Abstract A09: Single-cell RNA sequencing reveals phenotypic plasticity of drug tolerant, clonal populations of cancer cells
Authors: Lopez-Diaz Fernando , Lee Mei-Chong Wendy , Tariq Muhammad , Khan Shahid , Dayn Yelena , Vaske Charlie , Pourmand Nader , Emerson Beverly M. 
Published: May 2015

Cancer recurrence and metastatic tumors might arise not only from residual primary tumor cells but even from precancerous cells that are able to spread and seed distant organs. Thus, residual pre-neoplastic and neoplastic cells that persist following treatment constitute an ideal evolutionary niche for cancer evolution. We have investigated the role of the TGF-beta; signaling pathway, which is widely known as a pro-metastatic in late stages of tumor progression, in the context of the cellular response to genotoxic stress induced by chemotherapy drugs. We identified that TGF-beta signaling contribute to drug resistance by repressing the DNA damage response through a coordinate repression of transcription and translation of p53 and the stress response in precancerous and cancer cells. Indeed, TGF-beta/Smad signaling enacts an intrinsic survival pathway in metastatic cancer cells. However, the molecular events accompanying the evolution towards a drug tolerant phenotype are not well understood. Indeed, severe cellular stress might generate or select a subpopulation of reversibly stress-tolerant cells under conditions that are lethal to the majority of the population. Thus, drug tolerance could be 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&lt;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. Interestingly, despite of the high degree of heterogeneity, the whole gene expression profiles, of each cell rather than small gene sets can identify the response of cells to taxol. 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.

Citation Format: Fernando Lopez-Diaz, Mei-Chong Wendy Lee, Muhammad Tariq, Shahid Khan, Yelena Dayn, 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 AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A09.