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

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.

Fig. 1. NaNO₂ promoted the progression and lymphatic metastasis potential in 4-NQO induced TSCC mice model.

a Schematic of the in vivo animal experiment of NaNO₂ on TSCC initiation. Mice were dived into three group randomly, and the tongue and cervical lymph node tissues were harvested every 4 weeks after 16 weeks, n = 25. b Plasma levels of nitrite and cGMP in mice were measured at 32 weeks, n = 5. c H&E analysis the pathology of tongue tissues in 4-NQO induced mice, representative of the magnified images at start treatment point, 16, 20, 24, 28 and 32 weeks. d Representative mages from NaNO₂, H₂O and 4-NQO group at 32 weeks. e Masson staining analysis of the fiber layer in NaNO₂ and H₂O group at 32 weeks. f, g Schematic of the in vivo animal experiment of NaNO₂ on TSCC development, plasma levels of nitrite and cGMP were measured at the end point, n = 5. h, I Representative images for analysis the pathology of tongue tissue on TSCC development after NaNO₂ treatment, and the quantification of DOI. n = 3. j, k lymph node metastasis of TSCC were analyzed by IF using EdU and Pan-CK, and quantitative analysis of the total metastasis portion in mice after treated with NaNO₂. Scale bar = 25 µm. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ns P > 0.05.

Fig. 2. NaNO₂ enhanced the lymphatic metastasis potential of human TSCC.

a Schematic of the animal experiment of NaNO₂ effect on lymphatic metastasis in human TSCC. Tongue tissues and lymph nodes were collected at 12 weeks. b, c IF with GFP and Pan-CK were performed to tract the metastatic TSCC lesion in lymph node, and the percentage of lymph node metastasis in total mice were quantitated. d–f RNA-Seq analysis was performed in NaNO₂ treated group and H₂O group, data presented in Volcano map, KEGG pathway enrichment and GSEC enrichment. g, h IHC analysis using LYVE-1 to detected the LVD in CTSC-1 and CAL-27 xenograft tissue in tongue, and the quantification of LVD were evaluated. Scale bar = 25 µm. Data are presented as mean ± SEM. ***P < 0.001.

Fig. 3. Phenotypic changes were involved in TSCC during lymphatic metastasis after treatment with NaNO₂.

a, b IF staining of GFP and Pan-CK in metastasis lesion in both NaNO₂ group and H₂O group, and the portion of Pan-CK positive cells in GFP positive cells were quantitative, n = 3. c Cytokeratin genes and macrophage genes expression in CTSC-1 cells with or without NaNO₂ treatment by RNA-Seq data analysis. d, e RT-qPCR evaluated the RNA-Seq results in CTSC-1 and CAL-27 cells with or without NaNO₂. f Western blot analysis the protein level of CD68 and cytokeratin in CTSC-1 and CAL-27 cells. g IF double staining of CD68 and GFP in TSCC metastasis lesion in lymph node. h, i IHC detected the positive CD68 TSCC cells in primary TSCC of CTSC-1 and CAL-27 xenograft. Scale bar = 25 µm. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns P > 0.05.

Fig. 4. CD68 enhanced phenotypic changes and lymphatic metastasis in TSCC after NaNO₂ exposure.

a FACS analysis of the proportion of CD68 positive TSCC cells under the influence of NANO₂. b Representative FACS histogram of the indicated CTSC-1 cells and THP-1 cells after incubating with fluorescent phagocytic beads for 48 h. c Schematic of mice lymph node tissue extraction for TSCC migration assay. d-e Representative images of crystal violet straining in Transwell assay for CTSC-1 and CAL-27 with CD68 overexpression, quantification by acetic acid elution. n = 3. f The cells viability of CTSC-1 cells after cultured in mimic lymphatic fluid for 24 h, measured by Cell Tiger-GLO. n = 3. g Schematic of NK cells killing assay, created with gdp.renlab.cn. h NK cells were incubated with the indicated CTSC-1 target cells for 24 h, and the cell viability was assessed by Cell Tiger-GLO. n = 3. i-j Lymph node metastasis rate of mice implanted of indicated CTSC-1 cells after 7 weeks, and the representative IF results of GFP positive cells showing the metastasis lesion. k CD68 expression in clinical human TSCC samples. Scale bar = 25 µm. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. CD68 enhanced phenotypic changes and lymphatic metastasis in TSCC after NaNO₂ exposure.

a FACS analysis of the proportion of CD68 positive TSCC cells under the influence of NANO₂. b Representative FACS histogram of the indicated CTSC-1 cells and THP-1 cells after incubating with fluorescent phagocytic beads for 48 h. c Schematic of mice lymph node tissue extraction for TSCC migration assay. d-e Representative images of crystal violet straining in Transwell assay for CTSC-1 and CAL-27 with CD68 overexpression, quantification by acetic acid elution. n = 3. f The cells viability of CTSC-1 cells after cultured in mimic lymphatic fluid for 24 h, measured by Cell Tiger-GLO. n = 3. g Schematic of NK cells killing assay, created with gdp.renlab.cn. h NK cells were incubated with the indicated CTSC-1 target cells for 24 h, and the cell viability was assessed by Cell Tiger-GLO. n = 3. i-j Lymph node metastasis rate of mice implanted of indicated CTSC-1 cells after 7 weeks, and the representative IF results of GFP positive cells showing the metastasis lesion. k CD68 expression in clinical human TSCC samples. Scale bar = 25 µm. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. CREB1 transcriptionally upregulated CD68 expression in TSCC.

a GSEA showed activation of transcription factor activity signaling pathway related to DNA binding. b Luciferase assay verified the transcriptional regulation in CTSC-1 cells treated with NaNO_2. c-d The up-regulated transcript factors in CTSC-1 cells treated with NaNO₂, and CREB1 increase the luciferase activity of CD68. e The mRNA level of CREB1 was upregulated in CTSC-1 and CAL-27 cells treated with NaNO₂. f Western blot analysis the protein level of CREB1 and its phosphorylation activity. g JASPAR analysis the potential DNA binding cites of CREB1 to CD68 promoter. h Luciferase assay analysis the promoter activity of CD68 in different region after co-infection with CREB1. i CD68 transcription activity in CTSC-1 cell with CREB1-KO. j Western blot analysis the protein level of CREB1, p-CREB1 and CD68. k-l Protein level and mRNA level of CREB1 and CD68 after treated with NaNO₂ directly in CTSC-1 cells. (M) The mRNA level of CREB1 was upregulated in CTSC-1 after treated with cGMP. *P < 0.05, **P < 0.01, ***P < 0.001, ns > 0.05.

Fig. 6. Therapeutic inhibition of CREB1 reduced migration of TSCC to lymphatic node.

a-b The migration ability was evaluated after overexpression or deletion of CREB1 in CTSC-1 cells. n = 3. c-d The effect of CREB1 expression on the cell viability of CTSC-1 in mimic lymphatic flow and cell viability was detected. n = 3. e CREB1 expression level in tumor and normal tissues in 16 types of cancer from the TCGA with Kruskal-Wallis test, and 504 cases of HNSCC were analysis in this test. f CREB1 expression level in clinical TSCC samples showed that CREB1 was highly expressed in tumors and at the invasive front. g-h The CREB1 inhibitor 666-15 significantly downregulates the ability of CTSC-1 to migrate to lymph nodes. i The effect of 666-15 treatment on CD68 promoter activity CTSC-1 cells. *P < 0.05, **P < 0.01, ***P < 0.001, ns > 0.05. Scale bar=100 μm and 50 μm.