiplexed measurements on large numbers of single cells. The methods developed will allow researchers to leverage new comprehensive “atlases" of tumor cells for a wide range of studies from evaluating the effects of new drug treatments, to identifying tumor cells that predict patient outcomes. Within this advanced development grant, we move from established technical capabilities to two mature fully-working methods for single cell analysis.