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Cellular and Molecular Biology

Sodium nitrite orchestrates macrophage mimicry of tongue squamous carcinoma cells to drive lymphatic metastasis

British Journal of Cancer volume 132, pages 340–353 (2025)Cite this article

Subjects

Abstract

Background

Tongue squamous cell carcinoma (TSCC) is a malignant oral cancer with unclear pathogenesis that shows a tendency for early-stage lymphatic metastasis. This results in a poor prognosis, with a low 5-year survival rate. Dietary sodium nitrite (NaNO2) has proposed associations with disease, including cancer. However, a direct relationship between NaNO2 and TSCC has not been established.

Methods

In vitro and in vivo assays were used to investigate the role of NaNO2 in TSCC. Protein expression in TSCC specimens was detected by immunohistochemistry and immunofluorescence. The molecular mechanism was determined using RT-qPCR, western blot, RNA-seq, luciferase reporter assays, migration assays, and FACS analysis. More detail of methods can be found in the Materials and methods section.

Results

The data in this study showed that NaNO2 did not initiate carcinogenesis in the tongue but improved the lymphatic metastatic potential of TSCC cells in the specified experimental period. During metastasis to lymph nodes, monocyte-macrophage markers were upregulated in TSCC cells, whereas keratin markers were downregulated. Specifically, expression of the CD68 gene was high in TSCC cells following NaNO2-induced TSCC phenotypic switching. These phenotypic changes were associated with activation of transcription factor cyclic-AMP response binding protein (CREB1), which directly targets CD68 transcription. Furthermore, blocking CREB1 activity either through gene knockout or specific inhibitor treatment decreased the migration ability of TSCC cells and suppressed CD68 expression.

Conclusions

Our findings highlight the role of NaNO2 in enabling macrophage mimicry in TSCC cells through the CREB1-CD68 signaling pathway, which promotes lymphatic metastasis. Shedding light on drivers of lymphatic metastasis in TSCC and providing a new perspective on dietary strategies to improve outcomes of patients with TSCC.

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Fig. 1: NaNO2 promoted the progression and lymphatic metastasis potential in 4-NQO induced TSCC mice model.
Fig. 2: NaNO2 enhanced the lymphatic metastasis potential of human TSCC.
Fig. 3: Phenotypic changes were involved in TSCC during lymphatic metastasis after treatment with NaNO2.
Fig. 4: CD68 enhanced phenotypic changes and lymphatic metastasis in TSCC after NaNO2 exposure.
Fig. 5: CREB1 transcriptionally upregulated CD68 expression in TSCC.
Fig. 6: Therapeutic inhibition of CREB1 reduced migration of TSCC to lymphatic node.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. The RNA sequencing data has been uploaded to NCBI, accession to cite for the SRA data: PRJNA1045239, https://dataview.ncbi.nlm.nih.gov/object/PRJNA1045239?reviewer=eiqpu9osanh8ppg9fafhmc0gs.

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Acknowledgements

The authors are grateful to Jiang Qian (Laboratory Animal Center, Sun Yat-sen University) and Meilin Zheng (Laboratory Animal Center, School of Life Sciences, Sun Yat-sen University) for the valuable support in performing the animal experiments. We are also deeply grateful to all members of the Yan Zhang laboratory for their constructive advice and engaging discussions. This study was supported by grants from the National Natural Science Foundation of China (No. 31871413), Programs of Guangdong Science and Technology (No. 2019B1515210015), Shenzhen Science and Technology Innovation Committee (No. KJZD20230923114802005) and China Postdoctoral Science Foundation (No. 2023M744083).

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Authors and Affiliations

  1. MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, PR China

    Xiangwan Lu, Weifan Lin, Junheng Zheng, Wuheng Huang, Shuyi Yu, Haoxiang Chen & Yan Zhang

  2. Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, PR China

    Hua Wang

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Contributions

Conception and design, Project administration, Funding acquisition and Editing: Yan Zhang and Hua Wang; Experiment design, Methodology, Investigation: Xiangwan Lu and Junheng Zheng; Data Curation, Visualization and Formal analysis: Xiangwan Lu, Weifan Lin, Junheng Zheng, Wuheng Huang, Shuyi Yu, Haoxiang Chen; Manuscript drafting: Xiangwan Lu. All authors read and approved the submitted version. The work reported in the paper has been performed by the authors, unless clearly specified in the text.

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Correspondence to Hua Wang or Yan Zhang.

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The human specimen collection in this study was followed the protocol approved by the ethics committee of Guanghua Hospital, Sun Yat-sen University (approval No. 2010-9), and written informed consent was obtained from the patient in accordance with ethical guidelines. All animal experimental protocols were performed following by the Animal Ethics Committee of Sun Yat-sen University (approval No. 2022002118).

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Lu, X., Lin, W., Zheng, J. et al. Sodium nitrite orchestrates macrophage mimicry of tongue squamous carcinoma cells to drive lymphatic metastasis. Br J Cancer 132, 340–353 (2025). https://doi.org/10.1038/s41416-024-02923-0

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