The Role of the Microbiota in the Carcinogenesis and Progression of Squamous Cell Carcinoma: A Multi-Omic Investigation of Oral and Lung Tumours

Squamous cell carcinomas of the aerodigestive tract, including Lung Squamous Cell Carcinoma (LUSC) and Head and Neck Squamous Cell Carcinoma (HNSCC), share common etiological risk factors, anatomical continuity, and biological features. While genetic and environmental drivers are well established, increasing evidence suggests that the resident microbiota represents an active component of the tumour microenvironment (TME). However, its spatial organization, metabolic potential, and co-evolution with the host immune system during carcinogenesis remain poorly understood. This thesis investigates the hypothesis that malignant progression is associated with a non-random ecological succession of the local microbiome and its predicted interaction with host cellular and immune compartments across the continuum of disease. A multi-cohort, multi-omic study design was employed to characterise host–microbiome interactions in tissue, saliva, and plasma samples spanning pre-malignant, malignant, and post-treatment states. First, a discovery cohort of 72 LUSC patients was analysed using paired tumour and normal adjacent tissues profiled by 16S rRNA gene amplicon sequencing, complemented by spatially resolved transcriptomics and in situ validation. Second, to investigate early carcinogenic mechanisms, 66 oral potentially malignant disorder (OPMD) specimens underwent bulk RNA sequencing, enabling host molecular stratification and integrated metatranscriptomic analyses. Finally, disease trajectory was further examined in a progression cohort (n = 74) comprising oral premalignant lesion (OPL), HNSCC, and post-treatment Active Surveillance patients, using saliva shotgun metagenomics and multiplex plasma cytokine profiling. In LUSC, tumour and paired normal tissues exhibited broadly conserved microbial composition. However, tumour stage emerged as a key determinant of microbial structure, with late-stage tumours showing progressive enrichment of oral-associated taxa, including Fusobacterium, Prevotella, and Leptotrichia. Spatial analyses revealed that these bacteria preferentially localised within tumour-associated stromal niches enriched for malignant cells and cancer-associated fibroblasts, consistent with an immune-excluded microenvironment. Integrated host–microbiome analyses in OPMDs identified a high-risk hypoxia-associated molecular subtype characterised by the presence of metabolically active Fusobacterium nucleatum and distinct stromal and epithelial interactions. Across the disease continuum, oral carcinogenesis was marked by a dynamic ecological remodelling: the transition from OPL to HNSCC was characterised by a shift in metabolic pathway abundance. Pre-malignant lesions exhibited microbial functions linked to pro-carcinogenic inflammation, whereas invasive HNSCC displayed microbial adaptations consistent with metabolic stress tolerance and persistence within the TME. This microbial trajectory was linked with a host immune remodelling, shifting from a pro-inflammatory state in pre-malignancy to immune exhaustion in advanced cancer. Finally, post-treatment patients demonstrated a distinct microbial and immunological equilibrium indicative of tissue repair and immune reconstitution. This thesis demonstrates that the microbiome is an integral and dynamic component of the tumour microenvironment in aerodigestive tract squamous cell carcinomas. The results show that microbial changes during malignant progression are structured and follow defined ecological trajectories, characterised by spatial niche adaptation and metabolic remodelling that parallel host immune evolution. Collectively, these findings support a conceptual framework in which carcinogenesis is shaped by host–microbiome co-evolution and suggest that the microbiome may hold value for risk stratification or future therapeutic strategies.