Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells


Most human breast cancers have diversified genomically and biologically by the time they become clinically evident1,2,3. Early events involved in their genesis and the cellular context in which these events occur have thus been difficult to characterize. Here we present the first formal evidence of the shared and independent ability of basal cells and luminal progenitors, isolated from normal human mammary tissue and transduced with a single oncogene (KRASG12D), to produce serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice4. DNA barcoding5,6 of the initial cells revealed a dramatic change in the numbers and sizes of clones generated from them within 2 weeks, and the first appearance of many ‘new’ clones in tumours passaged into secondary recipients. Both primary and secondary tumours were phenotypically heterogeneous and primary tumours were categorized transcriptionally as ‘normal-like’. This system challenges previous concepts that carcinogenesis in normal human epithelia is necessarily a slow process requiring the acquisition of multiple driver mutations. It also presents the first description of initial events that accompany the genesis and evolution of malignant human mammary cell populations, thereby contributing new understanding of the rapidity with which heterogeneity in their properties can develop.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: De novo formation of tumours from normal human mammary BCs and LPs.
Figure 2: Phenotypic heterogeneity of primary and secondary de novo tumours.
Figure 3: Barcoding reveals a complex clonal landscape of primary and secondary tumours.
Figure 4: Early changes in clone growth in cells transduced with KRASG12D only.

Accession codes

Data deposits

Final transcriptome data has been deposited in the European Genome-phenome Archive ( under accession number EGAS00001001310.


  1. 1

    Stephens, P. J. et al. Oslo Breast Cancer Consortium (OSBREAC). The landscape of cancer genes and mutational processes in breast cancer. Nature 486, 400–404 (2012)

    CAS  Article  Google Scholar 

  2. 2

    Sørlie, T. et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc. Natl Acad. Sci. USA 98, 10869–10874 (2001)

    Article  ADS  Google Scholar 

  3. 3

    Curtis, C. et al. METABRIC Group. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 486, 346–352 (2012)

    CAS  Article  Google Scholar 

  4. 4

    Eirew, P. et al. A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability. Nature Med. 14, 1384–1389 (2008)

    CAS  Article  Google Scholar 

  5. 5

    Nguyen, L. V. et al. DNA barcoding reveals diverse growth kinetics of human breast tumour subclones in serially passaged xenografts. Nature Commun. 5, 5871 (2014)

    CAS  Article  ADS  Google Scholar 

  6. 6

    Nguyen, L. V. et al. Clonal analysis via barcoding reveals diverse growth and differentiation of transplanted mouse and human mammary stem cells. Cell Stem Cell 14, 253–263 (2014)

    CAS  Article  Google Scholar 

  7. 7

    Kannan, N. et al. Glutathione-dependent and -independent oxidative stress-control mechanisms distinguish normal human mammary epithelial cell subsets. Proc. Natl Acad. Sci. USA 111, 7789–7794 (2014)

    CAS  Article  ADS  Google Scholar 

  8. 8

    Kannan, N. et al. The luminal progenitor compartment of the normal human mammary gland constitutes a unique site of telomere dysfunction. Stem Cell Rep. 1, 28–37 (2013)

    CAS  Article  Google Scholar 

  9. 9

    Eirew, P. et al. Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution. Nature 518, 422–426 (2015)

    CAS  Article  ADS  Google Scholar 

  10. 10

    Parker, J. S. et al. Supervised risk predictor of breast cancer based on intrinsic subtypes. J. Clin. Oncol. 27, 1160–1167 (2009)

    Article  Google Scholar 

  11. 11

    Paquet, E. R. & Hallett, M. T. Absolute assignment of breast cancer intrinsic molecular subtype. J. Natl Cancer Inst. 107, 357 (2015)

    Article  Google Scholar 

  12. 12

    Keller, P. J. et al. Defining the cellular precursors to human breast cancer. Proc. Natl Acad. Sci. USA 109, 2772–2777 (2012)

    CAS  Article  ADS  Google Scholar 

  13. 13

    Kreso, A. et al. Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer. Science 339, 543–548 (2013)

    CAS  Article  ADS  Google Scholar 

  14. 14

    Bhang, H. E. et al. Studying clonal dynamics in response to cancer therapy using high-complexity barcoding. Nature Med. 21, 440–448 (2015)

    CAS  Article  Google Scholar 

  15. 15

    Logan, A. C. et al. Factors influencing the titer and infectivity of lentiviral vectors. Hum. Gene Ther. 15, 976–988 (2004)

    CAS  Article  Google Scholar 

  16. 16

    Imren, S. et al. High-level β-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells. J. Clin. Invest. 114, 953–962 (2004)

    CAS  Article  Google Scholar 

  17. 17

    Eirew, P., Stingl, J. & Eaves, C. J. Quantitation of human mammary epithelial stem cells with in vivo regenerative properties using a subrenal capsule xenotransplantation assay. Nature Protocols 5, 1945–1956 (2010)

    CAS  Article  Google Scholar 

  18. 18

    Lakhani, S. R., Ellis, I. O., Schnitt, S. J., Tan, P. H. & van de Vijver, M. J. WHO Classification of Tumours of the Breast 4th edn, Ch. 2 (World Health Organization, 2012)

  19. 19

    Tsuda, H., Akiyama, F., Kurosumi, M., Sakamoto, G. & Watanabe, T. ; Japan National Surgical Adjuvant Study of Breast Cancer (NSAS-BC) Pathology Section. Establishment of histological criteria for high-risk node-negative breast carcinoma for a multi-institutional randomized clinical trial of adjuvant therapy. Jpn. J. Clin. Oncol. 28, 486–491 (1998)

    CAS  Article  Google Scholar 

  20. 20

    Elston, C. W. & Ellis, I. O. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology 19, 403–410 (1991)

    CAS  Article  Google Scholar 

  21. 21

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Butterfield, Y. S. et al. JAGuaR: junction alignments to genome for RNA-seq reads. PLoS One 9, e102398 (2014)

    Article  ADS  Google Scholar 

  23. 23

    Li, H. et al. 1000 Genome Project Data Processing Subgroup. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009)

    Article  Google Scholar 

  24. 24

    Gascard, P. et al. Epigenetic and transcriptional determinants of the human breast. Nature Commun. 6, 6351 (2015)

    CAS  Article  ADS  Google Scholar 

Download references


We thank D. Wilkinson, G. Edin and M. Hale for technical support, E. Bovill, J. Boyle, S. Bristol, P. Gdalevitch, A. Seal, J. Sproul and N. van Laeken for access to discarded reduction mammoplasty tissue, T. Nielsen and N. Poulin for discussions, the Centre for Translational and Applied Genomics (BC Cancer Agency) for assistance with IHC, and T. MacDonald for assistance with rodent husbandry. This work was supported by grants from the Canadian Cancer Society Research Institute, the Canadian Breast Cancer Foundation and the Canadian Breast Cancer Research Alliance. L.V.N. received a Vanier Canada Graduate Scholarship from the Canadian Institutes of Health Research (CIHR), and N.K. was supported by a MITACS Elevate Fellowship. T.O. was supported by a Molecular Oncologic Pathology Fellowship from CIHR and the Terry Fox Foundation, and by grants from the Sumitomo Life Welfare and Culture Foundation, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, and the Takashi Tsuruo Memorial Fund. S.A. is supported by a Canada Research Chair.

Author information




L.V.N., D.P. and C.J.E. designed the project, drafted the manuscript and were assisted by S.L., C.L.C., W.K. and S. Balani in performing the experiments. M.M. and M.H. oversaw the generation of sequence data, and L.V.N., D.P., A.C. and M.B. analysed it. All authors contributed to the interpretation of the results, and read and approved the manuscript.

Corresponding author

Correspondence to Connie J. Eaves.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Quantification of human cells containing different vector reporters in tumours derived from triply transduced starting populations.

a, Lentiviral constructs used. CBR-Luc, click beetle red luciferase. b, Frequencies of donor samples producing at least one tumour subrenally from BCs or LPs exposed to different combinations of oncogene-encoding vectors. c, Representative FACS profiles of a cell suspension prepared from a tumour produced from cells transduced with all three genes and sorted for human EpCAM and HLA using human-specific antibodies. d, Percentage of cells expressing different lentiviral reporters in cells maintained in vitro for 72 h after transduction, and in primary and secondary tumours (G, GFP; Y, YFP; mCh, mCherry). e, Frequencies of donor samples producing at least one tumour under various transplantation conditions using BCs or LPs transduced with KRASG12D. f, Percentages of human cells in BC- and LP-derived tumours detected by FACS on the basis of their expression of human EpCAM and/or HLA.

Extended Data Figure 2 Molecular characterization of the tumours.

a, Examples of PCR evidence of all three vectors in DNA extracts obtained from a subset of tumours analysed with vector-specific primers. b, A representative Sanger sequencing chromatograph showing the expected point mutations in the tumour cells analysed. c, PCR evidence of the three vectors in FACS-purified doubly and triply transduced cells. d, e, Representative images of H&E- and IHC-stained sections of primary tumours (d, arising from cells transplanted subcutaneously) and secondary tumours (e, all arising subcutaneously) derived from either BCs or LPs. Scale bar, 50 μm. f, Relative expression (negative ΔCt values, mean ± s.e.m.) of gene transcripts typically associated with mesenchymal/basal or epithelial/luminal phenotypes, or associated with proliferation and cell growth.

Extended Data Figure 3 Threshold set for detection of barcoded clones for the two sequencing runs from which barcode data were acquired.

a, The relationship between the fractional read value (FRV) and the number of cells per clone. Spiked-in controls only and spiked-in controls added to experimental samples are shown as red and grey points, respectively. The shaded grey box represents distribution of false positive barcodes. b, Sensitivity and specificity data for controls compared with experimental samples for different sized clones.

Extended Data Figure 4 Clonal analyses of primary barcoded tumours.

a, Numbers of clones and frequencies of T-CFCs in primary tumours. b, Relative clone size distributions for individual primary tumours grouped by the cell type initially manipulated and the oncogene(s) used. Each column represents a single tumour. Each rectangle represents one clone. Its relative clone size is indicated by the shade of green, and its proportional contribution within each tumour is indicated by its length on the y axis.

Extended Data Figure 5 Clonal analyses of secondary barcoded tumours.

a, Numbers of clones and frequencies of T-CFCs in secondary tumours. b, Relative clone size distributions for individual secondary tumours grouped by the cell type initially manipulated. Each column represents a single tumour. Each rectangle represents one clone. Its relative clone size is indicated by the shade of green, and its proportional contribution within each tumour is indicated by its length on the y axis. c, Clonal landscape of replicate secondary tumours generated from single primary tumours in two separate experiments. Clones present in sibling tumours are shown above one another and unique clones are shown in the same horizontal bar. Increasing clone sizes are indicated by a grey intensity scale. d, Numbers of clones and T-CFC frequencies of combined primary and secondary tumours.

Extended Data Figure 6 Clonal analyses of transduced cells transplanted subrenally after 2 weeks in vivo.

a, Number of clones and frequency of CFCs in xenografts of transduced cells assessed after 2 weeks in vivo. b, Relative clone size distributions of individual 2-week transplants grouped by the cell type initially manipulated and the oncogene(s) used. Each column represents a single transplant. Each rectangle represents one clone. Its relative clone size is indicated by the shade of green, and its proportional contribution within each tumour is indicated by its length on the y axis.

Extended Data Table 1 Primary xenotransplant experiments performed subrenally with EP pellets and irradiated fibroblasts
Extended Data Table 2 Primary xenotransplant experiments testing different variables
Extended Data Table 3 Histopathological characterization of the de novo tumours
Extended Data Table 4 Details of all secondary xenotransplant experiments

Supplementary information

Supplementary Table 1

This file contains Luciferase activity values. (XLSX 11 kb)

Supplementary Table 2

This file contains lists of differentially expressed genes. (XLSX 113 kb)

Supplementary Table 3

This file shows Antibodies used for FACS and IHC analyses. (XLSX 9 kb)

Supplementary Table 4

This file contains Primers used for RT-qPCR. (XLSX 8 kb)

Supplementary Table 5

This file contains quality control report of RNA-seq data. (XLSX 15 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nguyen, L., Pellacani, D., Lefort, S. et al. Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells. Nature 528, 267–271 (2015).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing