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A marker-independent lineage-tracing system to quantify clonal dynamics and stem cell functionality in cancer tissue

Nature Protocols volume 14, pages 2648–2671 (2019)Cite this article

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Abstract

Lineage tracing is a powerful tool that can be used to uncover cell fates. Here, we describe a novel method for the quantitative analysis of clonal dynamics in grafted cancer tissues. The protocol involves the preparation and validation of cells for lineage tracing, establishment of grafts and label induction, analysis of clone-size distribution and fitting of the experimental data to a mathematical tumor growth model. In contrast to other lineage-tracing strategies, the method described here assesses stem cell functionality and infers tumor expansion dynamics independently of molecular markers such as putative cancer stem cell (CSC)-specific genes. The experimental system and analytical framework presented can be used to quantify clonal advantages that specific mutations provide, in both the absence and presence of (targeted) therapeutic agents. This protocol typically takes ~20 weeks to complete from cell line selection to inference of growth dynamics, depending on the grafted cancer growth rate.

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Fig. 1: Schematic overview of the protocol.
Fig. 2: Lentiviral transduction of cell culture with pLV-indLS2 construct.
Fig. 3: Generation and selection of pLV-indLS2+ single-cell clones.
Fig. 4: Validation of stability and neutrality of single-cell clone.
Fig. 5: Determination of the optimal in vivo tamoxifen concentration.
Fig. 6: Tumor isolation and sectioning.
Fig. 7: Unbiased in vivo lineage tracing of HT55 cells.
Fig. 8: App for clone-size data inference.
Fig. 9: CloneFit app for clone-size data inference, instructions.
Fig. 10: Data interpretation: model parameters.
Fig. 11: Stochastic tumor growth model.

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Data availability

Source data for Figs. 7, 8, 9 and 10 have been provided as Supplementary Data. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

Code availability

The custom clone-size quantification software can be downloaded from https://github.com/dmmiedema/Clone-Sizes-From-Image. The CloneFit app can be downloaded from https://github.com/dmmiedema/CloneFit. We have made available the programming code for the tumor growth model on GitHub: https://github.com/dmmiedema/Tumor-Growth-Model.

References

  1. Todaro, M. et al. Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell Stem Cell 1, 389–402 (2007).

    Article  CAS  Google Scholar 

  2. O’Brien, C. A., Pollett, A., Gallinger, S. & Dick, J. E. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445, 106–110 (2007).

    Article  Google Scholar 

  3. Ricci-Vitiani, L. et al. Identification and expansion of human colon-cancer-initiating cells. Nature 445, 111–115 (2007).

    Article  CAS  Google Scholar 

  4. Lenos, K. J. et al. Stem cell functionality is microenvironmentally defined during tumour expansion and therapy response in colon cancer. Nat. Cell Biol. 20, 1193–1202 (2018).

    Article  CAS  Google Scholar 

  5. Vermeulen, L. & Snippert, H. J. Stem cell dynamics in homeostasis and cancer of the intestine. Nat. Rev. Cancer 14, 468–480 (2014).

    Article  CAS  Google Scholar 

  6. Shimokawa, M. et al. Visualization and targeting of LGR5+ human colon cancer stem cells. Nature 545, 187–192 (2017).

    Article  CAS  Google Scholar 

  7. De Sousa e Melo, F. et al. A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer. Nature 543, 676–680 (2017).

    Article  Google Scholar 

  8. Flanagan, D. J., Hodder, M. C. & Sansom, O. J. Microenvironmental cues in cancer stemness. Nat. Cell Biol. 20, 1102–1104 (2018).

    Article  CAS  Google Scholar 

  9. Rodriguez, E. et al. Versatile and enhanced tumour modelling in mice via somatic cell transduction. J. Pathol. 232, 449–457 (2014).

    Article  CAS  Google Scholar 

  10. Vermeulen, L. et al. Defining stem cell dynamics in models of intestinal tumor initiation. Science 342, 995–998 (2013).

    Article  CAS  Google Scholar 

  11. Mascre, G. et al. Distinct contribution of stem and progenitor cells to epidermal maintenance. Nature 489, 257–262 (2012).

    Article  CAS  Google Scholar 

  12. Rulands, S. et al. Universality of clone dynamics during tissue development. Nat. Phys. 14, 469–474 (2018).

    Article  CAS  Google Scholar 

  13. Zhang, Z. & Lutz, B. Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein. Nucleic Acids Res. 30, e90 (2002).

    Article  Google Scholar 

  14. Fumagalli, A. et al. A surgical orthotopic organoid transplantation approach in mice to visualize and study colorectal cancer progression. Nat. Protoc. 13, 235–247 (2018).

    Article  CAS  Google Scholar 

  15. Roper, J. et al. Colonoscopy-based colorectal cancer modeling in mice with CRISPR-Cas9 genome editing and organoid transplantation. Nat. Protoc. 13, 217–234 (2018).

    Article  CAS  Google Scholar 

  16. Pavia-Jimenez, A., Tcheuyap, V. T. & Brugarolas, J. Establishing a human renal cell carcinoma tumorgraft platform for preclinical drug testing. Nat. Protoc. 9, 1848–1859 (2014).

    Article  CAS  Google Scholar 

  17. Lorenzatti Hiles, G. et al. A surgical orthotopic approach for studying the invasive progression of human bladder cancer. Nat. Protoc. 14, 738–755 (2019).

    Article  CAS  Google Scholar 

  18. Kim, M. P. et al. Generation of orthotopic and heterotopic human pancreatic cancer xenografts in immunodeficient mice. Nat. Protoc. 4, 1670–1680 (2009).

    Article  CAS  Google Scholar 

  19. Huynh, H., Soo, K. C., Chow, P. K., Panasci, L. & Tran, E. Xenografts of human hepatocellular carcinoma: a useful model for testing drugs. Clin. Cancer Res. 12, 4306–4314 (2006).

    Article  CAS  Google Scholar 

  20. van der Heijden, M. et al. Spatiotemporal regulation of clonogenicity in colorectal cancer xenografts. Proc. Natl. Acad. Sci. USA 116, 6140–6145 (2019).

    Article  Google Scholar 

  21. Driessens, G., Beck, B., Caauwe, A., Simons, B. D. & Blanpain, C. Defining the mode of tumour growth by clonal analysis. Nature 488, 527–530 (2012).

    Article  CAS  Google Scholar 

  22. Frick, P. L., Paudel, B. B., Tyson, D. R. & Quaranta, V. Quantifying heterogeneity and dynamics of clonal fitness in response to perturbation. J. Cell. Physiol. 230, 1403–1412 (2015).

    Article  CAS  Google Scholar 

  23. Gillespie, D. T. Exact stochastic simulation of coupled chemical reactions. J. Phys. Chem. 81, 2340–2361 (1977).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank L.E. Nijman and M.C. Lecca for their help with the animal procedures. This work was supported by the Academic Medical Center (Amsterdam), The New York Stem Cell Foundation, Cancer Research UK, and grants from KWF (UVA2011-4969, UVA2014-7245 and 10529), the Maurits en Anna de Kock Stichting (2015-2), Worldwide Cancer Research (14-1164), the Maag Lever Darm Stichting (MLDS-CDG 14-03), the European Research Council (ERC-StG 638193) and ZonMw (Vidi 016.156.308) to L.V. L.V. is a New York Stem Cell Foundation—Robertson Investigator.

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Author notes

  1. These authors contributed equally: Kristiaan J. Lenos, Sophie C. Lodestijn.

Authors and Affiliations

  1. LEXOR, Center for Experimental and Molecular Medicine, Cancer Center Amsterdam and Amsterdam Gastroenterology & Metabolism, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands

    Kristiaan J. Lenos, Sophie C. Lodestijn, Maarten F. Bijlsma, Daniël M. Miedema & Louis Vermeulen

  2. Oncode Institute, Amsterdam, The Netherlands

    Kristiaan J. Lenos, Sophie C. Lodestijn, Maarten F. Bijlsma, Daniël M. Miedema & Louis Vermeulen

  3. Preclinical Imaging, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA

    Scott K. Lyons

Authors
  1. Kristiaan J. Lenos
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  5. Daniël M. Miedema
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Contributions

K.J.L. and S.C.L. performed the experiments; D.M.M. and L.V. developed the quantitative models; D.M.M. developed the software; K.J.L., D.M.M., S.C.L., M.F.B. and L.V. analyzed the data; S.K.L. contributed reagents; K.J.L., D.M.M., M.F.B. and L.V. conceived and designed the research; K.J.L., S.C.L., D.M.M., M.F.B. and L.V. wrote the manuscript; L.V. directed the research. All authors approved the content of the manuscript.

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Correspondence to Daniël M. Miedema or Louis Vermeulen.

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The authors declare no competing interests.

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Lenos, K. J. et al. Nat. Cell Biol. 20, 1193–1202 (2018): https://doi.org/10.1038/s41556-018-0179-z

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Lenos, K.J., Lodestijn, S.C., Lyons, S.K. et al. A marker-independent lineage-tracing system to quantify clonal dynamics and stem cell functionality in cancer tissue. Nat Protoc 14, 2648–2671 (2019). https://doi.org/10.1038/s41596-019-0194-y

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