Al-Akkad Abu-Zeina, Walid; (2020) Decellularised Human Pancreata and Liver for the study of Pancreatic Ductal Adenocarcinoma: Development, Metastasis and Chemoresistance. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background and Aims: Over 80% of patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed with concurrent metastases. Over the last 50 years, conventional treatment approaches have had little impact on the course of this disease. Therefore, the development of new treatment strategies to control PDAC is needed. We propose the use of 3D extracellular matrix (ECM) scaffolds that could redefine in vitro models of PDAC and preclinical testing of novel therapies. Methods: Decellularised human pancreata and livers were characterised for the elimination of cellular material and preservation of ECM proteins and micro-architecture using histology, immunohistochemistry (IHC) and quantification kits. Both primary (PANC-1 and MIA PaCa-2) and metastatic pancreatic tumour cells (PK-1) were seeded onto 5 mm3 scaffolds, as well as 2D cultures. Histological analyses were used to confirm cell attachment and migration/invasion. Further, changes in protein expression (IHC) and gene expression (qPCR and RNAseq) were evaluated at day 14 post reseeding. Treatments with doxorubicin and Gemcitabine were performed; viability (AlamarBlue), protein expression (IHC) and gene expression (RNAseq) analyses were performed to test therapy-resistance in the 3D systems. Results: All primary PDAC cell lines were able to migrate and invade the pancreas scaffolds whereas several of these cells were only able to attach superficially onto the liver scaffolds. PK1 cells were able to exclusively migrate and invade the liver scaffolds and only attached superficially onto the pancreatic scaffolds. These differences were supported by significant deregulations in gene and protein expression (i.e. MMP9, WNT1, β-CATENIN) between pancreas scaffolds, liver scaffolds and 2D culture. Interestingly, both primary and metastatic cells were found significantly more resistant to all chemotherapy treatments in the 3D models when compared to 2D cultures, even though confocal microscopy confirmed the uptake of drugs into the cells. Conclusion: Our results suggest that primary and metastatic pancreatic cancer cells manifest a conserved invasive behaviour depending on the 3D ECM structure of origin. Moreover, there is an evident alteration in cell response to different cancer-therapies in the presence of a natural ECM niche. These observations provide a proof of concept for the development of an effective bio-engineered model for drug discovery, therapy screening and biomarker discovery.

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