P2.01-001 Enrichment-Free, Rapid Metabolic Assay for Detection of Tumor Cells in Pleural Effusion and Pheripheral Blood

Abstract

Current methods for circulating tumor cell (CTC) detection are mostly include an enrichment step and the subsequent immunostaining-based identification of CTCs by epithelial and leukocytes markers. These methods are limited by loss and damage of CTCs during the enrichment and fail to determine the malignancy and drug targets of putative CTCs. We describe an enrichment-free, metabolic-based assay for rapid detection of tumor cells in the pleural effusion and peripheral blood samples. All nucleated cells are plated on microwell chips that contain 200,000 addressable microwells. These cells are labeled with a fluorescent anti-CD45 antibody (leukocyte marker), a fluorescent glucose analog (2-NBDG) and a dead cell marker (EthD-1). The microwell chips are imaged by a computerized high-speed fluorescent microscope in three colors and the bright filed. A computation algorithm analyzes the images and identify candidate tumor cells that are viable, CD45 negative, and exhibit high glucose uptake (EthD-1-/CD45-/2-NBDGhigh). A micromanipultor is then utilized to retrieve single tumor cells based on recorded addresses for single-cell sequencing. EthD-1-/CD45-/2-NBDG>100 cells are identified as candidate tumor cells. Single-cell sequencing based on a small panel of driver oncogenes (EGFR, KRAS, PIK3CA) shows that >60% of candidate tumor cells are true tumor cells harboring mutations in the panel. Single-cell whole exome sequencing results show all candidate tumor cells have high mutation frequency in dirver oncogenece and tumor suppressors from Qiagen's Human Lung Cancer Panel. Meanwhile, CD45-/EthD-1-/2-NBDG>100 tumor cells show heterogenieity in cytokeratin (CK) expression, and only ∼40% of these tumor cells are found CK positive. We have developed a simple and functional-based method to rapidly identify tumor cells with high glucose uptake in the clinical liquid samples without enrichment. These tumor cells are addressable, enabling single-cell manipulation and sequencing. Clinical feasibility of this assay has been established by testing samples from a cohort of patients.