Prediction of the Potential Efficacy of Dlx5 in Immunotherapy for Hypopharyngeal Cancer through Integrated Bulk and Single-Cell RNA Sequencing

Background : Immunotherapy, as a personalized treatment strategy, has displayed promising potential in the management of head and neck squamous cell carcinoma. Nevertheless, the heterogeneity and initial resistance of hypopharyngeal squamous cell carcinoma present new obstacles to treatment, highlighting the urgent need for identifying novel predictive biomarkers to develop more targeted and effective treatment approaches. Method : We employed the CIBERSORT algorithm, which quantifies immune cell composition, along with Weighted Gene Co-expression Network Analysis (WGCNA) to identify gene modules associated with tumor immune infiltration of CD4+ T cells. We integrated single-cell sequencing technology to complement each other, conducting bidirectional screening to narrow down molecular associations with tumors. By constructing Protein-Protein Interaction (PPI) networks and conducting clinical Kaplan-Meier analysis, we identified crucial hub genes. We calculated tumor mutation rates, immune checkpoint expression, chemokine factors, and their corresponding receptor correlations to predict the efficacy of immunotherapy targeting DLX5. The R package "oncopredict" was utilized to compute drug sensitivity for each sample, inferring potential chemotherapeutic drugs targeting DLX5. Finally, we explored the precancerous phenotype of DLX5 in the Fadu cell line. Result: Bulk RNA sequencing and single-cell RNA sequencing revealed that in hypopharyngeal squamous cell carcinoma, the prognostically associated EGFR and DLX5 genes are upregulated. Immunological analysis showed a higher mutation rate of DLX5, which is significantly positively correlated with immune checkpoints and chemokine factors. Most importantly, three small molecule compounds (BI.2536_1086, MN.64_1854, Ulixertinib_2047) were identified, which could be potential drugs for treating hypopharyngeal cancer patients. Finally, high expression of DLX5 promoted proliferation, invasion, and migration of hypopharyngeal cancer cells. Conclusion： The association of Dlx5 with CD4+ T cells in hypopharyngeal cancer correlates with the immunological characteristics of the disease and the potential efficacy of immune checkpoint inhibitor therapy. These results indicate that DLX5 might respond well to immunotherapy, shedding light on the role of Dlx5 in hypopharyngeal cancer, providing crucial insights and offering vital information for the development of personalized immunotherapeutic strategies.