Abstract PR05: Single-cell RNA sequencing reveals phenotypic plasticity of drug tolerant clonal populations of cancer cells

Abstract

Abstract The notion that most cancers are ecosystems of evolving clones is now widely accepted. Hence, residual neoplastic cells that persist following treatment constitute an ideal evolutionary niche from which recurrent cancers arise and are a major obstacle to the successful treatment of human cancers. We have identified that TGFβ signaling contribute to drug resistance by coordinately repressing transcription and translation of p53 in precancerous and cancer cells. However, the molecular events accompanying the evolution towards a drug tolerant phenotype are not well understood. Indeed, severe cellular stress might generate a subpopulation of reversibly stress-tolerant cells under conditions that are lethal to the majority of the population. Drug tolerance attributed to heterogeneity of gene expression within the population to ensure survival of a minority under stress. To address these questions, we developed an experimental paradigm to generate and propagate populations of paclitaxel tolerant precancerous (MCF10A) and metastatic cancer (MDA-MB-231) cells that can resume proliferation after 1 week of consecutive treatment with lethal doses of the drug . We found that drug-tolerant cells reacquired Paclitaxel-sensitivity after expansion. Then we performed whole transcriptome sequencing to identify single nucleotide variants (SNVs) and gene expression profiles at the single-cell level associated with the evolution towards a reversible drug tolerant state in cancer cells. We evaluated untreated, stress-arrested and proliferating drug-tolerant cells from a small (n<64) clonal population. In addition to sequencing the mRNA of single cells, we also performed DNA and RNA sequencing of populations of untreated and drug-tolerant cells to aid in the identification of RNA variants. We performed differential gene expression profiling for single cells and population cells of the three groups to identify the transcriptional stress response and cytotoxic effects of Paclitaxel on gene expression. We find that within untreated, stress-arrested and drug-tolerant cell groups, specific transcriptional programs were enacted while generating high heterogeneity between single cells within and between groups. Drug-tolerant cells contained specific RNA variants encoded by genes involved in microtubule organization and stabilization as well as cell adhesion and cell surface signaling. Unexpectedly, Drug-tolerant cells from a single progenitor cell rapidly reacquired high degree of heterogeneity in gene expression similar to untreated cells, within a few doublings, while uniquely expressing SNVs and genes such as Integrin alpha 6, the histone demethylase KDM5A, and the IGF-1 receptor. Thus, single cell analyses reveal the dynamics of the response to stress in terms of cell-specific SNVs driving heterogeneity, the survival of a minority population through generation of specific genes and RNA variants and the efficient reconversion of drug-tolerant cells back to normalcy, suggesting that such heterogeneity within cancer cell populations might provide evolutionary advantage to increase the pool of drug tolerant cells poising a prospective path towards drug resistance. This abstract is also presented as poster B68. Citation Format: Fernando J. Lopez-Diaz, Mei-Chong Wendy Lee, Muhammad A. Tariq, Yelena Dayn, Shahid Y. Khan, Charlie Vaske, Nader Pourmand, Beverly M. Emerson. Single-cell RNA sequencing reveals phenotypic plasticity of drug tolerant clonal populations of cancer cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR05.