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Absence of host NF-κB p50 induces murine glioblastoma tumor regression, increases survival, and decreases T-cell induction of tumor-associated macrophage M2 polarization

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Abstract

High-grade gliomas harbor abundant myeloid cells that suppress anti-tumor immunity and support tumor growth. Targeting transcription factors, such as NF-κB p50, that mediate suppressive myeloid M2 polarization may prove therapeutic. GL261-Luc glioblastoma cells were inoculated into wild-type and p50−/− mice, followed by analysis of tumor growth, survival, tumor myeloid cells, and T cells. The absence of host p50 slows tumor growth and enables regression in 30% of recipients, leading to prolonged survival. Tumors developing in p50−/− mice possess a greater concentration of tumor-infiltrating myeloid cells (TIMs) than those in wild-type mice. TIMs are predominantly F4/80hi macrophages which, along with tumor-associated microglia, express increased pro-inflammatory M1 and reduced immune-suppressive M2 markers. In p50−/− mice, total tumor CD4 T cells are threefold more abundant, whereas CD8 T-cell numbers are unchanged, and both produce increased IFNγ and Granzyme B. Naïve splenic p50−/− CD8 T cells manifest increased activation, whereas naïve p50−/− and WT CD4 T cells show similar Th1, Th2, and Th17 polarization. Antibody targeting CD4, but not CD8, fully obviates the p50−/− survival advantage. Combined CD4 and CD8 T-cell depletion reverses myeloid M2 polarization in wild-type hosts, without affecting myeloid M1 polarization in p50−/− hosts. Finally, gliomas grow similarly in p50(f/f) and p50(f/f);Lysozyme-Cre mice, the latter having reduced p50 specifically in myeloid cells and tumor microglia. Thus, high-grade glioma T cells play a key role in directing M2 polarization of tumor myeloid cells, and reducing NF-κB p50 in both tumor myeloid cells and T cells may contribute to glioma therapy.

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Abbreviations

B6:

C57BL/6

BMDM:

Bone-marrow-derived macrophage

FC:

Flow cytometry

GBM:

Glioblastoma

IVIS:

In vivo imaging system

MR:

Mannose receptor

p50:

NF-κB p50

PBS:

Phosphate-buffered saline

qRT-PCR:

Quantitative real-time PCR

TAM:

Tumor-associated macrophage

TIM:

Tumor-infiltrating myeloid cell

WT:

Wild type

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Funding

This study was supported by grants from Alex’s Lemonade Stand Foundation, Hyundai Hope on Wheels, the Allegheny Health Network-Johns Hopkins Cancer Research Fund, the National Institutes of Health (T32 CA60441 and P30 CA006973), and by the Giant Food Children’s Cancer Research Fund.

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Authors and Affiliations

  1. Division of Pediatric Oncology, Department of Oncology, Johns Hopkins University, CRB I, Rm 253, 1650 Orleans St., Baltimore, MD, 21231, USA

    Theresa Barberi, Allison Martin, Rahul Suresh, David J. Barakat, Sarah Harris-Bookman, Charles G. Drake, Michael Lim & Alan D. Friedman

Authors
  1. Theresa Barberi
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  2. Allison Martin
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Contributions

TB, AM, RS, DJB, and SH-B conducted experiments. CGD, ML, and ADF guided the conduct of experiments. TB and ADF wrote the manuscript.

Corresponding author

Correspondence to Alan D. Friedman.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval and ethical standards

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Animal source

WT C57BL/6 (B6) mice were obtained from Charles River Laboratories, Nfkb1(p50)−/−, Lys-Cre, and ROSA26-FLPo mice from Jackson Laboratory (6097, 4781, 12930), and p50(flacZ/+) mice from the Knockout Mouse Project (CSD29053).

Cell line authentication

GL261-Luc cells were obtained from Perkin Elmer, who had authenticated it as C57BL/6 in origin by mouse STR analysis.

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Barberi, T., Martin, A., Suresh, R. et al. Absence of host NF-κB p50 induces murine glioblastoma tumor regression, increases survival, and decreases T-cell induction of tumor-associated macrophage M2 polarization. Cancer Immunol Immunother 67, 1491–1503 (2018). https://doi.org/10.1007/s00262-018-2184-2

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  • DOI: https://doi.org/10.1007/s00262-018-2184-2

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