GSE112274
Concurrent single cell RNA and targeted DNA sequencing on a microfluidics device allows high-throughput identification of transcriptome and genome alterations during the development of tyrosine kinase inhibitor resistance in EGFR-mutant lung cancer cells
Within the clonal heterogeneity of a tumor, the co-measurement of both genomic and transcriptomic signatures in single cells is of fundamental importance to accurately assess how the genetic information, for instance a somatic mutation, is correlated with a transcriptomic and cancer-related phenotype. However, there is currently no microfluidics-based high-throughput assay available that allows this analysis. To resolve this, we developed a new method for concurrent sequencing of the transcriptome and targeted genomic regions (CORTAD-seq) within the same single cell on a microfluidic high-throughput platform. Its operation is simple, at moderate reagent cost, with excellent data quality and good accessibility for a broad scientific community. We implemented this new method to dissect the cellular consequences of mutations that result in resistance to targeted therapy. Using different tyrosine kinase inhibitor (TKI) resistant PC9 lung cancer cell lines as our study model, we detected signatures of TKI resistant genes as well as expansion of T790M mutation and EGFR amplification in the resistant cells. Interestingly, we identify transcriptomic characteristics for resistance that are not dependent on the presence of T790M, the most common resistance mutation in EGFR, indicating that phenotypically similar resistance scenarios can emerge without T790M mutation and other genomic alterations or epigenetic modifications are required for the manifestation of the resistance phenotype. Cumulatively, the application of this method allows accurate evaluation of the genomic and transcriptomic changes in cancer during development of drug resistance at single cell level in uncovering potential new biomarker and therapeutic targets in resistant cancer. Overall design: A new method has been developed for concurrent single cell transcriptome and targeted genomic sequencing on a microfluidic platform. We utilized three PC9 lines that are responsive or resistant to TKI treatment as our study model. We implemented analysis pipeline for single cell RNAseq and targeted DNAseq studies. 546 single cells of PC9 cells were sequenced using the CoRTAD protocol. 507 cells with both the scRNAseq data and targeted DNA data passing the QC were selected for downstream analysis (20 cells in DMSO condition were also excluded from the downstream analysis).