Longo, Viviana (2026) Molecular and cellular investigation of gastric epithelial homeostasis following intrinsic and extrinsic environmental alterations. [Tesi di dottorato]

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Abstract

The stomach is a functionally complex organ with a dynamic and highly heterogeneous epithelial architecture, constantly undergoing regeneration while withstanding a perpetually harsh luminal environment. This intricate organization presents a significant challenge for modern biomedical research, as the high degree of cellular heterogeneity effectively masks the specific molecular and functional contributions of individual cell types to homeostasis and pathology, such as gastric carcinogenesis. To overcome this, advanced three-dimensional (3D) human gastric organoid (hGO) and polarized mucosoid models are critical ex vivo platforms for studying epithelial behaviour, cell lineage dynamics, and the complex interactions within the gastric microenvironment. This thesis shows a multi-faceted approach leveraging these advanced human gastric models to deconstruct the cellular and molecular mechanisms governing gastric integrity and homeostasis. This work focuses on three interconnected objectives: first, developing novel tools for the isolation and characterization of specific human gastric epithelial cytotypes; second, elucidating the molecular networks related to tumour-associated macrophages within the immunosuppressive environment of diffuse-type gastric cancer; and third, characterizing the cytotoxic and genotoxic effects of bile acids (BAs) on the highly susceptible gastric cardia epithelium, modelling the effects of bile reflux. To pursue the first objective, human gastric organoids (hGO) were employed to generate engineered and cytotype specific promoter-driven reporter systems to overcome cellular heterogeneity and to distinct epithelial putative progenitor cells. Then, to reach the second objective, in this thesis it has been shown that the Chloride Intracellular Channel 2 protein, expressed primarily in macrophages and endothelial cells of diffuse-type gastric cancer, supports an immunosuppressive immune phenotype by modulating critical intracellular signalling pathways, specifically linking CLIC2 absence to the downregulation of the IL6/STAT3 signalling axis. These results underwent peer review, which ultimately led to the publication of this research (Longo et al., 2025). Finally, the third objective has been achieved by establishing the specific cytotoxic profile of bile acids (BAs) in both submerged and polarized models, confirming that secondary and glyco-conjugated BAs are the most potent stressors, and critically, validated that the integrity of the epithelial barrier is fundamentally reliant on the functional protective capability of the apical mucus layer against chemical insult. The maintenance of gastric epithelial homeostasis is a fragile and highly coordinated process requiring precise intrinsic mechanisms of cellular renewal and fate determination, tightly regulated microenvironmental signalling that defines immune responses, and robust physical defences against external chemical challenges, such as bile reflux. This research collectively provides both essential methodological tools (validated reporter systems, patients-derived organoids and the polarized mucosoid platform) and key mechanistic insights, demonstrating that successful epithelial self-renewal, the regulation of resident macrophages signalling, and the functional integrity of the mucus barrier are interdependent elements governing the gastric tissue's resilience. By dissecting these components, this work strengthens the foundation for future studies aimed at unravelling the complex relationship between environmental stress, cellular identity, and the failure of homeostasis that precipitates gastric disease.

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