Validation of chemoresistance phenotypes in pleural mesothelioma across 2D, 3D, and in vivo models

Pleural mesothelioma (PM) is a highly aggressive cancer with limited treatment efficacy and poor prognosis. Conventional two-dimensional (2D) culture models fail to replicate the tumour microenvironment (TME), limiting their translational relevance. Here, we establish a three-dimensional (3D) spheroid model to investigate chemotherapy resistance across different PM subtypes. Compared to 2D cultures, 3D spheroids display enhanced resistance to cisplatin-pemetrexed, with elevated IC₅₀ values, reduced apoptosis, and altered cell cycle profiles. Seahorse metabolic analysis of 3D spheroids demonstrate a suppressed metabolic phenotype, characterised by reduced oxidative phosphorylation (OCR). However, the glycolytic capacity was not upregulated, consistent with the hypoxic and nutrient-limited conditions observed in mesothelioma lesions. In parallel, molecular profiling identifies subtype-specific miRNA signatures that closely align with patient-derived datasets. Proteomic analysis of 3D cultures identifies upregulation of PI3K/AKT and Notch/VEGF signalling, implicating these pathways in treatment resistance. Histological assessment of xenografts further confirms 3D model fidelity in capturing tumour fibrosis, necrosis, and response to therapy. These findings position the 3D spheroid system as a robust and physiologically relevant platform for modelling drug resistance and guiding therapeutic development in PM.