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Reply from Authors re: Xue-Ru Wu. Attention to Detail by Single-cell sequencing. Eur Urol. In press.

By: Zhao Yanga b , Song Wuc, Zhiming Caid and Chong Lib

European Urology, Volume 71 Issue 1, January 2017, Pages 15-16

Published online: 01 January 2017

Abstract Full Text Full Text PDF (99 KB)

Refers to article:

Single-cell Sequencing Reveals Variants in ARID1A, GPRC5A and MLL2 Driving Self-renewal of Human Bladder Cancer Stem Cells

Zhao Yang, Chong Li, Zusen Fan, Hongjie Liu, Xiaolong Zhang, Zhiming Cai, Liqin Xu, Jian Luo, Yi Huang, Luyun He, Chunxiao Liu and Song Wu

Accepted 20 June 2016

January 2017 (Vol. 71, Issue 1, pages 8 - 12)

Refers to article:

Attention to Detail by Single-cell Sequencing

Xue-Ru Wu

January 2017 (Vol. 71, Issue 1, pages 13 - 14)

We thank Dr. Xue-Ru Wu [1], one of the most prominent experts in the field of urinary bladder diseases, for his remarks on our recent paper describing single-cell sequencing of human bladder cancer stem cells (BCSCs) [2]. We agree with the majority of the points he raised, and would like to put some notes forward for additional consideration.

Bladder cancer is the most common urinary malignancy worldwide and has two typical types: non–muscle-invasive bladder cancer (NMIBC; ∼60%) and muscle-invasive bladder cancer (MIBC; ∼20%) [3]. Clinically, cystoscopy is the main diagnostic method, while transurethral resection combined with intravesical chemotherapeutic agents and radical cystectomy are the main therapeutic approaches; targeted therapy methods are available [4]. Resulting from distinct genetic lesions and signaling activation within tumors, NMIBC has good prognosis but a high recurrence rate, whereas MIBC patients have a poor prognosis [4]. Thus, bladder cancer is one of the most expensive types of tumor to deal with in terms of clinical therapeutics [3].

As Dr. Wu mentioned, high-throughput sequencing has revealed genetic variations and pathogenesis for many tumor types, including bladder cancer. For example, researchers have found that the PI3K/AKT/mTOR pathway, the RTK/MAPK pathway, and chromatin regulatory genes are frequently altered in bladder cancer samples [5] and [6]. With the rapid development of high-throughput sequencing, researchers were not satisfied with paired sequencing of normal and tumor samples, and further analyzed bladder cancer samples of a unique type or under specialized conditions according to their interest. For instance, whole-exome sequencing of bladder cancer samples from 37 individuals, including 20 primary and 17 recurrent samples, demonstrated that MLL was exclusively mutated in recurrent bladder cancer samples, which rendered cancer cells insensitive to chemotherapeutics [7]. In addition, whole-exome sequencing of 72 bladder cancer samples including 16 matched pairs of primary or advanced tumors collected before and after chemotherapy indicated that chemotherapy-treated urothelial carcinoma was enriched in clonal mutations involving L1CAM, the APOBEC family, and integrin signaling pathways [8].

The origin of bladder cancer is a hot topic in this field, and increasing evidence supports the notion that CSCs are responsible for tumor initiation, metastasis, and drug resistance [9]. However, CSCs account for only a small proportion of tumor cells whose specific mutations are difficult to uncover via mixed cell sequencing. The recent development of single-cell DNA and RNA sequencing made it possible to perform profiling and analysis of the genome and transcriptome of a single cell, which is especially important for some rare cell types [2]. In our study of single-cell sequencing of BCSCs, phylogenetic analysis indicated that the cells originated from either bladder epithelial stem cells (BESCs) or bladder cancer non-stem cells (BCNSCs) [2]. We agree that the sample size (n = 3) was small in our study, although we sequenced a relatively high number of qualified single cells (n = 59). We are currently trying to gain insight into the origin of BCSCs via single-cell RNA sequencing, as Dr. Xu also proposed [1]. Moreover, the few shared somatic mutations between the two cancer samples showing BESC origin may be caused by their different tumor stage and grade or the heterogeneity between the two samples. To address this issue, more fastidious sample collection could be helpful, such as using NMIBC-only tumors for sequencing, to reduce the differences between samples.

We also greatly appreciate Dr. Xu's idea to reconcile our findings with previous data from mouse lineage tracing experiments that suggested divergent progenitor cells for bladder cancer variants [1]. Van Batavia et al [10] found that intermediate cells gave rise primarily to papillary lesions, whereas K5 basal cells were likely progenitors of carcinoma in situ (CIS), muscle-invasive lesions, and squamous cell carcinoma (SCC), depending on the genetic background. Treating BCSCs and BCNSCs as a whole, phylogenetic analysis indicated that bladder cancer cells were derived from BESCs, which corresponded to K5 basal cells in the Van Batavia study [10]. From this standpoint, our results support the notion that the majority of bladder cancers originate from BESCs/K5 basal cells in patients. To further integrate the data from single-cell sequencing of human bladder cancers with data from mouse lineage tracing assays, it would be worth collecting the four types of bladder cancer (papillary, CIS, muscle-invasive lesions, and SCC) together with basal, intermediate, and umbrella cells from corresponding normal bladder and perform single-cell sequencing for all these cell types. Moreover, we should also take into consideration that the chemical carcinogenesis mouse model based on N-butyl-N-(4-hydroxybutyl) nitrosamine may differ from the physiological tumorigenesis of bladder cancer in humans.

Put much more effort into uncovering the origin and development of bladder cancer will help to deepen our understanding of bladder cancer biology, to develop novel target therapies, and to eventually improve patient prognosis.

Conflicts of interest: The authors have nothing to disclose.

Funding/Support and role of the sponsor: This project was supported by the National Natural Science Foundation of China (Grant No. 81472413, 81672956 and 81602644).


  • [1] X.R. Wu. Attention to detail by single-cell sequencing. Eur Urol. 2017;71:13-14
  • [2] Z. Yang, C. Li, H. Liu, et al. Single-cell sequencing reveals variants in ARID1A. GPRC5A and MLL2 driving self-renewal of human bladder cancer stem cells. Eur Urol. 2017;71:8-12
  • [3] M.A. Knowles, C.D. Hurst. Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity. Nat Rev Cancer. 2014;15:25-41 Crossref
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  • [5] Y. Gui, G. Guo, Y. Huang, et al. Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder. Nat Genet. 2011;43:875-878 Crossref
  • [6] Cancer Genome Atlas Research Network. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature. 2014;507:315-322
  • [7] S. Wu, Z. Yang, R. Ye, et al. Novel variants in MLL confer to bladder cancer recurrence identified by whole-exome sequencing. Oncotarget. 2016;7:2629-2645
  • [8] Faltas BM, Prandi D, Tagawa ST, et al. Clonal evolution of chemotherapy-resistant urothelial carcinoma. Nat Genet. In press.
  • [9] C. Li, Y. Du, Z. Yang, et al. GALNT1-mediated glycosylation and activation of sonic hedgehog signaling maintains the self-renewal and tumor-initiating capacity of bladder cancer stem cells. Cancer Res. 2016;76:1273-1283
  • [10] J. Van Batavia, T. Yamany, A. Molotkov, et al. Bladder cancers arise from distinct urothelial sub-populations. Nat Cell Biol. 2014;16:982-991 Crossref


a Core Facility for Protein Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

b CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

c Department of Urological Surgery, The Affiliated Luohu Hospital of Shenzhen University, Shenzhen University, Shenzhen, China

d Department of Urological Surgery, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, China

e Beijing Jianlan Institute of Medicine, Beijing, China

Corresponding authors.

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