Ferroptosis-driven stromal immune evasion and melatonin receptor suppression as novel predictive biomarkers of immunotherapy response in oral squamous cell carcinoma: a single-cell transcriptomic study.

BACKGROUND: Immune checkpoint inhibitor (ICI) therapy for oral squamous cell carcinoma (OSCC) yields objective responses in fewer than 20% of patients, underscoring the urgent need for novel predictive biomarkers beyond PD-L1 expression and tumor mutational burden (TMB). Ferroptosis, an iron-dependent oxidative cell death program, has emerged as a dual-function regulator of tumor immunity. Whether ferroptotic dysregulation of the PDL stromal compartment and MT1/MT2 suppression constitute novel mechanistic determinants of ICI resistance in OSCC remains poorly understood and warrants mechanistic investigation.

METHODS: We performed a single-cell RNA sequencing (scRNA-seq) analysis of a publicly available multi-regional OSCC dataset (GEO: GSE198315; adjacent non-tumor [NT], tumor core [TC], and metastatic lymph node [mLN]; 22,371 quality-controlled cells), encompassing dimensionality reduction, pseudotemporal trajectory reconstruction, immune checkpoint gene profiling (PD-1/PD-L1/CTLA-4/TIM-3/LAG-3), ferroptosis-immune crosstalk inference, and intercellular communication modeling. To experimentally validate key computational findings, we performed in vitro RT-qPCR experiments in primary human PDL fibroblasts exposed to oxidative conditioned medium (OCM), quantifying the expression of GPX4, ACSL4, MTNR1A (MT1), MTNR1B (MT2), and CD274 (PD-L1) at 24 h and 48 h.

RESULTS: scRNA-seq revealed that OCM-exposed PDL cells exhibited a 3.2-fold elevation in the ferroptotic index, co-occurring with profound MT1/MT2 suppression and circadian axis disruption. Ferroptosis-susceptible PDL subpopulations showed coordinated upregulation of immunosuppressive ligands (PD-L1, CD47, and Galectin-9), reduced CD8+ T-cell infiltration scores, and enhanced myeloid-derived suppressor cell (MDSC) and tumor-associated macrophage (TAM) recruitment signals, suggesting a mechanistic association between PDL stromal ferroptosis and immune checkpoint-mediated immune evasion. In vitro RT-qPCR validation in primary human PDL fibroblasts (ATCC PCS-201-018) exposed to OCM confirmed significant downregulation of GPX4, SLC7A11 (xCT), MTNR1A (MT1), and MTNR1B (MT2), concurrent with the upregulation of ACSL4 and CD274 (PD-L1) at both 24 h and 48 h (all p < 0.05), directly corroborating the scRNA-seq-predicted ferroptosis-melatonin receptor-immune checkpoint co-regulatory axis.

CONCLUSION: We establish a mechanistically grounded framework suggesting that PDL stromal ferroptosis and MT1 suppression constitute novel components of the OSCC tumor immune evasion landscape. In vitro RT-qPCR validation supports the ferroptosis-melatonin receptor-immune checkpoint co-regulatory axis as a candidate therapeutic target to overcome immunotherapy resistance in oral cancer, pending prospective clinical validation.