Immunomodulatory effects of short-chain fatty acids and immune-supporting nutrients on slice cultures of head and neck tumors.

INTRODUCTION: Head and neck squamous cell carcinoma (HNSCC) has a highly immunosuppressive tumor microenvironment (TME), which limits the effectiveness of conventional and immunotherapies. Metabolites derived from the gut microbiota, such as short-chain fatty acids (SCFAs), and targeted nutritional interventions, including immunonutrition (IN), have been proposed as ways of influencing tumor immunity and cell viability. However, the effects of these factors on the complex TME of HNSCC remain incompletely understood. Patient-derived organotypic slice cultures (SC) therefore provide a clinically relevant model to study these interactions. METHODS: SC were generated from tumors of nine HNSCC patients and cultured under four conditions: control; SCFAs; IN (glutamine, alanine, and omega-3 fatty acids); and SCFAs combined with IN, for 4 days. Apoptotic activity was assessed via cleaved caspase-3 (CC3), and cytotoxic activity via Granzyme B (GrB) staining. Inflammatory markers (IL-1β, IL-6, TNFα and IFNγ) were quantified in cultured and treated tissue, as well as in the tissue's supernatant. Quantitative immunohistochemistry (IHC) - based image analysis and dot blot assays were combined with statistical evaluation of patient- and treatment-specific effects. RESULTS: Treatments with IN alone or in combination with SCFAs significantly reduced CC3 intensity, indicating decreased apoptosis. However, SCFA treatment alone increased CC3 intensity in SC of certain patients. GrB IHC intensity remained largely stable, with patient-specific differences driving the observed variability. Among the cytokines analyzed in the SC supernatants, TNFα and IL-1β were selectively modulated by IN and combined treatment, while IL-6 and IFN-γ remained largely unchanged. Analysis of cultured and treated tissue mirrored these trends, with TNFα and IFN-γ showing minimal variation and IL-6 being almost undetectable. These findings highlight significant heterogeneity in apoptotic and immune responses among patients. CONCLUSION: SCFAs and IN exert modest but selective effects on apoptosis and inflammatory pathways in HNSCC, whereas cytotoxic activity remains stable. These results support the potential of tailoring metabolic and nutritional interventions to individual patients to modulate the tumor immune microenvironment, and provide a rationale for integrating SCFAs and IN with immunotherapeutic strategies in HNSCC.