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Updated: Dec 8, 2025
Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment
Published on: April 22, 2019
Malignant cell-specific CXCL14 promotes tumor lymphocyte infiltration in oral cavity squamous cell carcinoma
Anuraag Parikh1, JuneHo Shin2, William Faquin3
1Otolaryngology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA.
Objectives:
To explore lymphocyte infiltration as a potential mechanism behind CXCL14-mediated tumor growth suppression in oral cavity squamous cell carcinoma (OSCC).
Methods:
We analyzed single cell RNA-sequencing (scRNA-seq) data from OSCC to identify expression changes among malignant cells in lymph nodes (LN) versus primary tumors. CXCL14 expression in murine OSCC cell lines was quantified using qRT-PCR. Short hairpin RNA knockdown of CXCL14 was performed in mouse oral cavity (MOC)1 cells, and CXCL14 overexpression was performed in MOC2 cells. Cells in each condition were injected into C57BL/6 mice with and without T cell depletion, and tumor volume was measured. At 30 days, tumors were dissociated and analyzed by flow cytometry for CD45+CD3+ T cells. CXCL14 expression was correlated with gene expression signatures of tumor infiltrating lymphocytes (TIL) in scRNA-seq data, as well as TCGA tumors.
Results:
scRNA-seq revealed CXCL14 as the most significantly downregulated gene among malignant cells in LNs relative to primary tumor, supporting a role in preventing invasion and/or metastasis. In a murine immunocompetent model, CXCL14 expression was higher in indolent MOC1 cells than in more aggressive MOC2 cells. Tumor growth in vivo was significantly increased by CXCL14 knockdown in MOC1 cells relative to control, with a corresponding decrease in TIL. In MOC2 cells, tumor growth was significantly reduced by CXCL14 overexpression relative to control and TIL were increased. Both effects were lost with T cell depletion. In a human tumor scRNA-seq cohort, we found that only malignant cell CXCL14, but not non-malignant cell or fibroblast CXCL14, was associated with TIL. Bulk CXCL14 from the TCGA cohort had no association with TIL.
Conclusions:
Higher CXCL14 expression by tumor cells is associated with reduced tumor growth and increased TIL, supporting immune-mediated suppression of tumor growth in OSCC. Given that CXCL14 is downregulated in LN metastases compared with primary tumors, our data raise the possibility that CXCL14-mediated immune infiltration may discourage invasion and metastasis. In human scRNA-seq data, only malignant cell-specific CXCL14 was associated with TIL, suggesting a critical context-dependent effect of CXCL14 expression.
Insights
CXCL14, a gene downregulated in oral cancer metastasis, suppresses tumor growth by recruiting immune cells. Its expression in malignant cells is key for this immune-mediated effect, suggesting a role in preventing cancer spread.
Area of Science:
- Oncology
- Immunology
- Molecular Biology
Background:
- Oral cavity squamous cell carcinoma (OSCC) is a significant global health concern.
- The mechanisms underlying tumor growth suppression in OSCC require further elucidation.
- CXCL14's role in OSCC progression and immune response is not fully understood.
Purpose of the Study:
- To investigate the role of lymphocyte infiltration in CXCL14-mediated tumor growth suppression in OSCC.
- To determine if CXCL14 expression in malignant cells influences tumor growth and immune cell infiltration.
- To explore the potential of CXCL14 as a therapeutic target for OSCC.
Main Methods:
- Analysis of single-cell RNA-sequencing (scRNA-seq) data from OSCC patients.
- Gene expression analysis of CXCL14 in murine OSCC cell lines using qRT-PCR.
- In vivo studies using genetically modified murine OSCC cells (CXCL14 knockdown/overexpression) in immunocompetent mice, with and without T cell depletion.
- Flow cytometry analysis of tumor-infiltrating lymphocytes (TILs).
- Correlation analysis of CXCL14 expression with TIL signatures in human scRNA-seq and TCGA datasets.
Main Results:
- CXCL14 was significantly downregulated in malignant cells within lymph nodes compared to primary tumors.
- CXCL14 knockdown increased tumor growth and decreased TILs in a murine model, while overexpression reduced tumor growth and increased TILs.
- These effects were dependent on T cells and were lost upon T cell depletion.
- In human data, only malignant cell CXCL14 expression correlated with TILs, not non-malignant cell or fibroblast CXCL14.
Conclusions:
- Elevated CXCL14 expression in OSCC tumor cells is associated with reduced tumor growth and increased TILs, indicating immune-mediated tumor suppression.
- Downregulation of CXCL14 in lymph node metastases suggests a potential role in hindering invasion and metastasis through immune infiltration.
- Malignant cell-specific CXCL14 expression is critical for its association with TILs, highlighting a context-dependent function in OSCC immunity.
