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Metformin enhances gefitinib efficacy by interfering with interactions between tumor-associated macrophages and head and neck squamous cell carcinoma cells

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Abstract

Background

Tumor-associated macrophages (TAMs) play an important role in drug resistance in many tumors, including head and neck squamous cell carcinoma (HNSCC). However, how TAMs interact with HNSCC cells to induce drug resistance, especially under hypoxic conditions, is unclear. In this study, we investigated the mechanism of TAM-induced gefitinib resistance in HNSCC cells and sought for novel therapeutic strategies.

Methods

The effects of hypoxia-treated HNSCC cells on the migration and polarization of macrophages were analyzed. Recombinant cytokine proteins and neutralizing antibodies were used as controls. In addition, we assessed the cytotoxic effects of gefitinib on HNSCC cells treated with M2-type macrophage conditioned medium, and carried out a cytokine antibody array analysis, thereby revealing the key factor CCL15. The relationship between serum CCL15 expression levels and prognosis in HNSCC patients was analyzed. In addition, we performed bioinformatic analyses to pursue the mechanisms of CCL15-induced gefitinib resistance. Finally, metformin was used to evaluate the sensitizing effects of gefitinib treatment on HNSCC cells in vitro and in vivo.

Results

We found that HNSCC cells recruited macrophages by secreting VEGF and polarized the macrophages to the M2 phenotype through IL-6. Conversely, we found that M2-type TAMs promoted HNSCC cell resistance to gefitinib through paracrine CCL15 signaling. The serum CCL15 levels in HNSCC patients showed a significant correlation with patient prognosis. Furthermore, we found that M2-type TAMs could suppress the sensitivity of HNSCC cells to gefitinib through the CCL15-CCR1-NF-κB pathway. In addition, we found that metformin not only inhibited CCL15 expression in M2-type TAMs enhanced by hypoxia, but also suppressed CCR1 surface expression in HNSCC cells. Encouragingly, we found that metformin sensitized HNSCC cells to gefitinib treatment in vitro and in vivo.

Conclusions

Based on our data we conclude that we have identified a novel interaction between M2-type TAMs and HNSCC cells that contributes to gefitinib resistance. We also found that metformin inhibited the cross-talk between macrophages and tumor cells, thereby eliciting therapeutic effects both in vitro and in vivo.

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Abbreviations

ARG-1:

argininase 1

CCL2/5/15:

C-C motif chemokine ligand 2/5/15

CCR1:

C-C motif chemokine receptor 1

CSF-1:

colony stimulating factor 1

EGFR:

epidermal growth factor receptor

EMT:

epithelial-mesenchymal transition

EOT:

eotaxin

ET-2:

endothelin 2

GSEA:

gene set enrichment analysis

H-CM:

hypoxic condition medium

HE:

hematoxylin and eosin

HIF:

hypoxia-inducible factors

HNSCC:

head and neck squamous cell carcinoma

IL-4/6/10/13:

interleukin 4/6/10/13

KEGG:

Kyoto Encyclopedia of Genes and Genomes

NC:

negative control

N-CM:

normoxic condition medium

NF-κB:

nuclear factor kappa-light-chain-enhancer of activated B cells

OSM:

oncostatin M

PDTC:

pyrrolidinedithiocarbamate ammonium

PMA:

phorbol 12-myristate 13-acetate

SDF-1:

stromal cell-derived factor 1

SEMA3A:

semaphorin 3A

TAMs:

Tumor-associated macrophages

TCGA:

the Cancer Genome Atlas

TEFB:

transcription factor EB

TGF:

β: tumor growth factor β

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Acknowledgments

This study was supported by the Jiangsu Natural Science Fund for Excellent Young Scholars [BK20160051], Natural Science Foundation of Jiangsu Province [BK20180136], Natural Science Foundation of Jiangsu Province [BK20180138], Key Research and Development Program of Jiangsu Province [BE2017741] and Nan**g Municipal Science and Technology Commission [201715039].

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Author notes

  1. **teng Yin and Shengwei Han contributed equally to this work.

Authors and Affiliations

  1. Department of Oral and Maxillofacial Surgery, Nan**g Stomatological Hospital, Medical School of Nan**g University, No 30 Zhongyang Road, Nan**g, 210008, China

    **teng Yin, Shengwei Han, Chuanhui Song, Huihui Zou, Zheng Wei, Wenguang Xu, Jianchuan Ran, Chuanchao Tang, Yufeng Wang, Qingang Hu & Wei Han

  2. Central Laboratory of Stomatology, Nan**g Stomatological Hospital, Medical School of Nan**g University, No 22 Hankou Road, Nan**g, 210093, China

    **teng Yin, Shengwei Han, Chuanhui Song, Huihui Zou, Zheng Wei, Wenguang Xu, Jianchuan Ran, Chuanchao Tang, Yufeng Wang, Yu Cai, Qingang Hu & Wei Han

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  1. **teng Yin
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Correspondence to Qingang Hu or Wei Han.

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Ethics statement

All experiments were approved by the Ethics Review Board of the Nan**g Stomatological Hospital (approval number: 2017-NKL012). All animal experiments and experimental protocols used were in accordance with the animal care and use committee of the medical school of Nan**g University.

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Yin, X., Han, S., Song, C. et al. Metformin enhances gefitinib efficacy by interfering with interactions between tumor-associated macrophages and head and neck squamous cell carcinoma cells. Cell Oncol. 42, 459–475 (2019). https://doi.org/10.1007/s13402-019-00446-y

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