To combat cancer, new tests are needed that can more accurately monitor the status of the disease. Non-invasive circulating tumor cell (CTC) analysis has held great promise for the detection, serial-monitoring, and targeted treatment of cancer. Hampering this promise is the extreme difficulty of isolating and enumerating CTCs, which constitute a tiny fraction of all circulating cells and can have a range of biomarker profile depending on the cancer subtype or epithelial-mesenchymal transition status of the cell. Although much work has been done to develop tools for CTC enrichment and analysis, existing approaches lack the ability to enrich CTCs beyond 1% purity. The presence of thousands of contaminating leukocytes in enriched CTC populations produced by current methods prevents accurate CTC enumeration and confounds downstream sequencing and molecular analysis of the CTCs. These problems have proven to be major limitations that have prevented wider dissemination of CTC analysis in the fight against cancer. To realize the full potential of CTCs in diagnostic applications, new methods for analyzing the many thousands of cells remaining following initial CTC enrichment are needed. Specifically, a platform is needed that can unambiguously, and with minimal bias, identify the full spectrum of CTC subtypes and enable additional molecular characterization on the individual cells that are recovered. Information like this has never before been available to researchers or clinicians and could revolutionize the way in which such liquid biopsies are used in the clinical setting to inform treatment decisions. Th focus of this SBIR proposal is to apply a novel droplet-based microfluidic technology we have developed, PACS, to address the shortcomings of current approaches to CTC identification and molecular characterization following initial enrichment from blood. The PACS platform allows single-cells to be identified and sorted at high-throughput levels without fixation or antibodies. Specifically, this method will interrogate >100,000 individual cells using multiplexed PCR reactions to identify CTCs based on the presence of transcripts, genetic mutations, non-coding RNAs or other nucleic acid biomarkers. PACS will also simultaneously isolate user-defined populations of cells, either pooled or individually, for additional downstream transcriptomic or genomic analysis. Our principal aims for this project will be to improve upon the capabilities of the system we've already developed and configure it for rare CTC identification and analysis, where extreme sensitivity is essential. This research will lay the foundation for a complete CTC diagnostic workflow and ready us for Phase 2 where we will validate the use of PACS as a back-end to existing CTC enrichment platforms with clinical blood samples from cancer patients.