Implementing human derived ex vivo model to explore cholangiocarcinoma:
Cholangiocarcinoma (CCA) is an aggressive hepatobiliary malignancy with increasing incidence and persistently poor prognosis. The UK incidences have rapidly increased with 2500 cases being diagnosed annually. Mortality from CCA has doubled between 2001-2017, with areas of high deprivation disproportionally affected. The Northwest and North Wales have some of the highest incidence rates. Late-stage diagnosis often precludes curative resection and chemoresistance means therapeutic options are limited. As a modern civic university improving our understanding and treatment of HPB cancers is a 21st century challenge our Strategic Plan seeks to address.
In 2016 I secured an LJMU ECR fellowship to enable me to visit the Olinga lab (Groningen) to train in precision cut liver slices. Since 2020, I have secured £299,043 external funding from NC3Rs and North West Cancer Research to establish Precision cut tumour slices (hPCTS) in Liverpool. These patient derived, ex vivo 3D cellular structures can recapitulate critical aspects of cancer biology and crucially retain all aspects of the tumour microenvironment , to enable exploration of HPB cancers and investigate therapeutic drug response, cellular bioenergetics, validation of novel drug targets and aid in biomarker discovery.
This has been facilitated by fruitful collaborations with one of the UKs largest HPB units based at Liverpool University Hospital Trust, Mr Hasan Malik and Mr Rob Jones (Consultant HPB surgeons) and UoLiv colleagues, Prof. Chris Goldring (Drug Safety Science), Dr Lekh Dahal (Immunopharmacology) and Dr Amy Chadwick (Bioenergetics) enabling me to secure an Honorary Senior Lecturer role within Pharmacology and Therapeutics at UoLiv. I have established links with Prof. Ray Yeung (Washington) and Revivocell. In collaboration with Dr Stewart Chidlow (Mathematics, LJMU) and Dr Ross Kelly (Syngenta), I have utilised mathematical modelling to optimise our incubation parameters which has generated a recent PLOSone publication. I believe this translational model can bridge the gap from in vitro to in vivo models and man. The versatility of this technique means that it can easily be adapted for use on healthy and disease tissue from a variety of organs and species providing ample avenues for future exploration and collaboration and supporting my upward research trajectory. I anticipate at least four publications from the Ph.D. student’s work over the next five years, helping sustain activity in this area.
Developing Novel, Safe, Effective and Affordable Anti-malarial Drugs:
Every 60 seconds, a child dies of malaria. This equates to 1,300 children losing their lives every day. With an estimated 247 million cases and 619,000 attributable deaths reported globally (2021), malaria remains a significant clinical problem and represents a challenge to which LJMU could be involved in developing novel solutions.
In collaboration with colleagues in the medicinal chemistry research group (Dr Fyaz Ismail, Dr Alastair Fielding and Prof. Satya Sarker at LJMU, I was awarded an ECR Ph.D. Studentship (£68,090) to determine the safety profile of novel anti-malarial compounds using a variety of in vitro and ex vivo ADMET techniques in order to facilitate safer drug design and led to the publication of 2 research articles (del Casino et al., 2018 and Fielding, et al., 2017).
This project developed from my UG research project exploring the metabolism of Isoquine, a novel 4-aminoquinoline anti-malarial in collaboration with Prof. Paul O’Neill (UoLiv) and Prof. Steve Ward (Liverpool School of Tropical Medicine) which was subsequently published in J. Med. Chem (2003). These studies progressed to pre-clinical drug safety testing with GSK and are currently under further development.
Investigation the role of Nrf2-Keap1-ARE in hepatocellular defence:
Drug induced liver injury (DILI) is a major complication during drug development, with adverse reactions constituting a significant health and economic issue. The Keap1-Nrf2-ARE signalling pathway plays an essential mechanism for defence against chemical stress. My interests lie in understanding the chemical and cellular signalling systems that determine DILI, from modification of target protein(s) through to pathological outcome.
My Ph.D. studies in collaboration with Prof. Chris Goldring and Prof. Kevin Park (Liverpool University) explored liver defensive mechanisms in particular the ability of paracetamol and other model hepatoxins to induce the Nrf2-Keap1 signalling cascade in vivo, which subsequently formed a high quality publications (Goldring et al., 2004 and Randle et al., 2008).
During my Pfizer funded post-doctoral studies in collaboration with Dr Peter J. O’Brien and Dr Paul Hayter (Safety Sciences, Pfizer PGRD), I focused on the Nrf2-Keap1-ARE signalling pathway and defence gene induction in in vitro systems. The aim was to develop a fluorescent hepatotoxicity screen for the Cellomics HCS platform to assess the validity of transcription factor activation as a screen for determining oxidative stress in new chemical entities (Phillips, 2005). We also attempted to define the chemical and molecular mechanisms of cysteine modification that regulate the Nrf2-Keap1-ARE pathway and the consequent levels of defence gene induction. These studies were published in Hepatology (Copple, 2008).
My Wellcome Trust funded post-doctoral Research project in collaboration with Dr Neil Kitteringham and Prof. Kevin Park (MRC Centre for Drug Safety Science, UoLiv) was to use shotgun proteomics to define the role of Nrf2 in the hepatic adaptive response. Initially, we determined the basal cellular defence profile in an Nrf2 transgenic mouse colony, which I established and managed. I subsequently induced the Nrf2-Keap1-ARE signalling cascade using a series of model hepatotoxins during both acute and chronic toxicity These studies we later published in J. Proteome Research (Kitteringham et al, 2010) and Scientific Reports (Eakins et al., 2015).
Exploring hepatoprotection with α-Adrenergic Antagonists:
This research and its continuation has the potential to significantly impact on the clinical treatment of paracetamol overdose patients. My Ph.D. was in collaboration with Dr Dom Williams, Dr Neil Kitteringham (University of Liverpool) and Dr Yuri Clement (University of Trinidad and Tobago) investigated protective mechanisms within the liver, ‘examining the role of adrenergic modulation of chemical-induced hepatotoxicity’. Previous studies had suggested an important role for adrenoceptors in modulating the susceptibility of the liver to hepatotoxicity. My research has shown that α1-adrenergic antagonist, prazosin can provide hepatoprotection against paracetamol induced liver injury. This protection was not due to alterations in the metabolism of the hepatotoxicant. It appears that α1 antagonist can counteract stress induced vasoconstriction with the hepatic microvasculature, helping to maintain a nutritive blood flow to the liver and aiding hepatocellular regeneration.
In conjunction with Dr. Jean Satish, I developed an assay using fluorescently labelled red blood cells and FACS analysis to determine hepatic congestion which was published in Br. J. Pharmacology (Randle et al., 2008). Preliminary pre-clinical imaging supports our hypothesis that maintenance of hepatic blood flow underlies this hepatoprotective mechanism.