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Phenotype molding of stromal cells in the lung tumor microenvironment

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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Fig. 1: Overview of the 52,698 single cells from lung tumors and distal non-malignant lung samples.
Fig. 2: Endothelial cell clusters.
Fig. 3: Fibroblast clusters in lungs and lung tumors.
Fig. 4: B-cell and myeloid-like cell clusters in lungs and lung tumors.
Fig. 5: T-cell clusters in lungs and lung tumors.
Fig. 6: Distribution of stromal cells in tumor samples and their role as markers of patient survival.

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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).

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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.

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Correspondence to Diether Lambrechts or Bernard Thienpont.

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Supplementary Figures 1–20 and Supplementary Tables 1–3

Reporting Summary

Supplementary Table 4

Gene expression data for all 52 clusters

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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Lambrechts, D., Wauters, E., Boeckx, B. et al. Phenotype molding of stromal cells in the lung tumor microenvironment. Nat Med 24, 1277–1289 (2018). https://doi.org/10.1038/s41591-018-0096-5

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