Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells (original) (raw)

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Data deposits

Final transcriptome data has been deposited in the European Genome-phenome Archive (www.ebi.ac.uk/ega) under accession number EGAS00001001310.

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Acknowledgements

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

Author notes

  1. Long V. Nguyen and Davide Pellacani: These authors contributed equally to this work.

Authors and Affiliations

  1. Terry Fox Laboratory, BC Cancer Agency, 675 West 10th Avenue, Vancouver, V5Z 1L3, British Columbia, Canada
    Long V. Nguyen, Davide Pellacani, Sylvain Lefort, Nagarajan Kannan, Maisam Makarem, Claire L. Cox, William Kennedy, Philip Beer, Sneha Balani, Sonja Babovic & Connie J. Eaves
  2. Department of Medical Genetics, University of British Columbia, Vancouver, V6T 2B5, British Columbia, Canada
    Davide Pellacani & Connie J. Eaves
  3. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, V6T 2B5, British Columbia, Canada
    Nagarajan Kannan, Tomo Osako & Samuel Aparicio
  4. Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, V5Z 1L3, British Columbia, Canada
    Tomo Osako & Samuel Aparicio
  5. Department of Microbiology & Immunology, Centre for High-Throughput Biology, University of British Columbia, 2125 East Mall, Vancouver, V6T 1Z4, British Columbia, Canada
    Annaick Carles, Michelle Moksa, Misha Bilenky & Martin Hirst
  6. Canada’s Michael Smith Genome Sciences Centre, BC Cancer Agency, 675 West 10th Avenue, Vancouver, V5Z 1L3, British Columbia, Canada
    Misha Bilenky & Martin Hirst
  7. Biomedical Physiology and Kinesiology, Simon Fraser University, 8888 University Drive, Burnaby, V5A 1S6, British Columbia, Canada
    Ivan Sun & Miriam Rosin
  8. Cancer Control Unit, BC Cancer Agency, 675 West 10th Avenue, Vancouver, V5Z 1L3, British Columbia, Canada
    Ivan Sun & Miriam Rosin

Authors

  1. Long V. Nguyen
  2. Davide Pellacani
  3. Sylvain Lefort
  4. Nagarajan Kannan
  5. Tomo Osako
  6. Maisam Makarem
  7. Claire L. Cox
  8. William Kennedy
  9. Philip Beer
  10. Annaick Carles
  11. Michelle Moksa
  12. Misha Bilenky
  13. Sneha Balani
  14. Sonja Babovic
  15. Ivan Sun
  16. Miriam Rosin
  17. Samuel Aparicio
  18. Martin Hirst
  19. Connie J. Eaves

Contributions

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 toConnie 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 KRAS G12D. 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 Δ_C_t 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

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Extended Data Table 2 Primary xenotransplant experiments testing different variables

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Extended Data Table 3 Histopathological characterization of the de novo tumours

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Extended Data Table 4 Details of all secondary xenotransplant experiments

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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). https://doi.org/10.1038/nature15742

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