Abstract

Cancer cells are embedded in the tumor microenvironment (TME), a complex ecosystem of stromal cells. Here, we present a 52,698-cell catalog of the TME transcriptome in human lung tumors at single-cell resolution, validated in independent samples where 40,250 additional cells were sequenced. By comparing with matching non-malignant lung samples, we reveal a highly complex TME that profoundly molds stromal cells. We identify 52 stromal cell subtypes, including novel subpopulations in cell types hitherto considered to be homogeneous, as well as transcription factors underlying their heterogeneity. For instance, we discover fibroblasts expressing different collagen sets, endothelial cells downregulating immune cell homing and genes coregulated with established immune checkpoint transcripts and correlating with T-cell activity. By assessing marker genes for these cell subtypes in bulk RNA-sequencing data from 1,572 patients, we illustrate how these correlate with survival, while immunohistochemistry for selected markers validates them as separate cellular entities in an independent series of lung tumors. Hence, in providing a comprehensive catalog of stromal cells types and by characterizing their phenotype and co-optive behavior, this resource provides deeper insights into lung cancer biology that will be helpful in advancing lung cancer diagnosis and therapy.

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Acknowledgements

We thank M. De Waegeneer, T. Van Brussel, G. Peuteman, E. Vanderheyden and B. Tembuyser for technical assistance. This work was supported by a VIB TechWatch Grant to D.L. and B.T., Foundation Against Cancer grants to S.Aerts (2016-070) and E.W., ERC Consolidator Grants to S.Aerts (724226_cis-CONTROL) and D.L. (CHAMELEON), Funds for Research - Flanders grants to H.D. (1701018N) and D.L. (G065615N), an Austrian Science Fund (FWF) grant to A.P. (J3730-B26) and KU Leuven grants to D.L. and S.Aerts (PFV/10/016 SymBioSys), and to B.T. (BOFZAP).

Author information

Affiliations

  1. VIB Center for Cancer Biology, Leuven, Belgium

    • Diether Lambrechts
    • , Bram Boeckx
    • , Ayse Bassez
    • , Andreas Pircher
    • , Peter Carmeliet
    •  & Bernard Thienpont
  2. Laboratory for Translational Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium

    • Diether Lambrechts
    • , Bram Boeckx
    •  & Ayse Bassez
  3. Respiratory Oncology Unit (Pneumology) and Leuven Lung Cancer Group, University Hospitals KU Leuven, Leuven, Belgium

    • Els Wauters
    •  & Johan Vansteenkiste
  4. Laboratory of Pneumology, Department of Chronic Diseases, Metabolism and Ageing, KU Leuven, Leuven, Belgium

    • Els Wauters
    •  & Johan Vansteenkiste
  5. Laboratory for Computational Biology, Department of Human Genetics, KU Leuven, Leuven, Belgium

    • Sara Aibar
    •  & Stein Aerts
  6. VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium

    • Sara Aibar
    • , Oliver Burton
    • , Adrian Liston
    •  & Stein Aerts
  7. Histopathology Expertise Center, VIB Leuven Center for Cancer Biology, VIB, Leuven, Belgium

    • David Nittner
  8. Department of Oncology, KU Leuven, Leuven, Belgium

    • David Nittner
  9. Laboratory of Genetics of Autoimmunity, Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium

    • Oliver Burton
    •  & Adrian Liston
  10. Department of Thoracic Surgery, University Hospitals KU Leuven, Leuven, Belgium

    • Herbert Decaluwé
  11. Department of Chronic Diseases, Metabolism and Ageing, KU Leuven, Leuven, Belgium

    • Herbert Decaluwé
    •  & Paul De Leyn
  12. Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, KU Leuven, Leuven, Belgium

    • Andreas Pircher
    •  & Peter Carmeliet
  13. Translational Cell & Tissue Research, Department of Imaging & Pathology, KU Leuven, Leuven, Belgium

    • Kathleen Van den Eynde
    • , Birgit Weynand
    •  & Erik Verbeken
  14. State Key Laboratory of Ophthalmology, Zhongsan Ophthalmic Center, SunYat-Sen University, Guangzhou, China

    • Peter Carmeliet
  15. Laboratory for Functional Epigenetics, Department of Human Genetics, KU Leuven, Leuven, Belgium

    • Bernard Thienpont

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Contributions

D.L. and B.T. designed and supervised the study and wrote the manuscript. E.W. supervised sample collection and clinical annotation, with important help from H.D., A.P., K.V.d.E., B.W., E.V., P.D.L. and J.V. B.T. performed data analysis, with significant contributions from B.B., S.Ai., S.Ae. and A.B. D.N. and B.T. performed immunohistofluorescence analyses. O.B., A.L., P.C. and S.Ae. contributed critical data interpretation. All of the authors have read or provided comments on the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Diether Lambrechts or Bernard Thienpont.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–20 and Supplementary Tables 1–3

  2. Reporting Summary

  3. Supplementary Table 4

    Gene expression data for all 52 clusters

  4. Supplementary Table 5

    Gene expression data for tumor-derived and non-malignant lung-tissue-derived cells, per cluster having >100 cells from both sources

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DOI

https://doi.org/10.1038/s41591-018-0096-5