Clonal transcriptomics identifies mechanisms of chemoresistance and empowers rational design of combination therapies

Intratumour heterogeneity provides a fuel for cancer evolution enabling tumour cell populations to adapt to selective pressures imposed by the tumour microenvironment or therapeutic interventions. To uncover functionally heterogeneous subpopulations of tumour cells with different treatment sensitivity and to understand how clonal lineage-dependent transcriptomic diveristy contributes, we developed WILDseq, a hihg-complexity expressed barcode library for simultanous mapping of each cells' clonal identity and transcriptional states. We developed WILD-seq, a novel barcoding toolkit combining lineage tracing and single-cell RNA sequencing (scRNA-seq). Our WILD-seq toolkit contains our lentiviral barcoding system, experimental workflow and methods, and computational pipelines. Single cells are uniquely labelled with a hertiable WILD-seq barcode which can be captured as an expressed transcript by scRNA-seq on an oligo(dT)-capturing platform. The barcode cassette consists out of two 12 nt barcodes each with a hamming distance of 5 allowing to correct up to 2 errors per barcode. The overall library consists of ~1,600,000 potential barcode combinations. To ensure robust WILD-seq barcode capture, we further developed a one-step PCR enrichment strategy to specifically enrich WILD-seq barcode transcripts out of the pooled amplified cDNA of the transcriptome. We applied our WILD-seq approach to study the nature of clonal response to therapeutic pressure in two syngeneic mammary carcinoma models (4T1 and D2A1-m2) using a taxane-based chemotherapy and a first-in-class small-molecule inhibitor of BET bromodaomines JQ1. In total, we analysed 75,509 transcriptomes including tumour cells and cells from the tumour microenvironment with specific expression of the WILD-seq barcode in the tumour cell clusters. Our work provides a full pipeline from barcoding to data analysis and guidelines on how to customize the protocol allowing researchers to apply the tool for their scientific question.