Skip to main content

Thank you for visiting nature.com. 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.

  • Article
  • Published:

Single-cell dissection of transcriptional heterogeneity in human colon tumors

Nature Biotechnology volume 29, pages 1120–1127 (2011)Cite this article

Subjects

Abstract

Cancer is often viewed as a caricature of normal developmental processes, but the extent to which its cellular heterogeneity truly recapitulates multilineage differentiation processes of normal tissues remains unknown. Here we implement single-cell PCR gene-expression analysis to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single (n = 1) cell, we demonstrate that the transcriptional diversity of cancer tissues is largely explained by in vivo multilineage differentiation and not only by clonal genetic heterogeneity. Finally, we show that the different gene-expression programs linked to multilineage differentiation are strongly associated with patient survival. We develop two-gene classifier systems (KRT20 versus CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard ratios superior to those of pathological grade and comparable to those of microarray-derived multigene expression signatures.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Single-cell PCR gene-expression analysis of human normal colon epithelium.
Figure 2: Single-cell PCR gene-expression analysis of human colon tumor tissues.
Figure 3: Analysis of a monoclonal human colon cancer xenograft obtained from injection of a single (n = 1) cell in NOD/SCID/IL2Rγ−/− mice.
Figure 4: KRT20 and top-crypt genes can be used as prognostic markers in colorectal cancer patients.

Similar content being viewed by others

References

  1. Reya, T., Morrison, S.J., Clarke, M.F. & Weissman, I.L. Stem cells, cancer, and cancer stem cells. Nature 414, 105–111 (2001).

    Article  CAS  Google Scholar 

  2. Jordan, C.T., Guzman, M.L. & Noble, M. Cancer stem cells. N. Engl. J. Med. 355, 1253–1261 (2006).

    Article  CAS  Google Scholar 

  3. Dalerba, P., Cho, R.W. & Clarke, M.F. Cancer stem cells: models and concepts. Annu. Rev. Med. 58, 267–284 (2007).

    Article  CAS  Google Scholar 

  4. Shackleton, M., Quintana, E., Fearon, E.R. & Morrison, S.J. Heterogeneity in cancer: cancer stem cells versus clonal evolution. Cell 138, 822–829 (2009).

    Article  CAS  Google Scholar 

  5. Campbell, L.L. & Polyak, K. Breast tumor heterogeneity: cancer stem cells or clonal evolution? Cell Cycle 6, 2332–2338 (2007).

    Article  CAS  Google Scholar 

  6. Marusyk, A. & Polyak, K. Tumor heterogeneity: causes and consequences. Biochim. Biophys. Acta 1805, 105–117 (2010).

    CAS  Google Scholar 

  7. Kirkland, S.C. Clonal origin of columnar, mucous, and endocrine cell lineages in human colorectal epithelium. Cancer 61, 1359–1363 (1988).

    Article  CAS  Google Scholar 

  8. Odoux, C. et al. A stochastic model for cancer stem cell origin in metastatic colon cancer. Cancer Res. 68, 6932–6941 (2008).

    Article  CAS  Google Scholar 

  9. Vermeulen, L. et al. Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proc. Natl. Acad. Sci. USA 105, 13427–13432 (2008).

    Article  CAS  Google Scholar 

  10. Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011).

    Article  CAS  Google Scholar 

  11. Warren, L., Bryder, D., Weissman, I.L. & Quake, S.R. Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc. Natl. Acad. Sci. USA 103, 17807–17812 (2006).

    Article  CAS  Google Scholar 

  12. Guo, G. et al. Resolution of cell fate decisions revealed by single-cell gene-expression analysis from zygote to blastocyst. Dev. Cell 18, 675–685 (2010).

    Article  CAS  Google Scholar 

  13. White, A.K. et al. High-throughput microfluidic single-cell RT-qPCR. Proc. Natl. Acad. Sci. USA 108, 13999–14004 (2011).

    Article  CAS  Google Scholar 

  14. Jiao, Y.F., Nakamura, S., Sugai, T., Yamada, N. & Habano, W. Serrated adenoma of the colorectum undergoes a proliferation versus differentiation process: new conceptual interpretation of morphogenesis. Oncology 74, 127–134 (2008).

    Article  Google Scholar 

  15. Wielenga, V.J. et al. Expression of CD44 in Apc and Tcf mutant mice implies regulation by the WNT pathway. Am. J. Pathol. 154, 515–523 (1999).

    Article  CAS  Google Scholar 

  16. Prall, F. et al. CD66a (BGP), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues. J. Histochem. Cytochem. 44, 35–41 (1996).

    Article  CAS  Google Scholar 

  17. Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).

    Article  CAS  Google Scholar 

  18. Becker, L., Huang, Q. & Mashimo, H. Immunostaining of Lgr5, an intestinal stem cell marker, in normal and premalignant human gastrointestinal tissue. Scientific World Journal 8, 1168–1176 (2008).

    Article  CAS  Google Scholar 

  19. Merlos-Suarez, A. et al. The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. Cell Stem Cell 8, 511–524 (2011).

    Article  CAS  Google Scholar 

  20. Sahoo, D., Dill, D.L., Gentles, A.J., Tibshirani, R. & Plevritis, S.K. Boolean implication networks derived from large scale, whole genome microarray datasets. Genome Biol. 9, R157 (2008).

    Article  Google Scholar 

  21. Hoglund, P. et al. Mutations of the down-regulated in adenoma (DRA) gene cause congenital chloride diarrhoea. Nat. Genet. 14, 316–319 (1996).

    Article  CAS  Google Scholar 

  22. Fischer, H., Stenling, R., Rubio, C. & Lindblom, A. Differential expression of aquaporin 8 in human colonic epithelial cells and colorectal tumors. BMC Physiol. 1, 1 (2001).

    Article  CAS  Google Scholar 

  23. Koslowski, M., Sahin, U., Dhaene, K., Huber, C. & Tureci, O. MS4A12 is a colon-selective store-operated calcium channel promoting malignant cell processes. Cancer Res. 68, 3458–3466 (2008).

    Article  CAS  Google Scholar 

  24. Noah, T.K., Kazanjian, A., Whitsett, J. & Shroyer, N.F. SAM pointed domain ETS factor (SPDEF) regulates terminal differentiation and maturation of intestinal goblet cells. Exp. Cell Res. 316, 452–465 (2010).

    Article  CAS  Google Scholar 

  25. Gregorieff, A. et al. The ets-domain transcription factor Spdef promotes maturation of goblet and paneth cells in the intestinal epithelium. Gastroenterology 137, 1333–1345 (2009).

    Article  CAS  Google Scholar 

  26. van der Flier, L.G. et al. Transcription factor achaete scute-like 2 controls intestinal stem cell fate. Cell 136, 903–912 (2009).

    Article  CAS  Google Scholar 

  27. Ezhkova, E. et al. Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells. Cell 136, 1122–1135 (2009).

    Article  CAS  Google Scholar 

  28. Park, I.K. et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 423, 302–305 (2003).

    Article  CAS  Google Scholar 

  29. Sangiorgi, E. & Capecchi, M.R. Bmi1 is expressed in vivo in intestinal stem cells. Nat. Genet. 40, 915–920 (2008).

    Article  CAS  Google Scholar 

  30. Zeng, Y.A. & Nusse, R. Wnt proteins are self-renewal factors for mammary stem cells and promote their long-term expansion in culture. Cell Stem Cell 6, 568–577 (2010).

    Article  CAS  Google Scholar 

  31. Beider, K., Abraham, M. & Peled, A. Chemokines and chemokine receptors in stem cell circulation. Front. Biosci. 13, 6820–6833 (2008).

    Article  CAS  Google Scholar 

  32. Jensen, K.B. et al. Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis. Cell Stem Cell 4, 427–439 (2009).

    Article  CAS  Google Scholar 

  33. Dalla-Favera, R., Wong-Staal, F. & Gallo, R.C. Onc gene amplification in promyelocytic leukaemia cell line HL-60 and primary leukaemic cells of the same patient. Nature 299, 61–63 (1982).

    Article  CAS  Google Scholar 

  34. Hoey, T. et al. DLL4 blockade inhibits tumor growth and reduces tumor-initiating cell frequency. Cell Stem Cell 5, 168–177 (2009).

    Article  CAS  Google Scholar 

  35. Park, S.Y., Gonen, M., Kim, H.J., Michor, F. & Polyak, K. Cellular and genetic diversity in the progression of in situ human breast carcinomas to an invasive phenotype. J. Clin. Invest. 120, 636–644 (2010).

    Article  CAS  Google Scholar 

  36. Losi, L., Baisse, B., Bouzourene, H. & Benhattar, J. Evolution of intratumoral genetic heterogeneity during colorectal cancer progression. Carcinogenesis 26, 916–922 (2005).

    Article  CAS  Google Scholar 

  37. Dalerba, P. et al. Phenotypic characterization of human colorectal cancer stem cells. Proc. Natl. Acad. Sci. USA 104, 10158–10163 (2007).

    Article  CAS  Google Scholar 

  38. Oien, K.A. Pathologic evaluation of unknown primary cancer. Semin. Oncol. 36, 8–37 (2009).

    Article  CAS  Google Scholar 

  39. Lugli, A., Tzankov, A., Zlobec, I. & Terracciano, L.M. Differential diagnostic and functional role of the multi-marker phenotype CDX2/CK20/CK7 in colorectal cancer stratified by mismatch repair status. Mod. Pathol. 21, 1403–1412 (2008).

    Article  CAS  Google Scholar 

  40. Sahoo, D., Dill, D.L., Tibshirani, R. & Plevritis, S.K. Extracting binary signals from microarray time-course data. Nucleic Acids Res. 35, 3705–3712 (2007).

    Article  CAS  Google Scholar 

  41. Jorissen, R.N. et al. Metastasis-associated gene expression changes predict poor outcomes in patients with dukes stage B and C colorectal cancer. Clin. Cancer Res. 15, 7642–7651 (2009).

    Article  CAS  Google Scholar 

  42. Smith, J.J. et al. Experimentally derived metastasis gene expression profile predicts recurrence and death in patients with colon cancer. Gastroenterology 138, 958–968 (2010).

    Article  CAS  Google Scholar 

  43. Guastadisegni, C., Colafranceschi, M., Ottini, L. & Dogliotti, E. Microsatellite instability as a marker of prognosis and response to therapy: a meta-analysis of colorectal cancer survival data. Eur. J. Cancer 46, 2788–2798 (2010).

    Article  CAS  Google Scholar 

  44. Bardia, A. et al. Adjuvant chemotherapy for resected stage II and III colon cancer: comparison of two widely used prognostic calculators. Semin. Oncol. 37, 39–46 (2010).

    Article  CAS  Google Scholar 

  45. Dalerba, P. et al. Reconstitution of human telomerase reverse transcriptase expression rescues colorectal carcinoma cells from in vitro senescence: evidence against immortality as a constitutive trait of tumor cells. Cancer Res. 65, 2321–2329 (2005).

    Article  CAS  Google Scholar 

  46. Ringner, M. What is principal component analysis? Nat. Biotechnol. 26, 303–304 (2008).

    Article  CAS  Google Scholar 

  47. O'Doherty, U., Swiggard, W.J. & Malim, M.H. Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J. Virol. 74, 10074–10080 (2000).

    Article  CAS  Google Scholar 

  48. Wang, G.P. et al. DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer. Nucleic Acids Res. 36, e49 (2008).

    Article  Google Scholar 

  49. Ishizawa, K. et al. Tumor-initiating cells are rare in many human tumors. Cell Stem Cell 7, 279–282 (2010).

    Article  CAS  Google Scholar 

  50. Quintana, E. et al. Efficient tumour formation by single human melanoma cells. Nature 456, 593–598 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by National Institutes of Health (NIH) grants U54-CA126524 and P01-CA139490 (to S.R.Q. and M.F.C.), the NIH Director's Pioneer Awards (to S.R.Q.) and a grant from the Ludwig foundation (to M.F.C.). P.D. was supported by a training grant from the California Institute for Regenerative Medicine (CIRM) and by a BD Biosciences Stem Cell Research Grant (Summer 2011). T.K. was supported by a fellowship from the Machiah Foundation. D.S. was supported by NIH grant K99-CA151673, by Department of Defense grant W81XWH-10-1-0500 and a grant from the Siebel Stem Cell Institute and the Thomas and Stacey Siebel Foundation. We wish to thank R. Tibshirani, D. Witten, L. Warren, R.A. White III, E. Gilbert, P. Lovelace, M. Palmor, C. Donkers and S.P. Miranda for helpful discussion and technical support in many moments during the completion of this study.

Author information

Author notes

  1. Piero Dalerba, Tomer Kalisky, Debashis Sahoo, Michael F Clarke and Stephen R Quake: These authors contributed equally to this work.

Authors and Affiliations

  1. Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California, USA

    Piero Dalerba, Debashis Sahoo, Pradeep S Rajendran, Michael E Rothenberg, Sopheak Sim, Darius M Johnston, Dalong Qian, Maider Zabala, Shigeo Hisamori, Yohei Shimono & Michael F Clarke

  2. Department of Medicine, Division of Oncology, Stanford University, Stanford, California, USA

    Piero Dalerba & Michael F Clarke

  3. Department of Bioengineering, Stanford University, Stanford, California, USA

    Tomer Kalisky, Anne A Leyrat, Jennifer Okamoto, Darius M Johnston, Norma F Neff, Jianbin Wang & Stephen R Quake

  4. Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University, Stanford, California, USA

    Michael E Rothenberg

  5. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Jennifer Okamoto, Darius M Johnston & Stephen R Quake

  6. Tissue Bank, Stanford University, Stanford, California, USA

    Janet Bueno

  7. Department of Surgery, Stanford University, Stanford, California, USA

    Andrew A Shelton & Brendan Visser

  8. Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands

    Marc van de Wetering & Hans Clevers

Authors
  1. Piero Dalerba
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tomer Kalisky
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Debashis Sahoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pradeep S Rajendran
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael E Rothenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anne A Leyrat
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sopheak Sim
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jennifer Okamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Darius M Johnston
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Dalong Qian
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Maider Zabala
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Janet Bueno
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Norma F Neff
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Jianbin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Andrew A Shelton
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Brendan Visser
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Shigeo Hisamori
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Yohei Shimono
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Marc van de Wetering
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Hans Clevers
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Michael F Clarke
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Stephen R Quake
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.D., T.K., D.S., M.F.C. and S.R.Q. conceived the study and designed the experiments. P.S.R., M.E.R., A.A.L., M.Z., N.F.N., M.v.d.W. and H.C. provided intellectual guidance in the design of selected experiments. P.D., T.K., D.S., P.S.R., A.A.L., S.S., J.O., D.M.J., D.Q., J.W., and S.H. performed the experiments. P.D., T.K., D.S., N.F.N., Y.S., M.F.C. and S.R.Q. analyzed the data and/or provided intellectual guidance in their interpretation. J.B., A.A.S. and B.V. provided samples and reagents. P.D., T.K., D.S., M.F.C. and S.R.Q. wrote the paper.

Corresponding authors

Correspondence to Michael F Clarke or Stephen R Quake.

Ethics declarations

Competing interests

S.R.Q. is a founder, consultant and shareholder of Fluidigm. S.R.Q. and M.F.C. are founders, consultants and shareholders of QuantiCell. The authors have filed a patent application based on the results described in this study.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–4, Supplementary Methods and Supplementary Figures 1–24 (PDF 19216 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dalerba, P., Kalisky, T., Sahoo, D. et al. Single-cell dissection of transcriptional heterogeneity in human colon tumors. Nat Biotechnol 29, 1120–1127 (2011). https://doi.org/10.1038/nbt.2038

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2038

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research