Ney, Alexander; (2023) Decellularised human pancreatic tissue constructs as a platform for Pancreatic Neuroendocrine Tumours (pNETS) biomarker discovery. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background and aims: Pancreatic neuroendocrine tumours (pNETs) are rare epithelial neoplasms which account for ~4% of primary pancreatic malignancies. Disease rarity and biological heterogeneity challenge pNET research which further complicates their diagnosis and management. Understanding complex interactions between pNETs and their micro-environment is key for advancing biomarker discovery. Culturing cancer cells in their native extracellular matrix (ECM) mimic in vivo, tissue specific cell-ECM interactions and tissue remodelling. We aimed to characterise a novel, clinically relevant 3-dimensional in vitro disease model and identify products of pNET cell-ECM interactions as biomarkers of pNETs. / Methodology: Whole human pancreata were decellularised by perfusion. The elimination of cellular material, preservation of tissue architecture and composition were determined by DNA quantification, histology and LC-MS/MS, respectively. Decellularised tissue explants were re-cellularised with BON-1 and QGP-1 cells, and their viability and proliferation in the 3D culture model was studied using proliferation assays. Preservation of the neuroendocrine phenotype and secretory functions were studied by qRT-PCR and confirmed by immunohistochemistry (IHC). The adaptation of the cell proteome to the native culture substrate (versus monolayer cultures) was studied by LC-MS/MS with isobaric tag labelling (iTRAQ). Biological pathway enrichment and functional interactions were mapped using iPathwayGuide and STRING bioinformatics tools. The expression of top upregulated proteins was validated in surgically resected pNETs and fine needle biopsies using IHC. / Results: Decellularisation by perfusion preserved the native tissue architecture and key constituents of the native ECM. Cells grown in this model showed stable proliferation and propagation over 28 days in culture with invasion of the culture matrix, with preservation of their neuroendocrine phenotype and secretory function. LC-MS/MS identified 2,149 proteins of which 503 were differentially expressed (487 unregulated and 116 down-regulated) in 3D cultured cells. Biological pathway enrichment analysis (KEGG) and gene set enrichment analysis (GSEA) demonstrated enrichment of cell-ECM interaction pathways (i.e., ’ECM-receptor interaction’, ‘focal adhesion’, and ‘proteoglycans in cancer’), and up-regulation of proteins associated with cancer. Tumour expression of the cell-ECM linker tetraspanin-7, ECM remodelling enzymes (e.g., P4HA1, PLOD2) and deposition of ECM constituents (COL6A) which were upregulated in 3D grown cells, was confirmed in patient samples. / Conclusions: pNET cells cultured in the native pancreatic ECM are a clinically relevant model for the study of tumour cell-ECM interactions. Our results demonstrated successful clinical translation of the findings in patient tumour sections, suggesting a role for these proteins as pNET biomarkers and potential points for therapeutic targeting. This 3-dimensional culture model offers a platform for future studies of pNET biomarkers discovery and testing of novel approaches in treatment of pNETs.

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