Tong et al. Cancer Commun (2018) 38:56 https://doi.org/10.1186/s40880-018-0326-8 Cancer Communications ORIGINAL ARTICLE Open Access Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells Mengying Tong1†, Ziqian Deng1,2†, Mengying Yang1, Chang Xu1, Xiaolong Zhang1, Qingzheng Zhang1, Yuwei Liao1, Xiaodi Deng1, Dekang Lv1, Xuehong Zhang1, Yu Zhang1, Peiying Li1, Luyao Song1, Bicheng Wang2,3, Aisha Al‑Dherasi1, Zhiguang Li1* and Quentin Liu1,2* Abstract Background: Breast cancer stem cells (BCSCs) are considered responsible for cancer relapse and drug resistance. Understanding the identity of BCSCs may open new avenues in breast cancer therapy. Although several discover‑ ies have been made on BCSC characterization, the factors critical to the origination of BCSCs are largely unclear. This study aimed to determine whether genomic mutations contribute to the acquisition of cancer stem-like phenotype and to investigate the genetic and transcriptional features of BCSCs. Methods: We detected potential BCSC phenotype-associated mutation hotspot regions by using whole-genome sequencing on parental cancer cells and derived serial-generation spheres in increasing order of BCSC frequency, and then performed target deep DNA sequencing at bulk-cell and single-cell levels. To identify the transcriptional pro‑ gram associated with BCSCs, bulk-cell and single-cell RNA sequencing was performed. Results: By using whole-genome sequencing of bulk cells, potential BCSC phenotype-associated mutation hotspot regions were detected. Validation by target deep DNA sequencing, at both bulk-cell and single-cell levels, revealed no genetic changes specifcally associated with BCSC phenotype. Moreover, single-cell RNA sequencing showed profound transcriptomic variability in cancer cells at the single-cell level that predicted BCSC features. Notably, this transcriptomic variability was enriched during the transcription of 74 genes, revealed as BCSC markers. Breast cancer patients with a high risk of relapse exhibited higher expression levels of these BCSC markers than those with a low risk of relapse, thereby highlighting the clinical signifcance of predicting breast cancer prognosis with these BCSC markers. Conclusions: Transcriptomic variability, not genetic mutations, distinguishes BCSCs from non-BCSCs. The identifed 74 BCSC markers have the potential of becoming novel targets for breast cancer therapy. Keywords: Breast cancer, Cancer stem cell, Genomics, Sequencing, Transcriptomics Background initiation, progression, and drug resistance [1–5]. Tere- Traditional breast cancer therapies that target bulk cell fore, approaches targeting BCSCs have important clini- populations often have substantial short-term efects, cal implications [6]. Although several discoveries have and the existence of breast cancer stem cells (BCSCs) is been made on BCSC characterization [7–9], the origin a major barrier for achieving curability. BCSCs are cells of BCSCs is still unclear. Currently, two models are usu- that have the ability to self-renew, and they are consid- ally proposed to explain BCSCs. Te frst model [10, 11] ered responsible for key aspects of tumors, such as tumor is clonal evolution, in which tumor cells progressively accumulate mutations, some of which confer the ability *Correspondence: [email protected]; [email protected] of self-renewal and allow tumor cells with these muta- †Mengying Tong and Ziqian Deng contributed equally to this work tions to become BCSCs and out-compete other tumor 1 Center of Genome and Personalized Medicine, Institute of Cancer Stem cells. Tis model is a canonical hardwired BCSC hierar- Cell, Dalian Medical University, Dalian 116044, Liaoning, P. R. China Full list of author information is available at the end of the article chy, and BCSC dedication is largely defned by intrinsic © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tong et al. Cancer Commun (2018) 38:56 Page 2 of 16 genetic properties. In the second model [12, 13], BCSCs sphere was sucked up by a micro-pipette, dissociated do not necessarily acquire mutations and are instructed with a small amount of trypsin-EDTA, and mechanically by dedicated niche signals following competition dynam- dispersed. Te resulting single cells were then re-sus- ics. Breast cancer cells can be reprogrammed into BCSCs pended in serum-free medium. Te spheres were passed through plasticity in tumor microenvironment. every 7–10 days, and only spheres bigger than 50 μm in To determine the factors critical to the origination of diameter were included in the analyses. BCSCs, we assumed that genetic mutations contrib- utes to the acquisition of cancer stem-like phenotypes, ALDEFLUOR assay by fuorescence activated cell sorting e.g., BCSCs specifcally carry heritable genetic changes. (FACS) We then tested this hypothesis using next-generation Te ALDEFLUOR kit (STEMCELL, Vancouver, British sequencing (NGS) analysis, including whole-genome Columbia, Canada, Cat. 01700) was used for isolating sequencing (WGS), target deep DNA sequencing, and the cell population with high aldehyde dehydrogenase transcriptome sequencing at both bulk-cell and single- (ALDH) enzymatic activity. Cells were suspended in an cell levels. ALDEFLUOR assay bufer containing ALDH substrate 6 bodipy aminoacetaldehyde (BAAA, 1 mol/L per 1 × 10 Materials and methods cells) and incubated for 45 min at 37 °C. As negative con- Cell culture trols, for each example of cells, an aliquot was treated Te human breast cancer cell line MDA-MB-231 was with 50 mmol/L diethylaminobenzaldehyde (DEAB), a obtained from the American Type Culture Collection specifc ALDH inhibitor. Te ALDH-positive subpopula- (ATCC). Te cell line was authenticated at ATCC before tion was isolated by FACS. purchase by the standard short tandem repeat DNA typing and cultured in its standard medium as recom- Transwell invasion assay mended by ATCC. Parental cells (MDA-MB-231) were obtained by trypsini- zation and resuspended in pure DMEM. Spheres bigger Sphere formation assay than 50 μm were obtained through a 40-μm cell strainer Te single-cell suspension was obtained by trypsiniza- (Meilun Biotechnology, Dalian, Liaoning, China). Ten tion. Clumped cells were excluded with a 40-μm sieve. the spheres were centrifuged (1000 rpm, 5 min), dissoci- Single cells were plated in ultra-low attachment 6-well ated with trypsin-EDTA, and mechanically dispersed in plates (Corning, NY, USA) at a low density (1000 cells/ pure DMEM. For every chamber, 30,000 cells were placed well). Te cells were maintained in serum-free Dulbecco’s onto 1% matrigel (BD Biosciences)-coated membrane modifed eagle medium/nutrient mixture F12 (DMEM/ in the upper chamber (24-well insert, 8 μm, Corning, F12, Gibco, Waltham, MA, USA) supplemented with Cat. 3422). Medium with 10% fetal bovine serum (FBS, B27 (Invitrogen, Waltham, MA, USA), 20 ng/mL epider- Gibco) was used as an attractant in the lower cham- mal growth factor (EGF, Sigma, Darmstadt, Germany), ber. After being incubated for 24 h, cells that invaded and 20 ng/mL basic fbroblast growth factor (bFGF, BD through the membrane were fxed with 4% paraformal- Biosciences, Franklin Lakes, NJ, USA) for 7–10 days. For dehyde and stained with 0.1% crystal violet. Te stained sphere passage, the spheres were collected by centrifuga- cell images were captured under a microscope (Olym- tion (1000 rpm, 5 min), dissociated with trypsin-ethylene pus, Tokyo, Japan), and cells were counted for fve ran- diamine tetraacetic acid (EDTA), and mechanically dis- dom felds at ×10 magnifcation. Results are presented as persed. Te resulting single cells were then centrifuged mean ± standard deviation from at least three independ- (1000 rpm, 5 min) to remove the enzyme and re-sus- ent experiments. pended in serum-free medium. Te spheres were passed every 7–10 days, and only spheres bigger than 50 μm in RNA extraction, reverse transcription‑PCR, diameter were included in the analyses. and quantitative real‑time PCR Total RNA was extracted by using TRIzol reagent (Life Serial sphere formation assay Technologies, Waltham, MA, USA). cDNA was gen- Te single-cell suspension of MDA-MB-231 cells was erated by reverse transcription-PCR using EasyScript obtained by trypsinization. Cells were seeded in an ultra- One-Step gDNA Removal and cDNA Synthesis Super- low attachment 96-well plate (1 cell/well). Te cells were Mix Kit (TransGen Biotech, Beijing, China) according to maintained in serum-free DMEM/F12 supplemented the manufacturer’s instructions. Quantitative real-time with B27, and 20 ng/mL EGF, 20 ng/mL bFGF. Only wells polymerase chain reaction (qRT-PCR) was performed that initially contained a single cell were used for subse- by using the chamQ Universal SYBR qPCR Master Mix quent studies. For sphere passage, the single cell-derived (Vazyme, Najing, Jiangsu, China) in a MX3000p cycler Tong et al. Cancer Commun (2018) 38:56 Page 3 of 16 (Stratagene, La Jolla, CA, USA). Changes of mRNA lev- Reads