Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing

Significance Tumor cells are heterogeneous, and much variation occurs at the single-cell level, which may contribute to therapeutic response. Here, we studied drug resistance dynamics in a model of tolerance with a metastatic breast cancer cell line by leveraging the power of single-cell RNA-Seq technology. Drug-tolerant cells within a single clone rapidly express high cell-to-cell transcript variability, with a gene expression profile similar to untreated cells, and the population reacquires paclitaxel sensitivity. Our gene expression and single nucleotide variants analyses suggest that equivalent phenotypes are achieved without relying on a unique molecular event or fixed transcriptional programs. Thus, transcriptional heterogeneity might ensure survival of cancer cells with equivalent combinations of gene expression programs and/or single nucleotide variants. The acute cellular response to stress generates a subpopulation of reversibly stress-tolerant cells under conditions that are lethal to the majority of the population. Stress tolerance is attributed to heterogeneity of gene expression within the population to ensure survival of a minority. We performed whole transcriptome sequencing analyses of metastatic human breast cancer cells subjected to the chemotherapeutic agent paclitaxel at the single-cell and population levels. Here we show that specific transcriptional programs are enacted within untreated, stressed, and drug-tolerant cell groups while generating high heterogeneity between single cells within and between groups. We further demonstrate that drug-tolerant cells contain specific RNA variants residing in genes involved in microtubule organization and stabilization, as well as cell adhesion and cell surface signaling. In addition, the gene expression profile of drug-tolerant cells is similar to that of untreated cells within a few doublings. Thus, single-cell analyses reveal the dynamics of the stress response in terms of cell-specific RNA variants driving heterogeneity, the survival of a minority population through generation of specific RNA variants, and the efficient reconversion of stress-tolerant cells back to normalcy.