Article | Published:

Translational Therapeutics

Increased expression of the immunosuppressive interleukin-35 in patients with non-small cell lung cancer

British Journal of Cancer (2019) | Download Citation

Abstract

Background

The immunosuppressive role of the cytokine IL-35 in patients with non-small cell lung cancer (NSCLC) is poorly understood. In this study, we analysed the localisation and regulation of IL-35 in the lung of patients with non-small cell lung cancer (NSCLC) to further elucidate the immune-escape of cancer cells in perioperative course of disease.

Methods

Interleukin 35 (IL-35) was measured by ELISA in postoperative serum from 7 patients with NSCLC as well as 8 samples from healthy controls. Immunohistochemistry, FACS analysis, real-time PCR, as well as western blot from samples of the control (CTR), peri-tumoural (PT) and the tumoural (TU) region of the lung derived from patients with NSCLC and 10 controls were performed.

Results

Here we found elevated levels of IL-35 in the TU region as well as postoperative serum from patients with lung adenocarcinoma. Consistently, we found an increased expression of IL-35+Foxp-3+ cells, which associated with ARG1 mRNA expression and decreased TNFA in the TU region of the lung of patients with NSCLC as compared to their CTR region. Furthermore, in the CTR region of the lung of patients with NSCLC, CD68+ macrophages were induced and correlated with IL-35+ cells. Finally, IL-35 positively correlated with TTF-1+PD-L1+ cells in the TU region of NSCLC patients.

Conclusions

Induced IL-35+Foxp3+ cell numbers in the TU region of the lung of patients with NSCLC associated with ARG1 mRNA expression and with TTF-1+PD-L1+ cells. In the tumour-free CTR area, IL-35 correlated with CD68+ macrophages. Thus inhibitors to IL-35 would probably succeed in combination with antibodies against immune checkpoints like PD-L1 and PD-1 currently used against NSCLC because they would inhibit immunosuppressive macrophages and T regulatory cells while promoting T cell-mediated anti-tumoural immune responses in the microenvironment as well as the TU region of NSCLC patients.

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Acknowledgements

The authors thank Rebekka Springel and Sonja Trump for the qPCR analysis and their excellent experimental help, as well as all the supporting team in the Molecular Pneumology Department, the Thoracic Surgery and the Institute of Pathology in Erlangen. This work was financially supported by an IZKF grant number A59 awarded to S.F. D.I.T. was supported by a 2-year 50% time rotation fellowship at MP, financed by the IZKF in Erlangen.

Author contributions

D.I.T. and H.S. were involved in the recruitment of the patients of the cohort and performed surgery. Further, D.I.T. made a 2-year period rotation at the MP and contributed to cell isolation, tissue storage, and FACS analysis of the patients as reported in Fig. 2. D.I.T. did also the IL-35 serum analysis and contributed to the patient clinical analysis. R.S. did the qPCR analysis of the patients as reported in Fig. S2, contributed to cell culture, and analysed the IL-35 immunohistochemistry. L.H. contributed to all figures, supplementary figures and tables and to the recruitment of all patients. S.M. contributed to patients’ sample recruitment and did the immunohistochemistry. K.K. contributed to most of the patient tissue sample recruitment and performed the FACS analysis reported in Fig. 2. J.F. was involved in patient sample recruitment and storage and performed the analysis presented in Figs. S2 and S3. S.F. analysed the data reported in Fig. S4. S.F. and L.H. wrote the manuscript. The pathologists C.-I.G. made the diagnosis and did the scanning of the slides and R.J.R. did the TMA, additionally. All authors have read and approved the final manuscript.

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Affiliations

  1. Department of Molecular Pneumology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany

    • Lisanne Heim
    • , Katerina Kachler
    • , Raphaela Siegmund
    • , Denis I. Trufa
    • , Susanne Mittler
    • , Juliane Friedrich
    •  & Susetta Finotto
  2. Department of Thoracic Surgery, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany

    • Denis I. Trufa
    •  & Horia Sirbu
  3. Institute of Pathology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany

    • Carol-Immanuel Geppert
    •  & Ralf J. Rieker

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Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

This study was performed at the Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) and was approved by the ethics review board of the University of Erlangen (Re-No: 56_12B; DRKS-ID: DRKS00005376). Patients who suffered from NSCLC underwent surgery and gave their approval to be enrolled in this study in an informed written consent. The patient studies were conducted in accordance with the ethical guidelines of the Declaration of Helsinki.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

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This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0).

Corresponding author

Correspondence to Susetta Finotto.

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DOI

https://doi.org/10.1038/s41416-019-0444-3