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Research Article

Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters

Kevin J. Cheung, Veena Padmanaban, Vanesa Silvestri, Koen Schipper, Joshua D. Cohen, Amanda N. Fairchild, Michael A. Gorin, James E. Verdone, Kenneth J. Pienta, Joel S. Bader, and Andrew J. Ewald
PNAS February 16, 2016 113 (7) E854-E863; first published February 1, 2016; https://doi.org/10.1073/pnas.1508541113
Kevin J. Cheung
aDepartment of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
bDepartment of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
cDepartment of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
dTranslational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109;
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  • For correspondence: kcheung@fredhutch.org andrew.ewald@jhmi.edu
Veena Padmanaban
aDepartment of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
bDepartment of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
cDepartment of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
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Vanesa Silvestri
aDepartment of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
bDepartment of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
cDepartment of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
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Koen Schipper
aDepartment of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
bDepartment of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
cDepartment of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
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Joshua D. Cohen
aDepartment of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
bDepartment of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
cDepartment of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
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Amanda N. Fairchild
aDepartment of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
bDepartment of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
cDepartment of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
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Michael A. Gorin
eThe James Buchanan Brady Urological Institute and Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
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James E. Verdone
eThe James Buchanan Brady Urological Institute and Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
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Kenneth J. Pienta
eThe James Buchanan Brady Urological Institute and Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
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Joel S. Bader
fDepartment of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD 21205
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Andrew J. Ewald
aDepartment of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
bDepartment of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
cDepartment of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
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  • For correspondence: kcheung@fredhutch.org andrew.ewald@jhmi.edu
  1. Edited by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved December 23, 2015 (received for review April 30, 2015)

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Significance

Conventional models of cancer progression propose that single cells leave the primary tumor, enter the circulation, and seed clonal metastases. However, metastases can contain multiple clones, raising the question: How do polyclonal metastases form? We demonstrate that cancer cells seed distant organs as cohesive clusters, composed of two molecularly distinct subpopulations, whose proportions vary systematically during metastasis. We establish that collective dissemination is a frequent mechanism for metastasis and identify a molecular program in the most invasive, keratin 14+ (K14+) cancer cells, regulating cell–cell adhesion, cell–matrix adhesion, and immune evasion. We demonstrate that this metastatic phenotype is dependent upon K14 expression. Understanding the molecular basis of collective dissemination may therefore enable novel prognostics and therapies to improve patient outcomes.

Abstract

Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14+ cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14+ epithelial tumor cell clusters disseminate collectively to colonize distant organs.

  • collective invasion
  • collective dissemination
  • polyclonal metastasis
  • breast cancer
  • keratin 14

Footnotes

  • ↵1To whom correspondence may be addressed. Email: kcheung{at}fredhutch.org or andrew.ewald{at}jhmi.edu.
  • Author contributions: K.J.C., V.P., V.S., K.S., A.N.F., M.A.G., K.J.P., J.S.B., and A.J.E. designed research; K.J.C., V.P., V.S., K.S., J.D.C., A.N.F., M.A.G., and J.E.V. performed research; K.J.C., J.D.C., M.A.G., J.E.V., K.J.P., and J.S.B. contributed new reagents/analytic tools; K.J.C., V.P., V.S., K.S., J.D.C., A.N.F., M.A.G., J.E.V., J.S.B., and A.J.E. analyzed data; and K.J.C. and A.J.E. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: RNA-seq data have been uploaded to the NCBI Sequence Read Archive, www.ncbi.nlm.nih.gov/sra (accession nos. SRP066319 and SRP066316).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1508541113/-/DCSupplemental.

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K14+ tumor clusters form polyclonal metastases
Kevin J. Cheung, Veena Padmanaban, Vanesa Silvestri, Koen Schipper, Joshua D. Cohen, Amanda N. Fairchild, Michael A. Gorin, James E. Verdone, Kenneth J. Pienta, Joel S. Bader, Andrew J. Ewald
Proceedings of the National Academy of Sciences Feb 2016, 113 (7) E854-E863; DOI: 10.1073/pnas.1508541113

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K14+ tumor clusters form polyclonal metastases
Kevin J. Cheung, Veena Padmanaban, Vanesa Silvestri, Koen Schipper, Joshua D. Cohen, Amanda N. Fairchild, Michael A. Gorin, James E. Verdone, Kenneth J. Pienta, Joel S. Bader, Andrew J. Ewald
Proceedings of the National Academy of Sciences Feb 2016, 113 (7) E854-E863; DOI: 10.1073/pnas.1508541113
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