Di Palma, Rosita (2026) Modeling Idiopathic Pulmonary Fibrosis from 2D to 3D cultures: Functional characterization of AGR2-related pathways in human lung models. [Tesi di dottorato]

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	Documento PDF 38PNRR_Biologia_Di_Palma_Rosita_PhD_Thesis_Partial.pdf Visibile a [TBR] Amministratori dell'archivio Download (9MB) | Richiedi una copia

Abstract

Idiopathic pulmonary fibrosis (IPF) is increasingly recognized as an epithelial‑driven disease in which aberrant repair, chronic stress responses and a distorted microenvironment converge to promote irreversible scarring of the lung parenchyma. This thesis investigates the role of the ER protein disulfide isomerase AGR2 and the miR‑143‑3p/GSK3β axis as key regulators of epithelial stress and epithelial–stromal crosstalk in IPF and develops a suite of human cell‑based models to study these pathways at multiple levels of complexity. After bioinformatic re‑analysis of bulk and single‑cell transcriptomic datasets, AGR2 is identified as a hub gene upregulated in IPF lung tissue and enriched in stressed AT1‑like and aberrant epithelial populations that are transcriptionally connected with fibroblasts, macrophages and endothelial cells. In parallel, miR‑143‑3p emerges as a putative post‑transcriptional regulator of AGR2 within a network involving Wnt/β‑catenin and GSK3β signalling. To functionally interrogate these networks, the thesis first establishes an inflammatory 2D fibroblast model and fibroblast–epithelial co‑cultures, showing that LPS/TNF‑α stimulation and fibroblast activation state modulate AGR2‑related pathways, collagen deposition and microRNA expression. Building on this, advanced 3D systems are developed, including human lung alveospheres generated from primary alveolar epithelial cells and donor or IPF fibroblasts, as well as lung organoids derived from cryopreserved primary lung cell suspensions. These models reveal that highly activated IPF fibroblasts drive the emergence of AGR2high, CK8⁺/CK17⁺ aberrant epithelial structures resembling basaloid and bronchiolized lesions, whereas donor fibroblasts support more physiological alveolar‑like spheroids. Organoid cultures further demonstrate that biobanked material retains multipotent progenitors capable of generating bronchioalveolar‑, bronchial‑ and alveolar‑like organoids, which can be expanded over serial passages. Finally, the thesis provides proof‑of‑concept that AGR2 and associated pathways can be modulated using pharmacological including GSK3β inhibitors and explores the relevance of these strategies in lung cancer models in which AGR2 and GRP78 contribute to chemoresistance. Collectively, the work establishes a coherent experimental framework that links patient‑derived omics data to mechanistic studies in progressively more complex human models, supporting a view of IPF as a disorder of dysregulated epithelial plasticity within an altered niche and laying the groundwork for future precision‑oriented therapeutic interventions targeting AGR2‑centred networks.

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