Multi-functional barcoding enables high resolution study of clonal dynamics (ATAC-seq)

The remarkable evolutionary capacity of cancer poses major challenges to current therapeutic efforts, and results from the vast clonal heterogeneity and ability of individual cancer cells to adapt to diverse selective pressures. More complete mechanistic understanding of the basis of therapeutic resistance has been limited by difficulties in coupling information regarding genetic and epigenetic alterations present within individual cells to their respective transcriptomic and functional outputs. To this end, we developed a novel high-complexity expressed barcode system, ClonMapper, that integrates DNA barcoding with single-cell RNA-sequencing and clonal isolation to characterize thousands of clones within a mixed cancer cell population. In applying this system to the chronic lymphocytic leukemia cell line HG3 in the setting of resistance to fludarabine-based chemotherapy, we discover pretreatment sub-populations with distinct expression profiles (i.e. upregulated Wnt, Notch and CXCR4 signaling) that not only confer distinct treatment survivorship trajectories, but also provide the basis for long-term clonal equilibrium between co-existing clones in the absence of treatment. Characterization of individual clones comprising these sub-populations revealed remarkable genetic heterogeneity and the persistence of unique transcriptomic signatures throughout treatment exposure. These data reveal the diverse clonal characteristics and therapeutic responses of a heterogeneous cancer cell population and highlight the unprecedented resolution that can be achieved using ClonMapper. Overall design: Bulk ATAC-seq of FACS-sorted subpopulations of HG3 cell line