Biology subjects

Paish, H.

Publications and source records attributed to Paish, H..

3 recordsLinked to original sources

bioRxiv2024

Ex-vivo mouse precision cut tumour slices for modelling hepatocellular carcinoma; A 3Rs solution for at-scale drug screening

Disease modelling is vital for improving knowledge of disease mechanisms and for development of new therapeutic molecules and strategies. Modelling the intact living tumour microenvironment (TME) is increasingly considered to be vital not only for gaining a better understanding of the biology of cancer but for examining the efficacy of novel oncology drugs. To date, pre-clinical mouse models of cancer have represented the mainstay methodology for studying the evolving TME and for determining the effects of potential therapeutic molecules on tumour evolution and growth. Regarding drug screening, in vivo mouse models are expensive, require the use of large cohorts of mice and involve the administration of drugs with unknown toxicities to animals which often result in adverse effects that can cause animal suffering and the discontinuation of drug investigations. Hepatocellular carcinoma (HCC) is a primary cancer of the liver for which there is an urgent need for improved systemic treatments due to the disease usually being diagnosed at an advanced stage and current treatments having limited efficacy. To provide a practical solution to the screening of drugs for their likely efficacy in HCC we have developed an ex-vivo model in which orthotopic tumours are excised from the liver and subsequently processed to generate precision-cut tumour slices (PCTS) which provide an intact culture model of the HCC-TME. We describe simplified culture conditions that maintain the viability and metabolic activity of live PCTS which maintain the architecture, cellular complexity, drug sensitivity and responsiveness to immunotherapy of the original tumour. Importantly, we show that HCC derived PCTS can be miniaturised to 96-well scale and modified to express soluble luciferase, which in combination enabled non-destructive screening of a library of 26 drugs at two doses using just 5 tumours as the source for PCTS. This screen identified two small molecules, salinomycin and rottlerin, that have potent anti-tumour activities in HCC-PCTS and subsequently validated salinomycin as effective in vivo. In summary, we report a 3Rs (reduction, refinement and replacement) solution for study of HCC biology and for 96-well-scale screening of potential therapeutic agents in the context of an intact, metabolically active TME.

cancer biology↗

bioRxiv2019

mTORC1-Plin3 pathway is essential to activate lipophagy and protects against hepatosteatosis.

During postprandial state, the liver is exposed to high levels of dietary fatty acids (FAs) and carbohydrates. FAs are re-esterified into triglycerides, which can be stored in lipid droplets (LDs) in the liver. Aberrant accumulation of LDs can lead to diseases such as alcoholic liver disease and non-alcoholic fatty liver disease, the latter being the most common liver pathology in western countries. Improved understanding of LD biology has potential to unlock new treatments for these liver diseases. The Perilipin (Plin) family is the group of proteins that coat LDs, controlling their biogenesis, stabilization, and preventing their degradation. Recent studies have revealed that autophagy is involved in LD degradation and, therefore, may be crucial to avoid lipid accumulation. Here, we show that a phosphorylated form of Plin3 is required for selective degradation of LDs in fibroblasts, primary hepatocytes and human liver slices. We demonstrate that oleic acid treatment induces the recruitment of the autophagy machinery to the surface of LDs. When Plin3 is silenced, this recruitment is suppressed resulting in accumulation of lipid. Plin3 pulldowns revealed interactions with the autophagy initiator proteins Fip200 and Atg16L indicating that Plin3 may function as a docking protein involved in lipophagy activation. Of particular importance, we define Plin3 as a substrate for mTORC1-dependent phosphorylation and show that this event is decisive for lipophagy. Our study therefore reveals that Plin3, and its phosphorylation by mTORC1, is crucial for degradation of LDs by autophagy. We propose that stimulating this pathway to enhance LD autophagy in hepatocytes will help protect the liver from lipid-mediated toxicity thus offering new therapeutic opportunities in human steatotic liver diseases.

cell biology↗

bioRxiv2018

A novel bioreactor technology for modelling fibrosis in human and rodent precision-cut liver slices.

Summary boxO_LIWhat is already known about this subject?\nO_LICurrently there are no effective anti-fibrotic drugs to treat liver fibrosis and there is an urgent unmet need to increase our knowledge of the disease process and develop better tools for anti-fibrotic drug discovery.\nC_LIO_LIPreclinical in vitro cell cultures and animal models are widely used to study liver fibrosis and test anti-fibrotic drugs, but have shortfalls; cell culture models lack the relevant complex cell-cell interactions of the liver and animal models only reproduce some features of human disease.\nC_LIO_LIPrecision Cut Liver Slices (PCLS) are structurally representative of the liver and can be used to model liver fibrosis and test anti-fibrotic drugs. However, PCLS are typically cultured in elevated, non-physiological oxygen levels and only have a healthy lifespan of 48h.\nC_LI\nC_LIO_LIWhat are the new findings?\nO_LIWe have developed a novel bioreactor culture system that increases the longevity of functional PCLS to up to 6 days under normoxic conditions.\nC_LIO_LIBioreactor cultured PCLS can be used to model fibrogenesis in both normal and fibrotic PCLS using a combination of biochemical and histological outputs.\nC_LIO_LIAdministration of an Alk5 inhibitor effectively limits fibrogenesis in normal rodent and human PCLS and in rodent PCLS with established fibrosis.\nC_LI\nC_LIO_LIHow might it impact on clinical practice in the foreseeable future?\nO_LIThe extended longevity of bioreactor cultured PCLS represent a novel pre-clinical tool to investigate the cellular and molecular mechanisms of liver fibrosis.\nC_LIO_LIBioreactor cultured human PCLS offer a clinically relevant system to test efficacy of anti-fibrotic drugs.\nC_LI\nC_LI\n\nAbstractO_ST_ABSObjectiveC_ST_ABSPrecision cut liver slices (PCLS) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two-dimensional mono or co-cultures. The objective of this study was to develop a bioreactor system to increase the healthy lifespan of PCLS and model fibrogenesis.\n\nDesignPCLS were generated from normal rat or human liver, or 4-week carbon tetrachloride-fibrotic rat liver and cultured in our patented bioreactor. PCLS function was quantified by albumin ELISA. Fibrosis was induced in PCLS by TGF{beta}1 and PDGF{beta}{beta} stimulation. Alk5 inhibitor therapy was used. Fibrosis was assessed by fibrogenic gene expression, Picrosirius Red and Smooth Muscle Actin staining, hydroxyproline assay and collagen 1a1, fibronectin and hyaluronic acid ELISA.\n\nResultsBioreactor cultured PCLS are viable, maintaining tissue structure and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell cultured PCLS rapidly deteriorate and albumin secretion is significantly impaired by 48 hours. TGF{beta}1 and PDGF{beta}{beta} stimulation of rat or human PCLS induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts and histological fibrosis. Fibrogenesis slowly progresses over 6-days in cultured fibrotic rat PCLS without exogenous challenge. Alk5 inhibitor limited fibrogenesis in both TGF{beta}1 and PDGF{beta}{beta} stimulated PCLS and fibrotic PCLS.\n\nConclusionWe describe a new bioreactor technology which maintains functional PCLS cultures for 6 days. Bioreactor cultured PCLS can be successfully used to model fibrogenesis and demonstrate efficacy of an anti-fibrotic therapy.

cell biology↗