Biomedical Sciences

Supervisory Team; Dr Abhishek Gupta, Senior Lecturer in Physiology and Pharmacology, Aaron Vance, Lecturer in Engineering, Professor Neil Ashwood, Consultant Orthopaedic Surgeon

About the Project:

This project requires a candidate with interdisciplinary knowledge and/ or the commitment to explore beyond their previous experience.

This research endeavour presents a captivating opportunity to delve into revolutionary antimicrobial osteogenic biomaterials through Laser Powder Bed Fusion (L-PBF), marking a potential breakthrough in bone tissue engineering. By utilising the versatility of L-PBF technology and incorporating antimicrobial agents into osteogenic biomaterials, this study aims to meet the critical demand for personalised implants capable of both bone regeneration and innate resistance to microbial infections, especially crucial in orthopaedic procedures. The combination of osteogenic attributes with antimicrobial functionality will hold significant promise for customising implant materials and greatly enhancing patient outcomes.

The project materials innovation phase will identify emerging antimicrobial agents such as antimicrobial peptides, silver nanoparticles, or antibiotic-eluting polymers, and seek to embed them within osteogenic biomaterial matrices. Through rigorous experimentation, including the optimisation of L-PBF parameters and comprehensive characterisation, the research will aim to craft antimicrobial osteogenic biomaterials distinguished by heightened structural integrity, biocompatibility, and antimicrobial effectiveness. This pioneering approach will not only tackle current clinical challenges but also set the foundation for innovative strategies to combat implant-related infections, potentially diminishing the need for antibiotic therapy and revision surgeries.

Moreover, the exploration of antimicrobial osteogenic biomaterials via L-PBF will offer a compelling avenue for personalisation, facilitating the translation of research outcomes into practical applications tailored to individual patient needs. The opportunity to collaborate with clinical partners will provide invaluable insights into real-world challenges and further refine biomaterial development to match clinical requirements. The use of Finite Element Analysis (FEA) and Computational Fluid Dynamics (CFD) will enable the simulation of mechanical behaviour and fluid dynamics surrounding the fabricated implants under physiological conditions, providing crucial insights into their long-term functionality, and ensuring suitability for clinical use. Furthermore, this research will hold promise for commercialisation, with opportunities for patentable innovations. By bridging the gap between research and clinical practice, this ambitious project aims to advance both scientific understanding and patient care, driving innovation in bone tissue engineering and beyond.

For more information: For an informal discussion please contact via direct email to Dr Abhishek Gupta (a.gupta@wlv.ac.uk)

Supervisory Team

Dr Omar Hafid

School of Life Sciences, FSE, University of Wolverhampton, Wulfruna Street, Wolverhampton, UK.

Research Institute of Healthcare Science (RIHS), Faculty of Science and Engineering, University of Wolverhampton, Wulfruna Street, Wolverhampton, UK.

Dr Aikaterini Karakoula

School of Pharmacy, Faculty of Science and Engineering (FSE), University of Wolverhampton, Wulfruna Street, Wolverhampton, UK.

Research Institute of Healthcare Science (RIHS), Faculty of Science and Engineering, University of Wolverhampton, Wulfruna Street, Wolverhampton, UK.

Professor Matthew Brookes

Research Institute of Healthcare Science (RIHS), Faculty of Science and Engineering, University of Wolverhampton, Wulfruna Street, Wolverhampton, UK.

Background

The gastrointestinal tract is in contact with a huge variety of diverse pathogenic and commensal microbiota. Therefore, a balance between immunity and immune tolerance is required. Crohn’s disease (CD) is a chronic relapsing incurable inflammatory bowel disease (IBD) affecting 165/100000 people in the UK. The role of the gut microbiota is increasingly considered to be an important factor in the aetiology of IBD.

The cytokine milieu in the intestine is also an important factor in the maintenance of the immune balance and in IBD this balance is dysregulated resulting in mucosal inflammation. For example, in CD we found that the levels of proinflammatory cytokines are elevated, whereas levels of anti-inflammatory cytokines were reduced.

Dysbiosis or changes in bacterial diversity plays a major role in the progression of CD and associated with changes in the cytokine profile. While manipulating gut flora with probiotics has been effective in prevention of inflammation and maintenance of remission, probiotics are still ineffective in treating an established inflammation.

The application of biotherapeutics such as monoclonal antibodies against certain cytokines have revolutionised the treatment of various diseases including IBD. However, different gut bacteria may enhance different cytokine profile in the gut, particularly, during inflammatory conditions which may affect the efficacy of the biological drug.

Most investigations on the interaction between the gut immune system and the gut bacteria have been carried out on animal models and information on this interaction from human studies are sparse. In addition, we still do not fully know the effect of biological drugs on the bacterial composition and in relation to the systemic cytokine profile in CD patients.

Hence, we will investigate bacterial diversity and immune responses in patients with CD who are under biological treatment.

Aims

We aim to characterise the persistent dysbiosis in CD and examine concurrent changes in both gut flora and serum cytokine profiles in response to Infliximab.

Research Plan

Blood and stool samples will be obtained from IBD patients at three time points: before treatment, 6-8 weeks post treatment and 9 months post treatment. Blood and stool samples will also be obtained from healthy participants as a control group. Systemic TH1 and TH2 cytokines will be determined using Multiplex Bead-Based Immunoassay and flow cytometry techniques. Bacterial DNA will be extracted from the stool samples and the bacterial diversity will be assessed by 16s RNA gene sequence and microbial bioinformatic analysis.

The project will include a wide range of molecular biology and analytical techniques, including NGS, microbial DNA qPCR array, flow cytometry, immunofluorescence, Multiplex Bead-Based Immunoassay, 16S rRNA sequencing and bioinformatic analysis.

Anticipated outcome

Generated data will further our understanding of the bacterial composition/diversity in the human gut and whether changes in this diversity during inflammatory bowel disease is associated with a specific systemic cytokine profile. Results may also lead to optimisation of probiotic preparations for CD treatment.

For an informal discussion please contact via direct email to Dr h.omar6@wlv.ac.uk

Supervisory Team:

Dr Omar Hafid

School of Life Sciences, FSE, University of Wolverhampton, Wulfruna Street, Wolverhampton, UK. Research Institute of Healthcare Science (RIHS), Faculty of Science and Engineering, University of Wolverhampton, Wulfruna Street, Wolverhampton, UK.

Dr Abhishek Gupta

School of Pharmacy, Faculty of Science and Engineering (FSE), University of Wolverhampton, Wulfruna Street, Wolverhampton, UK.

Research Institute of Healthcare Science (RIHS), Faculty of Science and Engineering, University of Wolverhampton, Wulfruna Street, Wolverhampton, UK.

Professor Matthew Brookes

Research Institute of Healthcare Science (RIHS), Faculty of Science and Engineering, University of Wolverhampton, Wulfruna Street, Wolverhampton, UK.

Background

Colorectal cancer (CRC) is a major cause of cancer-related mortality worldwide with an estimated 1.3 million cases diagnosed every year. Tumour bearing individuals are characterised by defects in immune mechanisms that normally eliminate incipient carcinoma. Growing evidence linked inflammation and CRC suggesting that pro-inflammatory microenvironment promotes tumour progression and invasion.

Inflammation is a complex process implicating the interaction of many inflammatory mediators. These include recruitment and activation of various inflammatory immune cells such as neutrophils, basophils, T cells, macrophages and dendritic cells (DC) as well as production of pro-inflammatory cytokines and mediators of inflammation. Interactions between all or some of these mediators can cause damage to the intestinal mucosa and may promote proliferation and metastasis of tumours.

Anaemia is associated with colorectal cancer but its treatment with oral supplementation of iron may prove inappropriate because the accumulation of iron in the intestinal tract can involve the generation of oxidative radicals and inflammatory mediators. This may also lead to infiltration of further inflammatory cells; in particular tumour associated macrophages (TAMs) promoting tumour progression as TAMs may provide tumours with iron rather than withholding it. Infiltration of DC may also play a major role in inducing inflammation. DC are the most potent antigen presenting cells that bridge the innate and adoptive immune systems and can initiate a cytotoxic immune response against cancer cells. However, the tumour may produce factors that induce DC migration to lymph nodes leading to tumour metastasis, especially at early stages of the disease. In addition, L-selectin facilitate the entry of T cells into secondary lymphoid tissues via high endothelial venules. However, we don’t know the effect of oral iron supplementation on DC recruitment into tumours or their migratory function.

Aims

The aim is to compare in normal and tumour tissues as well as serum from iron deficient patients who received either oral or systemic iron supplementation before surgery the following:

1. Infiltration of immune cells and their phenotype in colorectal tumour tissues.

2. Compartmentalisation of immunity and inflammatory markers in colorectal tumour tissues.

3. Iron haemostasis related cytokines in tumour tissues and cytokine profile in the serum.

4. Functional studies to investigate the effects of oral versus systemic iron supplementation on dendritic cell migration, T cell phenotype and activation and on Wnt signalling in peripheral blood mononuclear cells from healthy donors.

A wide range of techniques and protocols will be used in the course of this project including; RT-PCR, qPCR, ELISA, flow cytometry, luminex technology, immunohistochemistry and immunofluorescence microscopy.

Anticipated Outcome

The outcome may reveal whether oral iron supplementation is related to tumour development via changes in the immune profile either systemically or in the tumour microenvironment. The data could be harnessed to devise logical, evidence-based therapy targeting the innate and adaptive immune systems in colorectal cancer patients with iron deficiency anaemia.

For an informal discussion please contact via direct email to Dr h.omar6@wlv.ac.uk

Supervisory Team: Dr. Opeolu Ojo (DOS), Dr. Gavin McNee (Second Supervisor)

About the Project:

Several studies have indicated the link between type 2 diabetes and Parkinson's disease (PD), and how one disease could lead to the development of the other. Specifically, it has been reported that oxidative stress associated with type 2 diabetes could lead death of brain cells observed in PD. Epidemiological studies have also indicated that 32% of people with type 2 diabetes will eventually develop PD. However, studies investigating how PD can contribute to the development of type 2 diabetes are lacking. PD is also associated with significant oxidative stress and the role that oxidative stress characterising PD plays in development of insulin resistance is not yet fully understood.

This project aims at investigating whether physiological changes associated with early stages of PD (including oxidative stress and differential expression of the alpha synuclein gene - a key biomarker for PD) could trigger metabolic changes observed in type 2 diabetes.

This project will employ cutting techniques in cellular and molecular biology to examine how oxidative stressors characterising PD affect metabolic processes in cells involved in glucose homeostasis (such as beta cells, adipocytes and muscle cells). Moreover, changes in glucose uptake and metabolism in cells transfected to over-express alpha synuclein gene will be studied. In addition, this project will observe changes in glucose homeostasis (and other parameters) in fruit fly model of PD in addition to protective effects of some novel incretin-based therapies.

For more information: For an informal discussion please contact via direct email to Dr Opeolu Ojo (o.ojo2@wlv.ac.uk)

Supervisory Team

Director of Studies: Professor Olivia Corcoran, Senior Lecturer in Forensic Analysis, SOLS

Second Supervisor: Dr Hamid Omar, Reader in Immunology, RIHS

Third Supervisor: Dr Ahmed Eissa, Senior Lecturer in Organic Chemistry, SOLS

About the Project:

Cancer metabolomics to earlier diagnose gut and lung cancer in clinical and 3D biomaterials.

A hallmark of cancer progression is the fast metabolic reprogramming of cells in epithelial diseases such as gut and lung cancer. Yet, the 5-year prognosis for gut and lung cancer patients remains stubbornly low. The University of Wolverhampton’s Research Institute for Health Sciences (RIHS) has been successfully externally funded over many years to study the genetic basis of metastasis in brain cancers, from a genomics and proteomics perspective.

This project aims to develop a state-of-the-art metabolic profiling platform thereby extending the global analysis for small molecule prognostic markers for, and therapeutic targets against, epithelial cancers that may metastasise to the brain, ultimately resulting in clinical benefit.

Biomaterials (hydrogels and porous polymer materials) serving as scaffolds for 3D cell culture and tissue engineering are developed in Eissa’s group using modern synthetic chemistry and bioconjugation methods. The supervisory team’s recent work to build an in-house metabolomics database has shown that GC-MS and LC-HRMS technologies are capable of quantifying thousands of small molecule signatures for many diagnostic samples including human lung tissue and faecal samples from a myriad of gut diseases (Corcoran and Omar, 2024). The aim of the proposed work is to extend the 3D scaffold into fast-growing cancer cells to identify prognostic metabolic markers for, and therapeutic targets against, epithelial tumours that may metastasise to the gut, lung and brain.

This interdisciplinary project will create a range of complex architecture materials to serve as scaffolds for culturing gut and lung cells and, ultimately, tissue in 3D. This will involve progressive emulsion templating and additive manufacturing 3D printing technologies. One produced, accurately optimised and validated, scaffolds will be used to establish optimal in vitro tissue models of gut and lung cancers. Gold standard cancer drugs and novel plant-based natural products will be tested for anticancer potential. The outcome will be a robust analytical platform for investigating diverse cancer metabolic signatures in clinical and 3D in vitro models. This will have significant implications for RIHS, increasing the efficiency of the discovery process and translation of biomaterials and deliver a ‘step change’ in accelerating cancer therapy development.

Experience is required in subject areas including Chemistry, Immunology, Biomedical Science, Bioinformatics or a related field. This multidisciplinary project will involve working at the interface between materials chemistry and immunology. Prior experience is desirable but appropriate training in the full range of chemical and biomedical techniques will be provided to the successful candidate. Laboratory work will be in the Rosalind Franklin Building, which houses a broad range of state-of-the-art research facilities suitable for cell culture alongside recently installed state-of-the-art GC-MS and LC-HRMS. The project will involve collaborations with external research groups at the University of Warwick, Imperial College Department of Surgery & Cancer, and groups in Brazil, China and Mexico.

Applications are particularly welcomed from students of all backgrounds that are suitably qualified and highly motivated.

For more information: For an informal discussion please contact via direct email to (Professor) Olivia Corcoran (o.corcoran@wlv.ac.uk)

Supervisory Team:

Dr Ahmed M. Eissa (Senior Lecturer); Professor Weiguang Wang (Chair); Dr Mark Morris (Reader); Dr Vinodh Kannappans (Senior Research Fellow)

About the Project:

Bioprinting is an emerging technology that enables the generation of precisely controlled 3D cell models and tissue constructs. Bioprinting process requires bioinks that contain living cells and biomaterials that mimic the extracellular matrix (ECM) environment; supporting cell adhesion, proliferation and differentiation after printing. There are many different biomaterials (natural or synthetic or a combination of the two as hybrid materials) reported as bioinks for 3D bioprinting. An ideal bioink should possess appropriate mechanical and biological properties of the target tissues and organs, which are essential to ensure correct functionality of the bioprinted tissues and organs. Hydrogels of natural polymers have been widely used in 3D bioprinting as they provide the desired microenvironment mimicking the native ECM for cell attachment and proliferation; however, they usually process poor shape fidelity and limited rigidity. While synthetic polymers may lack the ability to promote cellular adhesion when compared to natural polymers, they are promising candidates as they allow tuneable mechanical and morphological properties. Precursors of porous polymer scaffolds that recapitulate both mechanical and biochemical properties of the native ECM could potentially be utilised as a novel alternative to existing bioinks to generate functional bioprinted tissues. Porosity enables cell infiltration, tissue growth and vascularisation within the structural constructs.

In this project we will utilise the bioprinting technology to develop novel hybrid scaffolds (hydrogels and macroporous polymers) to create an advanced ex vivo 3D tissue model in order to explore efficacy of novel drugs (e.g. anticancer). Recent work involved the synthesis of designed monomers and crosslinkers which can serve as a reactive ‘handle’ for subsequent chemical modification of the resulting biomaterials. The pore diameter and properties of materials can be tailored to a high extent, making them suitable for 3D cell culture, tissue engineering and regenerative medicine. Gelatine-based hydrogels will be synthesised and imbedded within the macroporous of polymer scaffolds to provide integrin-binding RGD motifs, promoting cell infiltration. Development of such novel hybrid polymer scaffolds will be trialed using modern polymerisation / chemical approaches.

This a multidisciplinary project that involves development of the polymeric material and optimisation of their morphological and mechanical characteristics as well as investigations of the use of these scaffolds in 3D cell culture and drug screening assays.

This project is part of ongoing research, so is suitable for either Chemistry or Biomedical Sciences PhD students as the direction of work can be adapted and shifted from one aspect to another with support from the team.

Relevant recent references:

• S. A. Richardson, et al., “Covalent Attachment of Fibronectin onto Emulsion‐Templated Porous Polymer Scaffolds Enhances Human Endometrial Stromal Cell Adhesion, Infiltration, and Function”, Macromolecular Bioscience 2019, 19(2), 1800351.
• A. M. Eissa, et al., “Enhanced Differentiation Potential of Primary Human Endometrial Cells Cultured on 3D Scaffolds”, Biomacromolecules 2018, 19, 8, 3343-3350.
• A. S. Hayward, A. M. Eissa, et al., “Galactose-functionalised polystyrene-based polyHIPE scaffolds for use in routine three dimensional culture of mammalian hepatocytes”, Biomacromolecules 2013, 14 (12), 4271–4277.

For more information: For an informal discussion please contact via direct email to Dr Ahmed Eissa (A.M.Eissa@wlv.ac.uk)

Supervisory Team:

Dr Ahmed M. Eissa (Senior Lecturer); Dr Mark Morris (Reader); Professor Weiguang Wang (Chair); Dr Vinodh Kannappans (Senior Research Fellow)

About the Project:

In this project, we will exploit the highly porous polymer scaffolds to create in vitro 3D tissue models. Recently, we have shown that emulsion-templated porous polymer scaffolds are capable of supporting 3D growth of many cell types including human pluripotent stem cells, human primary epithelial and stromal endometrial cells and human haematopoietic stem cells. Herein, we will broaden the scope of application of these scaffolds to address other biomedical problems including carcinoma by creating functional 3D cancer tumour models.

Polymerised high internal phase emulsions (polyHIPEs) will be first produced by emulsion templating whereby the continuous, oil-based phase of the emulsion is polymerised in the presence of the aqueous phase droplets, yielding a highly porous solid foam material. We will tailor the morphological and mechanical properties of our polyHIPE materials, making them more suitable for the culture of carcinoma tumours. We will also utilise different post-polymerisation approaches to functionalise our polyHIPE scaffolds by attaching interesting biomolecules/biomacromolecules that can facilitate notch signalling and therefore lays the foundation for biomimicry of the tissue niche. In collaboration with biomedical scientists and oncologists, the utility of the newly developed functional polyHIPE scaffolds will be fully characterised and exploited for the development of therapeutic strategies.

This a multidisciplinary project that involves development of the polymeric material and surface functionalisation with extracellular matrix proteins and/or polysaccharides as well as investigations of exploitation of its use for the development of cancer therapeutic strategies.

This project is part of ongoing research, so is suitable for either Chemistry or Biomedical Sciences PhD students as the direction of work can be adapted and shifted from one aspect to another with support from the team.

Relevant recent references:

• C. E. Severn, A. M. Eissa, et al., “Ex vivo culture of adult CD34+ stem cells using functional highly porous polymer scaffolds to establish biomimicry of the bone marrow niche”, Biomaterials 2019, 225, 1195332.
• J. L. Ratcliffe, M. Walker, A. M. Eissa, et al., “Optimized peptide functionalization of thiol-acrylate emulsion-templated porous polymers leads to expansion of human pluripotent stem cells in 3D culture”, Journal of Polymer Science Part A: Polymer Chemistry 2019, 57, 1974.
• S. A. Richardson, et al., “Covalent Attachment of Fibronectin onto Emulsion‐Templated Porous Polymer Scaffolds Enhances Human Endometrial Stromal Cell Adhesion, Infiltration, and Function”, Macromolecular Bioscience 2019, 19(2), 1800351.
• A. M. Eissa, et al., “Reversible surface functionalisation of emulsion-templated porous polymers using dithiophenol maleimide functional macromolecules”, Chemical Communication 2017, 53, 9789 - 9792.
• C. Chen, A. M. Eissa, et al., "Emulsion-templated porous polymers prepared by thiol-ene and thiol-yne photopolymerisation using multifunctional acrylate and non-acrylate monomers", Polymer 2017, 126, 395–401.
• A. S. Hayward, A. M. Eissa, et al., “Galactose-functionalised polystyrene-based polyHIPE scaffolds for use in routine three dimensional culture of mammalian hepatocytes”, Biomacromolecules 2013, 14 (12), 4271–4277.

For more information: For an informal discussion please contact via direct email to Dr Ahmed Eissa (A.M.Eissa@wlv.ac.uk)

Supervisory Team :

Dr Ahmed M. Eissa (Senior Lecturer); Professor Iza Radecka (Chair); Dr Leigh Jones (Senior Lecturer); Dr Abhishek Gupta (Senior Lecturer)

About the Project:

Antimicrobial materials are essential in many medical applications including wound-healing. Due to their morphological similarity to the extra cellular matrix of skin, porous polymer scaffolds hold great potential for skin tissue engineering. Over the past couple of decades, metallic nanoparticles (e.g. gold and silver nanoparticles) have been extensively explored in wound-healing applications as efficient antimicrobial agents. Nevertheless, the use of metallic nanoparticles has raised concerns as these particles can penetrate into the stratum corneum of skin, or even diffuse into the cellular plasma membrane. Therefore, there is a real need for the development of polymer scaffolds with controlled release of metallic nanoparticles and preferably programmable release of different antimicrobial / anti-inflammatory agents as well.

In this project, we will utilise the three-dimensional (3D) structured emulsion-templated macroporous polymers (known as polyHIPEs) as biocompatible scaffolds to enable the incorporation of both traditional and metal based antimicrobial agents with programmable release. Our approach will allow the creation of customised antimicrobial 3D structures for a broad range of tissue engineering applications, with particular emphasis in wound-healing applications. The incorporation of a uniform, continuous layers of metallic nanoparticles/antimicrobial agents in the polyHIPE scaffolds will be verified by XPS analysis. Electron microscopy will be used to investigate morphological features of the scaffolds. The mechanical properties of the scaffolds will also be studies in details using compression/tensile testing and DMA. The antimicrobial efficacy of the antimicrobial scaffolds against a range of gram +ve and gram –ve bacterial e.g. (Staphylococcus aureus and Escherichia coli) will be determined by industry-standard AATCC protocols. Cytotoxicity analyses of the antimicrobial scaffolds toward human epidermal keratinocytes and human dermal fibroblasts will be performed using quantitative analyses of cell viability and proliferation.

In this project will involve close collaboration with microbiologists and biomedical scientists where investigations of the use of these antimicrobial scaffolds for skin tissue engineering / wound-healing applications will take place.

This project is part of ongoing research, so is suitable for either Chemistry, Microbiology or Biomedical Sciences PhD students as the direction of work can be adapted and shifted from one aspect to another with support from the team.

Relevant recent references:

• A. M. Eissa, et al., “Reversible surface functionalisation of emulsion-templated porous polymers using dithiophenol maleimide functional macromolecules”, Chemical Communication 2017, 53, 9789 - 9792.
• C. Chen, A. M. Eissa, et al., "Emulsion-templated porous polymers prepared by thiol-ene and thiol-yne photopolymerisation using multifunctional acrylate and non-acrylate monomers", Polymer 2017, 126, 395–401.
• A. M. Eissa, et al., “Glycosylated nanoparticles as efficient antimicrobial delivery agents”, Biomacromolecules 2016, 17, 8, 2672-2679.

For more information: For an informal discussion please contact via direct email to Dr Ahmed Eissa (A.M.Eissa@wlv.ac.uk)

Supervisory Team:

Professor Weiguang Wang and Dr Vinodh Kannappans

About the Project:

Pancreatic ductal adenocarcinoma (PDAC) is the 4th most common malignancy with very dismal prognosis. The findings indicate that PDAC are heterogeneous tumours containing cancer stem cells (CSCs). CSCs are highly metastatic and resistant to conventional chemotherapies becoming the primary source of PDAC relapse. CSCs are commonly located in poorly vascularised hypoxic regions. Tumour hypoxia is typically associated with chemo- and radio-resistance. Our previous study demonstrated that both hypoxia and NFκB pathway activation were co-detected in cells expressing CSC markers isolated from the core region of tumour spheres(1). High NFκB activity can be induced by hypoxia and detected in chemoresistant cancer(2, 3). It may also play a pivotal role in hypoxia-induced CSC characteristics and chemoresistance. Elucidation of the relationship between hypoxia, NFκB and CSCs may shed light on PDAC CSC-targeting.

Due to the time and costs for new drug development, repositioning of old drugs for new indications is an emerging drug R&D strategy in recent years. Disulfiram (DS), an anti-alcoholism drug used in clinic for over 60 years, demonstrates excellent activity against a wide range of cancers without toxicity to normal cells(1, 3-5). DS also potentiates the cytotoxic effect of anticancer drugs and increases the therapeutic index(4, 6). The anticancer effect of DS is copper (Cu) and zinc (Zn) dependent(7). Cu and Zn plays a crucial role in redox reactions and triggers the generation of reactive oxygen species (ROS) which induce apoptosis. The transport of Cu into the cell is strictly mediated by the trans-membrane Cu transporter Ctr1. DS can be promptly converted into diethyldithiocarbamate (DDC) which is a strong divalent metal ion chelator, DS chelates Cu(II) and Zn(II) forming a Cu-DDC or Zn-DDC complex which improves the transport of Cu and Zn into cancer cells. Cu-DDC and Zn-DDC complexes are much stronger ROS inducers than Cu and Zn alone. The ROS induced by conventional anticancer drugs is commonly counterbalanced by ROS-activated NFκB which inhibits ROS-induced cytotoxicity. The Cu-DDC and Zn-DDC complex not only induces ROS it also inhibits NFκB activity in cancer cells(5, 8). It abolishes CSCs population in culture and completely reverses the chemoresistance and cross-resistance in chemoresistant cancer cells(3, 4, 6). The data from Professor Wang’s lab shows that Cu-DDC and Zn-DDC block the sphere reforming activity and potentiates cytotoxicity of gemcitabine, paclitaxel and 5-fluorouracil in PDAC, while also blocking cancer invasion at very low concentrations. Although the anticancer activity of DS has been known for more than two decades, its use as a cancer treatment is limited when administered orally. The half-life of DS in the bloodstream is less than 4 minutes(7), which may explain the lack of encouraging results from several clinical trials (http://www.clinicaltrials.gov/) using oral administration. Nanotechnology-based drug delivery system (NDDS) is a rapidly evolving interdisciplinary field. Nanoparticles (NPs) can protect drugs from metabolism on their way to the cancer. We have successfully encapsulated DS into liposome, PLGA and gold NPs demonstrating improved anticancer efficacy in several mouse cancer models. These promising pilot data prompt us to develop biodegradable long-circulating NAB-encapsulated Cu-DDC and Zn-DDC to target PDAC CSCs.

The demand for novel anti-PDAC treatments is urgent while drug development is slow and costly, mainly due to the large risk of toxicity of novel molecules. The repurposing of clinically available drugs into new treatment areas allows for the clinical development of safe drugs for new indications at much reduced time, risk and cost. DS shows very promising anti-CSC activity in other types of cancer. This project aims to translate its derivatives, Cu-DDC and Zn-DDC into PDAC treatment.

1. P. Liu et al., Liposome encapsulated Disulfiram inhibits NFκB pathway and targets breast cancer stem cells in vitro and in vivo. Oncotarget 5, 7471 - 7485 (2014).

2. S. Liu, M. S. Wicha, Targeting breast cancer stem cells. J Clin Oncol 28, 4006-4012 (2009).

3. P. Liu et al., Disulfiram targets cancer stem-like cells and reverses resistance and cross-resistance in acquired paclitaxel-resistant triple-negative breast cancer cells. British journal of cancer, (2013).

4. P. Liu et al., Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells. Br J Cancer 107, 1488-1497 (2012).

5. N. C. Yip et al., Disulfiram modulated ROS-MAPK and NFkB pathways and targeted breast cancer cells with cancer stem cell like properties. Br J Cancer 104, 1564-1574 (2011).

6. X. Guo et al., Disulfiram/copper complex inhibiting NFkappaB activity and potentiating cytotoxic effect of gemcitabine on colon and breast cancer cell lines. Cancer Lett 291, 104-113 (2010).

7. P. E. W. Tawari, Z.; Najlah, M.; Tsang, C. W.; Kannappan, V.; Liu, P.; McConville, C.; He, B.; Armesilla, A. L.; Wang, W., The cytotoxic mechanisms of disulfiram and copper(II) in cancer cells. Toxicology Research 4, 1439 - 1442 (2015).

8. X. Guo et al., Disulfiram/copper complex inhibiting NFkappaB activity and potentiating cytotoxic effect of gemcitabine on colon and breast cancer cell lines. Cancer letters 290, 104-113 (2009).

For more information: For an informal discussion please contact via direct email to Professor Weiguang Wang at w.wang2@wlv.ac.uk

Investigation of the anticancer activity and mechanisms of the PEGylated liposomal Cu-DDC and Zn-DDC in malignant mesothelioma cell lines and primary cultures

Supervisory Team:

Professor Weiguang Wang and Dr Vinodh Kannappans

About the Project:

Malignant mesothelioma (MM) is one of the most dreadful malignancies, which is commonly diagnosed at very late stage. The treatment outcomes in many other cancers have been significantly improved but the prognosis of MM remains very dismal. With most comprehensive treatment, MM patients can only survive for 9 – 12 months. Although it is a rare malignancy, the incidence of MM will significantly increase, especially in the developing nations. Due to the difficulty of getting rid of cancer tissues by surgery and the damaging effect of radiotherapy on the surrounding vital organs, chemotherapy becomes a major choice for MM treatment. Unfortunately, only two drugs (cisplatin and pemetrexed) are available for MM chemotherapy and MM cells are commonly resistant to all conventional anticancer drugs. Therefore, the current chemotherapy is more palliative rather than curative. The previous studies indicate that MM is heterogeneous containing cancer stem cells (CSCs). CSCs are highly invasive and resistant to conventional chemotherapies. CSCs are commonly located in poorly vascularised hypoxic regions. Tumour hypoxia is typically associated with chemo- and radio-resistance. Our previous study demonstrated that both hypoxia and NFκB pathway activation were co-detected in cells expressing CSC markers isolated from the core region of tumour spheres(1). High NFκB activity can be induced by hypoxia and detected in chemoresistant cancer, including MM(2, 3). Elucidation of the relationship between hypoxia, NFκB and CSCs may shed light on MM treatment.

The demand for novel anti-MM drug with low/non systemically toxic and highly efficacious is urgent but new drug development is a time (~15 years/drug) and cash ($1.5 billion/drug) consuming procedure. The repurposing of clinically available drugs into new treatment areas allows for the clinical development of safe compounds for new indications at much reduced time, risk and cost.

Disulfiram (DS), an antialcoholism drug without systemic toxicity to patients, shows very strong toxicity in CSCs from a wide range of cancer types including MM. In our pilot experiments, DS was highly toxic to MM cells and demonstrated curative effect on MM developed in mouse abdominal cavity. DS also blocked the expression of PD- L1, a protein compromising immune surveillance in MM patients. All of these findings indicate that DS is a very promising candidate for MM treatment. The studies from our and other groups indicate that the anticancer activity of disulfiram is copper (Cu) and zinc (Zn) dependent. DS can be promptly converted into diethyldithiocarbamate (DDC) which is a strong divalent metal ion chelator. DS chelates Cu(II) and Zn(II) forming a Cu-DDC or Zn-DDC complex which improves the transport of Cu and Zn into cancer cells. Cu-DDC and Zn-DDC complexes are strong reactive oxygen species (ROS) inducers. The ROS can also be induced by conventional anticancer drugs which are counterbalanced by ROS-activated NFκB which inhibits ROS-induced cytotoxicity and apoptosis. The Cu-D

it also inhibits NFκB activity in cancer cells(4, 5). It abolishes CSCs population in culture and completely reverses the chemoresistance and cross-resistance in chemoresistant cancer cells(3, 6, 7). Therefore, the functional anticancer compounds are Cu-DDC and Zn-DDC. This study intends to investigate the anti-MM activity of a novel injectable version of Cu-DDC and Zn-DDC, which is applicable in chest and abdominal cavities. We have developed PEGylated liposomal Cu-DDC and Zn-DDC (PEGlipo/Cu-DDC and PEGlipo/Zn-DDC) with promising drug loading contents and very stable shelf life in solution. The PEGlipo/Cu-DDC and PEGlipo/Zn-DDC showed very strong killing effect on other cancer cell lines. In this project, the effect of the new formulation of Cu-DDC and Zn-DDC on MM will be examined in cell culture and in mouse models. The molecular anticancer mechanisms, especially in CSCs and immune system, will be investigated.

Development of disulfiram derivatives in an anticancer setting will benefit MM patients and have significant financial implications for the NHS.

Relevant recent references:

1. P. Liu et al., Liposome encapsulated Disulfiram inhibits NFκB pathway and targets breast cancer stem cells in vitro and in vivo. Oncotarget 5, 7471 - 7485 (2014).

2. S. Liu, M. S. Wicha, Targeting breast cancer stem cells. J Clin Oncol 28, 4006-4012 (2009).

3. P. Liu et al., Disulfiram targets cancer stem-like cells and reverses resistance and cross-resistance in acquired paclitaxel-resistant triple-negative breast cancer cells. British journal of cancer, (2013).

4. N. C. Yip et al., Disulfiram modulated ROS-MAPK and NFkB pathways and targeted breast cancer cells with cancer stem cell like properties. Br J Cancer 104, 1564-1574 (2011).

5. X. Guo et al., Disulfiram/copper complex inhibiting NFkappaB activity and potentiating cytotoxic effect of gemcitabine on colon and breast cancer cell lines. Cancer letters 290, 104-113 (2009).

6. X. Guo et al., Disulfiram/copper complex inhibiting NFkappaB activity and potentiating cytotoxic effect of gemcitabine on colon and breast cancer cell lines. Cancer Lett 291, 104-113 (2010).

7. P. Liu et al., Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells. Br J Cancer 107, 1488-1497 (2012).

For more information: For an informal discussion please contact via direct email to Professor Weiguang Wang at w.wang2@wlv.ac.uk

Supervisory Team:

P. Goggolidou, Reader in Molecular Genetics, A. Karakoula, Senior Lecturer in Pharmacy

About the Project:

Certain types of brain tumours develop much more aggressively and rapidly than others. Glioblastoma is a tumour that is known for its aggressive growth and resistance to treatment. Recent studies on glioblastoma biopsies have shown that many tumour cells have a reduced number of cilia (Sarkisian and Semple-Rowland, 2019). The primary cilia are microtubule organelles that project from the surface of cells (Bergmann, 2018) and have various functions including transportation of fluid into and out of the cell, epithelial structural functions, and sending and receiving signals into the cellular environment. Studies have shown that cilia are intimately involved in cell division therefore, it is possible that mutations that disrupt ciliogenesis could promote tumorigenesis as a result of a loss of cell cycle control (Plotnikova et al., 2008; Basten and Giles, 2013). Currently, it is largely unknown how the primary cilia are involved in these brain tumours and whether the cilia play a role in regulating the progression of cell growth.

The aim of this study is to determine whether the cilia is indeed involved in these paediatric brain tumours and to establish whether there is a link between the stage of the tumour progression and the stage of ciliogenesis dysregulation.

This research will investigate the rate of ciliogenesis in paediatric patient-derived tumour cells (GBM and ependymoma) using confocal microscopy. This will be compared to the number of cilia and rate of ciliogenesis with frozen sections of tumours from patient biopsy samples. Using RNA sequencing and qPCR analysis, we are planning to identify if there are any specific genes involved in the processes being studied and measure the expression levels of genes associated with deregulated pathways in ciliogenesis.

The results will provide an insight into the involvement of cilia in paediatric brain tumour cell proliferation and identify novel potential therapeutic targets for this type of malignancy.

For more information: For an informal discussion please contact via direct email to Dr Goggolidou (p.goggolidou@wlv.ac.uk)

Supervisory Team

Dr Iain D. Nicholl, DOS, Senior Lecturer in Biomedical Science

Dr Evi Goggolidou, Co-supervisor, Reader in Biomedical Science

About the Project:

There is clear epidemiological evidence that aspirin has a chemoprotective effect in colorectal cancer (CRC) development. Prophylactic aspirin use is thus currently considered when managing individuals that carry an identified mutation in highly penetrant genes that predispose them to familial CRC, including Lynch Syndrome (caused by mutations in DNA Mismatch Repair Genes) and Familial Adenomatous Polyposis (caused by mutations in the APC gene). As aspirin acts as a non-steroidal inflammatory (NSAID) agent, its effect on reducing inflammation is considered to be a primary mechanism for this observed chemoprotective effect. However, there is emerging evidence that aspirin and other NSAIDS can reduce tumour cell growth through additional mechanisms unrelated to inflammation. For example, Greenspan et al (2011), have indicated that NSAIDS including ibuprofen and aspirin can inhibit the activation of b-catenin - a key player in the Wnt signaling pathway that can drive cellular proliferation - by increasing b-catenin phosphorylation by a component of the APC destruction complex (GSK-3b) which is traditionally interpreted to enhance b-catenin degradation.

We have examined this phenomenon in preliminary experiments using confocal analysis and find an increased level of phosphorylated b-catenin at the centrosome in SW480 CRC cells incubated with aspirin – an unexpected result. b-catenin has additionally and traditionally been identified as a core component of the Zonula Adherens protein complex, playing a role in cell-cell adhesion and mechano-transduction bridging the cadherins and actin cytoskeleton (Farago et al, 2021). More recent experimentation is revealing that b-catenin processing at the centrosome may impact on the Wnt signaling pathway (Vora et al, 2020).

We thus wish to explore phosphorylated b-catenin localisation in CRC derived cell lines treated with a range of NSAIDS in detail, using immunochemical approaches including immunofluorescence analysis and confocal microscopy. We will additionally seek to isolate the centrosome using classical biochemical fractionation and identify interacting partners of phosphorylated b-catenin employing immunoprecipitation and mass spectrometry. This approach will provide insight into a) the molecular action of NSAIDS as anti-cancer agents and b) the centorosme-related function of b-catenin and is likely to result in high quality publications given the novelty of the experimental approach.

Farago, B., I.D. Nicholl et al, (2021) Activated nanoscale actin-binding domain motion in the catenin–cadherin complex revealed by neutron spin echo spectroscopy PNAS (USA) 118 (13) e2025012118; https://doi.org/10.1073/pnas.2025012118

Greenspan EJ, Madigan JP, Boardman LA, Rosenberg DW. Ibuprofen inhibits activation of nuclear {beta}-catenin in human colon adenomas and induces the phosphorylation of GSK-3{beta}. Cancer Prev Res (Phila). 2011 Jan;4(1):161-71. doi: 10.1158/1940-6207.CAPR-10-0021.

Vora SM, Fassler JS, Phillips BT. Centrosomes are required for proper β-catenin processing and Wnt response. Mol Biol Cell. 2020 Aug 1;31(17):1951-1961. doi: 10.1091/mbc.E20-02-0139.

For more information: For an informal discussion please contact via direct email to Dr Iain Nicholl (I.Nicholl@wlv.ac.uk)