Multifunctional gRNA Barcodes Reveal Diverse Adaptive and Evolutionary Dynamics in Response to Cancer Therapy

Tumor expansion and chemoresistance are driven by the evolutionary dynamics of heterogeneous cancer cell populations. Beyond genetic variation, significant intratumoral epigenetic variation and plasticity of cell states underlie these heterogeneous phenotypes. Clonal fitness dynamics have been quantified by heritable DNA barcodes; however, it has not been possible to also analyze the gene expression states of lineages of interest ^1,2. In monitoring the emergence of chemoresistant cell populations, it is critical to identify and compare surviving cell lineages, to determine the different adaptive trajectories towards resistance. To address this challenge, we developed a multifunctional barcode lineage tracing tool, Control of Lineages by Barcode Enabled Recombinant Transcription (COLBERT) that allows for i) simultaneous measurement of clonal fitness and lineage-resolved single-cell RNA-sequencing of high diversity populations to characterize and determine lineages of interest and ii) isolation of specific lineages, as a starting point for downstream molecular and functional live-cell analyses ³. Here we show the capacity of this platform to isolate lineages in cancer cell lines and primary patient derived cells. We also analyze lineage-resolved single-cell transcriptome profiles for chronic lymphocytic lymphoma (CLL) cells treated with the first-line chemotherapeutic, fludarabine. The ability to concurrently track clonal fitness dynamics and generate lineage-specific genomic and transcriptomic data over longitudinal studies will provide unprecedented insight into cancer adaptation and other diseases with an evolutionary basis.

1. Bhang H. E. et al. Studying clonal dynamics in response to cancer therapy using high-complexity barcoding. Nat Med, 21, 440-8 (2015).
2. Hata A. N. et al. Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition. Nat Med. 22, 262-269. (2016)
3. Al’Khafaji, A. & Brock, A. Control of lineage-specific gene expression by functionalized gRNA barcodes. 178897. (2017).