Single-cell analyses of transcriptional heterogeneity PNAS PLUS during drug tolerance transition in cancer cells by RNA sequencing Mei-Chong Wendy Leea,1, Fernando J. Lopez-Diazb,1, Shahid Yar Khana,2, Muhammad Akram Tariqa,3, Yelena Daync, Charles Joseph Vasked, Amie J. Radenbaugha, Hyunsung John Kima, Beverly M. Emersonb,4, and Nader Pourmanda,4 aBiomolecular Engineering Department, University of California, Santa Cruz, CA 95064; bLaboratory of Regulatory Biology, The Salk Institute for Biological Studies, La Jolla, CA 92037; cTransgenic Core Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; and dFive3 Genomics, LLC, Santa Cruz, CA 95060 Edited by Lewis T. Williams, Five Prime Therapeutics, Inc., South San Francisco, CA, and approved September 29, 2014 (received for review March 12, 2014) The acute cellular response to stress generates a subpopulation of resistance. Multiplexed single-cell quantitative PCR (qPCR) reversibly stress-tolerant cells under conditions that are lethal to assays allow expression-based analysis of up to 96 targets in the majority of the population. Stress tolerance is attributed to a single experiment (13). Recently, a few groups have demon- heterogeneity of gene expression within the population to ensure strated that RNA-Seq of single cells using NGS technology is survival of a minority. We performed whole transcriptome se- feasible, reproducible, and usable for gene expression-based quencing analyses of metastatic human breast cancer cells sub- classification of cell subpopulations (14–17). A major advantage jected to the chemotherapeutic agent paclitaxel at the single-cell of RNA-Seq in single-cell studies is that the entire transcriptome and population levels. Here we show that specific transcriptional can be surveyed, rather than a limited number of genes. DNA programs are enacted within untreated, stressed, and drug- and RNA methodologies are not mutually exclusive and can be tolerant cell groups while generating high heterogeneity between combined to generate more biologically significant information. single cells within and between groups. We further demonstrate Paclitaxel (Taxol) is a chemotherapy drug commonly used to that drug-tolerant cells contain specific RNA variants residing in treat solid cancers including breast tumors (18). This toxin targets genes involved in microtubule organization and stabilization, as CELL BIOLOGY well as cell adhesion and cell surface signaling. In addition, the microtubules to interfere with the mitotic spindle, resulting in cell gene expression profile of drug-tolerant cells is similar to that of cycle arrest and ultimately apoptosis. Paclitaxel treatment kills untreated cells within a few doublings. Thus, single-cell analyses most tumor cells but, for the residual cancer cells, the mechanisms reveal the dynamics of the stress response in terms of cell-specific of resistance are unclear (18). An important question is whether RNA variants driving heterogeneity, the survival of a minority mutations that drive drug resistance are common in a population population through generation of specific RNA variants, and the or arise from unique mutations in individual cells. efficient reconversion of stress-tolerant cells back to normalcy. Significance single cell | paclitaxel | tumor heterogeniety | drug resistance | RNA-Seq Tumor cells are heterogeneous, and much variation occurs at the major barrier to successful cancer treatment is the re- single-cell level, which may contribute to therapeutic response. Acurrence of cancer cells with acquired resistance to che- Here, we studied drug resistance dynamics in a model of toler- motherapy (1–3). However, the molecular events underlying cancer ance with a metastatic breast cancer cell line by leveraging the cell evolution toward a drug-resistant phenotype are largely un- power of single-cell RNA-Seq technology. Drug-tolerant cells known. Recent studies using next-generation sequencing (NGS) within a single clone rapidly express high cell-to-cell transcript systems have attempted to identify the genetic changes that drive variability, with a gene expression profile similar to untreated tumorigenesis and resistance to treatments (4, 5). These sequenc- cells, and the population reacquires paclitaxel sensitivity. Our ing studies have revealed that many of the resistance-imparting gene expression and single nucleotide variants analyses suggest mutations identified are different from tumor to tumor. In addition that equivalent phenotypes are achieved without relying on to heterogeneity across tumors from different patients, intratumor a unique molecular event or fixed transcriptional programs. heterogeneity adds another level of complexity. Minor sub- Thus, transcriptional heterogeneity might ensure survival of populations of cancer cells can harbor aberrations that are asso- cancer cells with equivalent combinations of gene expression ciated with resistance to therapy and tumor progression (6–8). programs and/or single nucleotide variants. Thus, treatments may be effective against the majority of the tu- mor, but a small population of resistant cells can cause the per- Author contributions: M.-C.W.L., F.J.L.-D., B.M.E., and N.P. designed research; M.-C.W.L., sistence, recrudescence, or recurrence of cancer that is refractory F.J.L.-D., S.Y.K., M.A.T., and Y.D. performed research; M.-C.W.L., F.J.L.-D., C.J.V., A.J.R., and H.J.K. analyzed data; and M.-C.W.L., F.J.L.-D., B.M.E., and N.P. wrote the paper. to further treatment. Sequencing studies on bulk tumor tissue can only identify mutations present in subpopulations of a heteroge- The authors declare no conflict of interest. neous tumor in a limited capacity. By sequencing the transcriptome This article is a PNAS Direct Submission. of single cells in depth, low abundance mutations can be detected Data deposition: The sequencing short reads data have been deposited in the Sequence Read Archive at the National Center for Biotechnology Information, www.ncbi.nlm.nih. that will facilitate identifying the drivers of drug resistance. gov/sra (accession no. SRP040309). Recent advances have enabled the analysis of DNA and RNA 1M.-C.W.L. and F.J.L.-D. contributed equally to this work. within a single cell. The coupling of whole genome amplification 2Present address: The Wilmer Eye Institute, Johns Hopkins University, Baltimore, and DNA sequencing have allowed multiple groups to study the MD 21287. genetics of single cells, but not without significant amplification 3 – Present address: Department of Pathology, King Edward Medical University, Nelagumbad, biases (9 11). Moreover, single-cell exome sequencing con- Anarkali, Lahore, Pakistan 54000. firmed the clonal heterogeneity of a solid tumor identifying key 4To whom correspondence may be addressed. Email: [email protected] or mutations across much of the genome (12). DNA sequencing can [email protected]. identify mutations across the genome, but is unable to illuminate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. expressional differences that can contribute significantly to drug 1073/pnas.1404656111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1404656111 PNAS Early Edition | 1of10 Downloaded by guest on September 27, 2021 Here we leverage the power of single-cell RNA-Seq to identify A Ptx Ptx B Untreated single nucleotide variants (SNVs) and gene expression at the Drug free single-cell level in an insightful drug-tolerance experimental Naïve cells Ptx-Rcr cells paradigm. We evaluated three groups of cells from the human days 0 5 5+1 5 +n breast carcinoma cell line, MDA-MB-231: untreated cells, stressed cells that had been exposed to paclitaxel treatment for 5 d plus 1 d 100X drug free, and drug-tolerant cells from a small (n < 64) clonal Stressed population of cells that resumed proliferation after paclitaxel (5d Ptx +1day) treatment. In addition to sequencing the mRNA of single cells, we also performed DNA sequencing of a population of untreated Untreated Stressed Drug-Tolerant (1.0±0.5e-2) (2±1e-6) cells and RNA-Seq of a population from each of the three groups to facilitate the identification of SNVs and RNA variants. We also Single-cell (n=5-6) / populations (n>10,000) RNA- sequencing performed differential gene expression profiling for single cells and population cells of the three groups to identify the tran- 100X C MDA-MB-231 Drug- tolerant scriptional stress response and cytotoxic effects of paclitaxel on 100 (5d Ptx +22d) IC50=7 nM gene expression. 75 DT 50 Results Naïve 25 % cell number IC50=0.2 nM Generation of a Paclitaxel Tolerance Paradigm in Metastatic Human relative to control 0 Cancer Cells and Isolation of Single Cells. To investigate the mo- 1 10 0.1 40X 100 0.01 lecular events associated with the response of cancer cells to 0.001 Paclitaxel (nM) drug-treatment followed by drug withdrawal that may be po- MCF10A MDA-MB-231-DT clone tentially associated with drug tolerance, we exposed the pacli- D (5d Ptx +15d) 100 IC =0.6 nM taxel-sensitive (IC50 < 10 nM) (18) metastatic human breast 50 cancer cell line MDA-MB-231 to paclitaxel (100 nM) according 75 to the regimen diagrammed in Fig. 1A. After 5 d of drug expo- 50 DT % cell number 25 Naïve sure, most cells had died. Residual cells alive 1 d after paclitaxel relative to control IC50=0.08 nM removal were considered to be a stressed cell population, and the 0 1 200X 10 – 0.1 majority of these cells underwent apoptosis within 2 4 wk. A 100 0.01 small number of residual stressed cells resumed proliferation and 0.001 Paclitaxel (nM) established clones, and such cells were considered to be drug- B – Fig. 1. Reversible phenotypic equilibrium in response to paclitaxel in cancer tolerant cells (Fig. 1 ). A drug toxicity curve was also con- cells. (A) Regimen for expansion of paclitaxel (Ptx) stress-tolerant cells. structed using a range of paclitaxel concentrations (Fig. 1C). The Highly metastatic MDA-MB-231 naïve (yellow) cells were treated with Ptx IC50 was ∼7 nM with ∼20% viable cells.