Supporting data for "DECODING THE NEUROIMMUNE AXIS OF HEAD AND NECK CANCER TO ENHANCE IMMUNOTHERAPEUTIC OUTCOMES"

Head and Neck Squamous Cell Carcinoma (HNSCC) exhibits extensive innervation that correlates with its aggressiveness. Although tumors actively recruit nerves through neurogenesis, axonogenesis, and neuronal reprogramming, the underlying molecular mechanisms remain elusive. Concurrently, HNSCC sustains a profoundly immunosuppressive microenvironment, contributing to poor prognosis and immunotherapy resistance. While nerves and immune cells independently drive HNSCC progression, their dynamic bidirectional crosstalk – the neuroimmune axis – remains poorly understood. This thesis aimed to decode the HNSCC neuroimmune axis to elucidate novel immune evasion mechanisms and informing strategies to enhance immunotherapy. To achieve this, a multi-modal approach was employed integrating bioinformatics, molecular biology, and preclinical models. The research findings are organized into seven chapters, each exploring critical facets within the complex cancer-neuron-immune triad.<b>Chapter 1</b> presents a comprehensive literature review on the roles of neurons and their subtypes in tumor progression and immunomodulation. It highlights key gaps in the characterization of tumor-associated neurons and the molecular mediators of neuroimmune interactions in HNSCC, forming the foundation for the hypotheses and objectives outlined in <b>Chapter 2</b><b>.</b><b>Chapter 3</b> investigates neural infiltration in two tongue squamous cell carcinoma models utilizing immunodeficient and immunocompetent mice. A threefold increase in nerve density was observed in immunodeficient mice, which was associated with poor prognosis. The increased neural infiltration was consistently observed across sensory, sympathetic, and parasympathetic neural subtypes. These findings suggest that adaptive immunity regulates neural infiltration and cancer progression, highlighting the importance of the neuroimmune axis in HNSCC progression.<b>Chapter 4</b> examines the differential expression of tumor-derived neuropeptides and evaluates their correlations with neuroimmune factors, immune cell infiltration, and response to checkpoint immunotherapy. Through comprehensive profiling, PTHLH, NMB, GAST, APLN, and LYNX1 were identified as key neuropeptides that potentially mediate the neuroimmune axis in HNSCC.<b>Chapter 5</b> focuses on PTHLH, the most significantly upregulated neuropeptide in HNSCC, which showed extensive correlations with neuroimmune factors. Genetic ablation of PTHLH resulted in dysregulation of neurotrophic signaling pathways essential for intra-tumoral innervation, significant reduction in tumor burden, and enhanced antitumor immunity —evidenced by increased CD8+ and CD4+ T cell infiltration, as well as decreased immunosuppressive markers. These findings establish the tumor-derived neuropeptide PTHLH as a critical regulator of the neuroimmune axis in HNSCC.<b>Chapter 6</b> reveals that anti-PD1 therapy triggers CGRP secretion from tumor-associated neurons, which counteracts its anti-tumor effects in a preclinical tongue cancer model. Importantly, blocking CGRP and PD1 synergistically reduced tumor growth, indicating that anti-PD1-induced neuronal activation constitutes a novel resistance mechanism. Therefore, neuronal modulation provides a novel strategy to improve checkpoint immunotherapy in HNSCC.<b>Chapter 7</b> synthesizes the key findings and concludes that reciprocal neuroimmune interaction drives tumor progression and confers resistance to checkpoint immunotherapy in HNSCC. The identification of mechanistically distinct, therapeutically targetable molecules (PTHLH and CGRP) provides a rational foundation for development novel strategies that simultaneously disrupt protumorigenic neural signaling and enhance antitumor immunity, offering promising avenues to overcome current limitations in immunotherapy.