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Interleukin-33 pretreatment promotes metastatic growth of murine melanoma by reducing the cytotoxic capacity of CD8+ T cells and enhancing regulatory T cells

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Abstract

Interleukin-33 (IL-33) regulates innate and acquired immune response to pathogens, self-antigens and tumors. IL-33 effects on tumors depend on the dose and mode of administration along with the type of malignancy. We studied the effects of IL-33 on the development of primary and metastatic melanoma induced by B16-F1 cell line in C57BL/6 mice. Intraperitoneally applied IL-33 restricts primary tumor growth. When administered intranasally 3 days prior to the intravenous injection of the tumor cells, IL-33 promoted growth of B16-F1 melanoma metastases, while B16-F10 gave massive metastases independently of IL-33. To mimic natural dissemination, we next used a limited number (5 × 104) of B16-F1 cells intravenously followed by application of IL-33 intraperitoneally. IL-33 increased the size of metastases (10.96 ± 3.96 mm2) when compared to the control group (0.86 ± 0.39 mm2), without changing incidence and number of metastases. IL-33 increased expression of ST2 on both tumor and immune cells in metastases. Also, IL-33 enhanced eosinophils and anti-tumor NK cells in the lung. The striking finding was reduced cytotoxicity of CD8+ T cells derived from metastatic lung of IL-33 injected mice. IL-33 reduced the percentage of TNF-α+ and IFN-γ+ CD8+ T cells while increasing the frequency of CD8+ T cells that express inhibitory molecules (PD-1, KLRG-1 and CTLA-4). There was a significant accumulation of CD11b+Gr-1+ myeloid suppressor cells and FoxP3+, IL-10+ and CTLA-4+ regulatory T cells in the metastatic lung of IL-33 injected mice. The relevance of IL-33 for melanoma metastases was also documented in a significantly increased level of serum IL-33 in stage III melanoma patients.

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Abbreviations

AJCC:

American Joint Committee on Cancer

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

KLRG1:

Killer cell lectin-like receptor subfamily G member 1

TAP1:

Transporter associated with antigen processing 1

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Acknowledgements

The authors thank Professor Milan Knezevic for great help in pathohistological analyses and Aleksandar Arsenijevic, Aleksandar Ilic and Dusan Tomasevic for excellent technical assistance.

Funding

This work was funded by grants from the Ministry of Education, Science and Technological Development, Serbia (ON 175071, ON 175069 and ON 175103), a bilateral project with People’s Republic of China (06/2018) and by the Faculty of Medical Sciences of the University of Kragujevac, Serbia (MP 02/14).

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Authors and Affiliations

  1. Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, 69 Svetozara Markovica Street, 34000, Kragujevac, Serbia

    Andra Jevtovic, Jelena Pantic, Ivan Jovanovic, Marija Milovanovic, Nebojsa Arsenijevic, Miodrag L. Lukic & Gordana D. Radosavljevic

  2. Department of Otorhinolaryngology, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia

    Andra Jevtovic

  3. Institute for Medical Research, Military Medical Academy, Belgrade, Serbia

    Ivan Stanojevic & Danilo Vojvodic

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  1. Andra Jevtovic
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  2. Jelena Pantic
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  3. Ivan Jovanovic
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  4. Marija Milovanovic
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  5. Ivan Stanojevic
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  8. Miodrag L. Lukic
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  9. Gordana D. Radosavljevic
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Contributions

AJ performed most of the experiments, analyzed the data and drafted the manuscript. GDR contributed to the design and interpretation of the experiments, analyzed the data, generated figures and revised the manuscript. JP contributed to the design and interpretation of the experiments, assisted and/or performed some of the experiments, analyzed the data and revised the manuscript. IJ, MM and DV assisted and/or performed some of the experiments and analyzed the data. IS oversaw the consent and contributed to the collection of human samples. NA and MLL coordinated the design and execution of the experiments, data analysis, and manuscript drafting and revision. All authors approved the final version of the manuscript.

Corresponding authors

Correspondence to Miodrag L. Lukic or Gordana D. Radosavljevic.

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Conflict of interest

The authors declare that they have no conflict of interest.

Cell line authentication

The murine melanoma B16-F1 and B16-F10 cell lines, purchased from American Type Culture Collection (ATCC, Manassas, USA), were routinely cultured. Cell culture was performed under standardized protocols to ensure that phenotypically similar cells are implanted during each experiment. Viability of cells was determined by trypan blue exclusion and only cell suspensions with > 95% viable cells were used.

Ethical approval and ethical standards

All animal care procedures and experimental protocols were performed in accordance with institutional and the official guidelines of EU Directive 2010/63/EU, at the Faculty of Medical Sciences, University of Kragujevac, Serbia. The experiments were approved by the Animal Ethics Board of the Faculty of Medical Sciences, University of Kragujevac, Serbia (01-2588, 17/03/2014). Consented melanoma patients were recruited from the Clinics of Dermatovenerology and Plastic Surgery, Military Medical Academy, Belgrade, Serbia, while healthy donors (with no prior history of cancer) were recruited on periodical systematic examinations. The study was approved by the local Research Ethics Committee, Military Medical Academy (11-03/2014).

Animal source

C57BL/6 mice were bred in animal breeding facilities of the Faculty of Medical Sciences, University of Kragujevac, Serbia.

Informed consent

Written informed consent was obtained prior to sample collection from all individual participants included in the study in accordance with the Declaration of Helsinki.

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Jevtovic, A., Pantic, J., Jovanovic, I. et al. Interleukin-33 pretreatment promotes metastatic growth of murine melanoma by reducing the cytotoxic capacity of CD8+ T cells and enhancing regulatory T cells. Cancer Immunol Immunother 69, 1461–1475 (2020). https://doi.org/10.1007/s00262-020-02522-x

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