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Molecular Diagnostics

TGFβ carrying exosomes in plasma: potential biomarkers of cancer progression in patients with head and neck squamous cell carcinoma

British Journal of Cancer volume 128, pages 1733–1741 (2023)Cite this article

Subjects

Abstract

Objectives

Contributions of TGFβ to cancer progression are well documented. However, plasma TGFβ levels often do not correlate with clinicopathological data. We examine the role of TGFβ carried in exosomes isolated from murine and human plasma as a contributor to disease progression in head and neck squamous cell carcinoma (HNSCC).

Materials and methods

The 4-nitroquinoline-1-oxide (4-NQO) mouse model was used to study changes in TGFβ expression levels during oral carcinogenesis. In human HNSCC, TGFβ and Smad3 protein expression levels and TGFB1 gene expression were determined. Soluble TGFβ levels were evaluated by ELISA and TGFβ bioassays. Exosomes were isolated from plasma using size exclusion chromatography, and TGFβ content was quantified using bioassays and bioprinted microarrays.

Results

During 4-NQO carcinogenesis, TGFβ levels in tumour tissues and in serum increased as the tumour progressed. The TGFβ content of circulating exosomes also increased. In HNSCC patients, TGFβ, Smad3 and TGFB1 were overexpressed in tumour tissues and correlated with increased soluble TGFβ levels. Neither TGFβ expression in tumours nor levels of soluble TGFβ correlated with clinicopathological data or survival. Only exosome-associated TGFβ reflected tumour progression and correlated with tumour size.

Conclusions

Circulating TGFβ+ exosomes in the plasma of patients with HNSCC emerge as potential non-invasive biomarkers of disease progression in HNSCC.

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Fig. 1: Changes in TGFβ expression levels during carcinogenesis in 4-NQO-treated mice.
Fig. 2: Evaluation of TGFβ, P-Smad3 and T-Smad3 expression by IHC.
Fig. 3: Analysis of TGFB1 gene expression in the TCGA database for HNSCCs using the UCSC Xena Browser.
Fig. 4: TGFβ plasma levels in patients with HNSCC and normal donors (ND).
Fig. 5: The presence of TGFβ in plasma-derived exosomes of HNSCC patients.
Fig. 6: The microarray workflow developed for the capture of total exosomes or TGFβ+ exosomes from plasma.

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Data availability

The data that support the findings of this study are available on request from the corresponding author, TLW.

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Acknowledgements

The authors would like to thank late Professor Lee E. Weiss (The Robotics Institute, Carnegie Mellon University, Pittsburgh, PA) for building the custom inkjet bioprinter that enabled the fabrication of TβR microarrays, and Dr. Tomasz Szafarowski (Department of Otolaryngology, Faculty of Dental Medicine, Medical University of Warsaw) for collecting tissue samples.

Funding

This work was supported by National Institutes of Health grants R01-CA 168628 and U01-DE029759 to TLW. NL was supported by the Leopoldina Fellowships LPDS 2017-12 and LPDR 2019-02 from the German National Academy of Sciences Leopoldina and by the Walter Schulz Foundation. AG was supported by the Medical University of Warsaw MB/M/48[79]#. MJS was supported by National Science Centre, Poland UMO-2017/26/M/NZ5/00877#.

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Authors and Affiliations

  1. Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, 93053, Regensburg, Germany

    Nils Ludwig, Theresa Jordan, Gerrit Spanier, Juergen Taxis, Steffen Spoerl, Johannes K. Meier & Torsten E. Reichert

  2. Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA

    Nils Ludwig & Theresa L. Whiteside

  3. UPMC Hillman Cancer Center, Pittsburgh, PA, USA

    Nils Ludwig & Theresa L. Whiteside

  4. Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA

    Saigopalakrishna S. Yerneni

  5. Department of Pathology, University of Medical Sciences, Poznan, Poland

    Malgorzata Harasymczuk

  6. Chair and Department of Biochemistry, Faculty of Medicine, Medical University of Warsaw, Warsaw, Poland

    Mirosław J. Szczepański & Alicja Głuszko

  7. Department of Otolaryngology, Faculty of Dental Medicine, Medical University of Warsaw, Warsaw, Poland

    Wojciech Kukwa

  8. Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15260, USA

    Cynthia S. Hinck & Andrew P. Hinck

  9. Department of Biomedical Engineering and Engineering Research Accelerator, Carnegie Mellon University, Pittsburgh, PA, USA

    Phil G. Campbell

  10. Departments of Immunology and Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA

    Theresa L. Whiteside

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Contributions

Conceptualisation: NL, SSY and TLW; formal analysis: NL, SSY, MH, MJS, AG and TJ; funding acquisition: NL, MJS, AG and TLW; investigation: NL, SSY, MH, MJS, AG, TJ and CSH; methodology: NL, SSY and TLW; project administration: NL, MJS, GS, JT, SS, JKM, PGC, TER, APH and TLW; resources: NL, SSY, MJS, WK, PGC, TER, APH and TLW; supervision: NL, MJS, GS, PGC, TER, APH and TLW; visualisation: NL, SSY, MJS and AG; writing—original draft: NL; writing—review & editing: NL, SSY, MH, MJS, AG, WK, TJ, GS, JT, SS, JKM, CSH, PGC, TER, APH and TLW.

Corresponding author

Correspondence to Theresa L. Whiteside.

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The authors declare no competing interests.

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All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee at the Medical University of Warsaw (#AKBE/50/12) and the Institutional Review Board (IRB) of the University of Pittsburgh (IRB approvals #0403105 and #0506140).

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Ludwig, N., Yerneni, S.S., Harasymczuk, M. et al. TGFβ carrying exosomes in plasma: potential biomarkers of cancer progression in patients with head and neck squamous cell carcinoma. Br J Cancer 128, 1733–1741 (2023). https://doi.org/10.1038/s41416-023-02184-3

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