Cancer is a genetic disease characterised by excessive cell proliferation, migration and invasion, which result in tumour development and its subsequent spreading to other tissues and organs. Phenotypic alterations in cell morphology and function are underlined by changes in gene/protein structure and expression. Expression of many other proteins is aberrant – either elevated or reduced. For example, expression of matrix metalloproteinases is increased, resulting in modifications in extracellular matrix. This facilitates release of cancer cells from the primary tumour and promotes metastatic spread.

Many currently developed anti-cancer therapies exploit knowledge of the fundamental molecular and cellular mechanisms involved in tumour formation and progression, including changes in the function and expression of the crucial proteins.

Studies in the area of cancer disease mechanisms are led by Dr Neil Cross, Dr Sarah Haywood-Small, Dr Nick Peake, Dr Tanya Klymenko, Dr Rebecca Leyland, Dr Laura Cole, Prof Christine Le Maitre and Dr Nikki Jordan-Mahy.

Studies in the application of biophysical techniques to the study of cancer biomarkers in fingermarks, solid tumours and drug distribution and response in solid tumours and 3D models of solid tumours are led by Dr Neil Cross, Dr Laura Cole, Professor Simona Francese and Professor Malcolm Clench.

Research Areas of Interest

(a) Cancer Disease Mechanisms

(i) Development of 3D cell culture models for assessment of cancer therapeutics

A major issue of testing novel anti-tumour agents is predicting whether in vitro findings can be translated to an in vivo and subsequently patient situation. It is widely acknowledges that discrepancies between in vitro cell culture leads to considerable drug failure in translational research, highlighting the need for improved in vitro methods. We have developed several 3D methodologies at SHU aimed at improving in vitro cancer drug testing. Work was initially funded by an Nc3Rs award to develop both skin and cancer models (Clench (PI) Smith (Co-I) and Cross (Co-I) resulting a publication on the skin work in Proteomics. Cancer models were further refined resulting in an InnovateUK award (Cross (PI)) to develop novel Lung cancer cell lines and to develop novel 3D cell culture models to study drug activity. This work has resulted in the successful commercial release of the 3D Cancer OrganDot cell culture platform by our collaborators Bio-IVT along with Bio-IVT commercially developing two new lung cancer cell lines, all of which were validated at SHU. Related models have been developed for Multiple Myeloma and Uveal melanoma resulting in recent publications in Investigative Ophthalmology and Visual Sciences and Experimental Cell Research which were used to assess phenotypic plasticity/cancer stem cell behaviour, and targeted apoptotic responses respectively, as outlined further below. Furthermore, work with the Mass Spectrometry Imaging group (Clench, see below) have used the 3D models developed in Dr Cross' group to perform mass spectrometry imaging of proteins and metabolites.

(ii) Role of cancer stem cells in tumour development (Dr Neil Cross)

Recent evidence suggests that tumours comprise differentiated tumour cells (which have a limited life-span) and cancer stem cells/tumour initiating cells (which are undifferentiated cells with unlimited replicative ability and the ability to differentiate). The cancer stem cells hypothesis suggests that if cancer stem cells are selectively ablated, tumour growth will cease and eventually the tumour will regress. We are investigating whether cancer stem cells are present in solid tumours including prostate cancer and primary uveal melanomas. The aim is to determine drug combinations which selectively ablate these cancer stem cells with chemotherapeutic agents.

These studies have resulted in a recent publication (Doherty et al, 2017), and 2 PhD completions (Doherty, 2013, Wright, 2016) with 4 related earlier papers from these studies published between 2011 and 2013. These studies show for the first time that a) Uveal melanoma cells do not contain a cancer stem cell population, b) that their melanocytic phenotype is reversible in 3D cell culture, c) that pre-malignant lesions frequently contain oncogenic mutations in that were previously considered to be present in fully malignant disease and d) prostate cells contain a clearly defined hierarchical stem cell population that can easily be isolated and characterised. These studies are in collaboration with Dr Karen Sisley (Uveal melanoma), University of Sheffield and Dr Tom Sayers, National Cancer Institute, USA.

Doherty, RD, Hammond, DW, Rennie IG, Sisley, K, Cross, NA (2017) Phenotypic plasticity in uveal melanoma is not restricted to a tumour subpopulation and is unrelated to cancer stem cell characteristics. Investigative Ophthalmology and Visual Science 58(12):5387-5395. doi: 10.1167/iovs.17-22272.

(iii) Factors regulating the activity of the anti-tumour agent TRAIL (Dr Neil Cross)

Recent studies have focused on how tumours evade apoptotic signals from Tumour Necrosis Factor (TNF) superfamily members, with particular emphasis on the potential anti-tumour agent TRAIL (TNF-Related Apoptosis Inducing Ligand), its receptors (Death Receptors 4 and 5) and decoy receptors (Decoy receptors 1 and 2, and OPG (Osteoprotegerin). Current studies are focusing on mechanisms of TRAIL resistance in cancer cells, and whether combinations of agents such as histone deactylase (HDAC) inhibitors, nuclear export inhibitors and proteasome inhibitors can reverse TRAIL resistance. In our recent studies, we have also demonstrated that histone deacetylase inhibitor in particular can potently enhance tumour cell sensitivity to TRAIL in a range of tumour cells, leading to successful a PhD completion by Arhoma (2017) in collaboration with Dr Andy Chantry at University of Sheffield. This work generated a recent publication in Experimental Cell Research (Arhoma et al 2017), which included the first reliable and clonal myeloma 3D cell culture model, the identification of a novel Death Receptor sensitiser published in Biosciences Horizons (Khalid 2016) and one book chapter in Tumour Immunology and Immunotherpay (Sayers and Cross, 2014). Very recent data for 3 further studies showing that Nuclear export inhibitors enhance Death Receptor agonists activity in prostate, myeloma and osteosarcoma have complete data sets with publication in progress, and three further studies showing that inhibitors of EZH2 and G9a (epigenetic modifiers) are also potent sensitisers of Death receptor mediated apoptosis in myeloma in 3D cell culture are complete and awaiting drafting of manuscripts.

(iv) Use of gold nanoparticles in targeting the mitochondrial for anticancer therapeutics (Dr Neil Cross and Prof Neil Brickelbank)

Gold nanoparticles are attractive theranostic agents, in that they are excellent imaging contrast agents, and are capable of inducting anti-tumour activity. In our studies (Brickclebank and Cross) we have developed triphenylphosphine-conjugated gold nanoparticles for targeting to the mitochondrial of cancer cells. These nanoparticles can be excited by specific wavelengths of light to induce photothermal therapy. Our studies initially characterised the ligand's toxicity, and demonstrated mitochondrial targeting and photothermal toxicity. These studies have generated 4 papers between 2012 and 2019, with another to be submitted shortly. These studies have been expanded to show properties of Arsonium-conjugated gold nanoparticles, and to explore cytotoxicity of different phosphonium ligands in cancer cells. This work is being expanded into radiosensitivity studies for future projects.

(v) Nutraceuticals in the prevention and treatment of cancer (Dr Nikki Jordan Mahy, Professor Christine Le-Maitre)

We are currently researching a number of novel compounds derived from fruit and vegetables. This involves investigating the biological effects on tumour and non-tumour cells of agents derived foods. Specifically, we are interested in their potential to induce cell death and inhibit proliferation, and the molecular mechanisms of their actions, focussing on polyphenols and polyacetylenes. We have a particular interest in leukaemia, where Prof Lemaitre's group have generated 2 PhD completions 5 publications between 2011 and 2015 on the role of polyacetylenes and polyphenols. These studies identified and isolated bioactive agents from foods which act against tumour cells and induce tumour cell killing. Furthermore, Dr Jordan-Mahy's group (along with Dr Haywood-Small, Prof Lemaitre and Dr Cross) have shown that polyphenols enhance chemotherapeutic activities, and in some cases, antagonise chemotherapy activity in leukaemia, resulting in 1 PhD completion and 4 publications (Mahbub et al, 2013, 2015a, 2015b and 2017). These studies identify glutathione as a likely determinant of polyphenol-induced chemotherapy sensitivity, and highlight the potential importance of diet on chemotherapy responses. These studies are currently being continued by Dr Mahbub who is a visiting fellow at SHU whilst based in Saudi Arabia. The group has one additional study on synergistic drug interactions ready for imminent submission to Cell death and Discovery, and Dr Jordan-Mahy has recently initiated feeding studies on polyphenols, measuring distribution in blood.

(iv) Inflammation and tissue remodelling in the bowel cancer microenvironment (Dr Nick Peake)M

Cancers remodel their environment as disease progresses in order to invade and spread into surrounding tissues. Cancer cells are also highly responsive to their environment, making this interaction crucial in determining disease outcome. Following on from a research background in inflammatory signalling and tissue remodelling, I now lead projects investigating these events in the tumour microenvironment, with a focus on bowel cancer. This work has identified that the enzyme Transglutaminase-2 is prominently expressed in the tissue surrounding colorectal tumours, and that it contributes to nflammation and tissue remodelling (Cellura et al, 2015). My current research is studying these processes in 3D cell culture models of bowel cancer, investigating how extracellular vesicles are involved, and aiming to develop ways of treating advanced stages of disease which currently have a poor outcome. This involves extensive collaboration with scientific and clinical partners, including the University of Sheffield, Sheffield Teaching Hospitals, Queen Mary University London (QMUL), University of Southampton, Durham University, and Charles University Prague, and the early results from these studies have recently been presented at the UK Extracellular vesicle (UKEV) and International Society for Extracellular Vesicles (ISEV) annual meetings.

a) Biomechanical characterisation of the tumour microenvionment.

Cancer tissue is notably stiffer than normal tissue, and tissue stiffness drives aggressive cancer cell behaviour. Since the enzyme transglutaminase-2 is prominent in areas around cancers and causes protein cross-linking, I have been examining the impact of knocking down this enzyme and measuring impact on stiffness of 3D cultures developed using primary colon fibroblasts and colon adenocarcinoma cell lines. This work has been funded by Bowel & Cancer Research (Targeting matrix stiffness to treat colorectal cancer. Peake N (PI), Knight M (QMUL), Mirnezami A (University of Southampton). Bowel & Cancer Research £47720, 2015-16), and continues through collaborative projects with Dr Paul Hughes (Department of Engineering, University of Durham), recent SHU CK Strategic Investment funding to build biomechanical testing capacity here at SHU, and with ongoing funding applications with Dr Haywood Small to extend this work into mesothelioma disease models.

b) Extracellular vesicles - cellular cross-talk in the bowel.

Extracellular vesicles are tiny secreted packages released from most cells, and contain bioactive cargo capable of reprogramming recipient cells. Following on from observations that EVs contain key mediators (including transglutaminase-2 and regulatory miRNAs), I am now exploring how EVs alter cell behaviour in the bowel with a focus on colon-adipose cross-talk given the links between inflammatory bowel disease, fat and cancer. This work is supported by a BMRC studentship (Colon-derived extracellular vesicles and their impact on adipose cells - linking fat to colorectal disease. SHU BMRC funded PhD studentship £93,744. Peake N (DoS), Kelly D, Dalton C), and by summer studentships from Animal-free Research UK (Building a physiologically relevant model of fat development during inflammatory bowel disease, Animal Free Research UK Summer studentship award 2018, £1940; Building an adipose-rich tumour microenvironment model to study fat-cancer cross-talk, Animal Free Research UK Summer studentship award 2017, £1940).

c) Extracellular vesicles as drivers of metastasis in bowel cancer.

EVs are known to drive organotropism - the ability of cancers to establish metastases at preferential secondary sites in the body. We will take advantage of the world-leading mass spectrometry imaging expertise here at SHU to investigate how EVs shape the tumour microenvironment using bioengineered models of key metastatic sites for bowel cancer. This is supported by a PhD studentship award from Bowel & Cancer Research (Extracellular vesicles: small packages with a big role in bowel cancer metastasis. PhD studentship award, Bowel & Cancer Research £74,659. Supported by £21,683 through the SHU CK Collaborative PhD studentship scheme. Peake N (DoS), Cole L, Le Maitre C, Hunt S (University of Sheffield), Mirnezami A (University of Southampton) - 2019 to 2022).

(v) Mesothelioma Research in the BMRC (Dr Sarah Haywood Small)

Dr Haywood-Small established her mesothelioma research group in 2017. She is now a member of the Mesothelioma Research Network group (British Lung Foundation) and provides scientific support for the June Hancock Mesothelioma Research Fund (JHMRF). She leads several projects which involve using mass spectrometry as a bioanalytical technique for biomarker discovery, 3D cell culture models, tissue imaging and identification of mineral fibres in mesothelioma. The research is assisted by several industrial partners in the UK. Santia Asbestos Management Ltd is an asbestos testing service (Blidworth) and NewGene is an ISO 15189 accredited provider of molecular diagnostics using high throughput sequencing and genotyping technologies (Newcastle Upon Tyne). Finally, clinical research support is provided by consultant pathologists (Dr Kitsanta and Prof Survana) at the Royal Hallamshire Hospital.

a) Identification of Mineral Fibres and Biomarkers in Malignant Mesothelioma

This PhD research began in October 2017, it is match funded by the JHMRF and the BMRC Studentship Scheme (£90,000). Current laboratory investigations are focussing on the physical profile of selected mineral fibres and emerging biomarkers in mesothelioma using a combination of mass spectrometry, flow cytometry and histopathology techniques. Other aims include biomarker discovery using multiplex analytical techniques and to contribute to the knowledge base surrounding mesothelioma pathogenesis. Such techniques would ultimately involve future integration into the clinical workflow; diagnosis, prognosis and treatment monitoring of mesothelioma, as well as contributing to knowledge about the pathogenesis of the disease. This project is in partnership with Dr Amy Managh (Loughborough University), Dr Laura Cole (SHU) and Prof Malcolm Clench (SHU). Data has been presented at the IMSC in August 2018 (Florence, Italy) and submitted to the ASMS in June 2019 (Atlanta, USA). Two manuscripts are currently in preparation for publication.

b) Breathomics as a Non-Invasive Early Diagnostic Tool for Malignant Mesothelioma.

In Feb 2019, another PhD student joined the group funded by the Vice Chancellors Award Scheme, SHU (£90,000). This work is in collaboration with Dr Liz Allen (SHU), Dr Laura Cole (SHU) and Dr Vikki Carolan (SHU).

c) Enhancement of Death Receptor-Mediated Apoptosis in Malignant Mesothelioma.

Another PhD student will join the group in May 2019 funded by the Libyan Embassy (£90, 000) in collaboration with Dr Neil Cross (SHU) and Prof Adrian Dobbs (University of Greenwich).

d) Gaining Insights into Malignant Mesothelioma: Deconstructing the Tumour Microenvironment.

Research in collaboration with Dr Nick Peake (SHU) and Dr Munitta Muthana (University of Sheffield).

Given the latency period of mesothelioma and the overwhelming evidence linking this cancer to inflammation, it is not surprising that the immune system is gaining support as a key feature of this cancer. Therefore, it is crucial to ‘dissect or deconstruct’ the mesothelioma tumour environments, alongside their interaction with neighbouring cells. This research has built on existing projects to generate 3D mesothelioma models that can be used as a 'real' tumour to closely monitor cellular interactions with asbestos fibres and emerging therapeutics (SAHA, MCC950). Ultimately, these findings will enable us to re-educate the tumour microenvironment to enhance therapeutic approaches. Currently awaiting grant outcome in partnership with Dr Nick Peake to appoint a research assistant for six months.

e) Novel Photodynamic Therapy Agents (PDT) for Malignant Mesothelioma

We have designed, developed and tested novel therapeutic PDT reagents specifically targeted to malignant mesothelioma. Our network-level computational models and research laboratories are well positioned to understand the intricate mechanisms of drug response, resistance and toxicity for targeted small molecule drugs in this incurable cancer.

PDT is an efficient therapeutic modality, approved for the treatment of malignant and non-malignant diseases. PDT involves three main components, a photosensitising compound, visible light, and molecular oxygen, which in turn will become the prime component of selective tumour cell destruction. PDT has always been one of my research interests since 2006 (Haywood- Small et al., 2006).

The limited penetration of light is usually a disadvantage for PDT, but this could be advantageous for treating malignant mesothelioma. As an inter-operative treatment to the chest cavity, this approach will greatly reduce the adverse impact on surrounding and deeper tissues. PDT was introduced into the BMRC initially with several successful undergraduate and postgraduate research projects. In 2018, we started to combine expertise in the mesothelioma field (Dr Sarah Haywood-Small), with computational chemistry (Dr Alex Hamilton) and synthetic chemistry (Dr Simon Turega). Our next step is to develop a Novel ABC-PDT drug design initiative (A-Agent, B-Bridge and C-Cancer). For each component A, B and C- we have selected two candidates from the literature. We hypothesise that specific metabolic deficiencies in mesothelioma cells can be exploited therapeutically when combined with a novel photoactive Agent to selectively kill the cancer cells. This project is in collaboration Prof. Julia Weinstein (University of Sheffield). Currently awaiting grant outcome to appoint a research assistant for six months (Nov 2018 submission, JHMRF). Dr Turega is also awaiting the outcome of a PhD application.

(vi) The immunological response to cancer (Dr Rebecca Leyland)

Dr Rebecca Leyland established her research group in 2017 when she joined Sheffield Hallam University, having previously worked in several immunology-focused research intensive positions. Her group is focused on investigating the cellular and molecular mechanisms between the immune system and cancer development. She leads several projects which involve using flow cytometry, molecular biology, microscopy and 3D cell culture models. She has been a member of the British Society for Immunology (BSI) since carrying out her PhD at the National Institute for Medical Research in London and has been successful in obtaining studentship grants and travel grants from this society to support her work during her time at SHU. She was selected through a competitive process to attend a national sandpit event in Oxford ran by Cancer Research UK and Arthritis UK, where she developed further external collaborations to support her work. This research has been presented at several national and international conferences since starting SHU.

(a) Molecular mechanisms of B-cell differentiation and cancer formation

B-cells are an essential part of the immune response and function mainly by differentiating into antibody secreting plasma cells. This differentiation is co-ordinated by a specific network of transcription factors and microRNAs, which when dysregulated can induce oncogenic mechanisms and the development of cancer. Dr Rebecca Leyland's research aims to further explore this network in B-cell sub-populations focusing on microRNA-155. This work builds on previous research she led at the Babraham Institute in Cambridge and she maintains collaborations with Dr Martin Turner, Head of Lymphocyte Development and Signalling group at the Babraham Institute, and Dr Elena Vigorito who is based on the MRC Biostatical unit in Cambridge. Dr Rebecca Leyland has authored four high impact publications in this area in previous positions and during her time at SHU has further published a review with Dr Richard Chahwan, University of Exeter, and is currently addressing reviewers' comments for a senior author manuscript for publication in Life Science Alliance journal. A £700 travel grant was successfully obtained from the BSI to support the presentation of this work at the European Congress for Immunology in Amsterdam in 2018 and she was invited to present this work at a B-cell Immunology School in Germany during February 2019. A grant was submitted to the BMRC funding round in March 2017 to continue research in this area and upon publication of the latest manuscript, it is planned to apply for an external grant.

(b) Intrinsic cancer mechanisms which inhibit the immune system in 2D and 3D cell culture systems

This PhD research began in October 2018 and is funded by the BMRC Studentship Scheme (£78,500). Current investigations are focused on the contribution of programmed death ligand-1 (PD-L1) to the development of a variety of cancers in 2D and 3D cell culture systems and the response to immune-targeted (immunotherapy) drugs, many which have been recently approved by the FDA. Other aims include the development of a 3D cell culture model incorporating both cancer cells and immune cells to further dissect the role of immunotherapy drugs on the tumour microenvironment. This research area compliments previous research Dr Rebecca Leyland carried out at MedImmune/AstraZeneca, where she was involved in inventing, leading and developing several immunotherapy drugs from pre-clinical to clinical stages (Leyland et al., 2017). This current work is in collaboration with Dr Neil Cross (SHU) and Dr Nikki Jordan-Mahy (SHU) and the research is planned to be presented at the British Association for Cancer Research (BACR) conference in June 2019.

(c) Modulation of immuno-inhibitory biomarkers by anti-cancer therapeutics.

Several anti-cancer modulating agents have been shown to induce changes in the tumour microenvironment and alter cell surface expression of immuno-inhibitory biomarkers, including but not restricted to PD-L1. These projects aim to investigate these mechanisms and the potential use for combination therapy in cancer patients. This work also compliments the PhD project currently ongoing in Dr Rebecca Leyland's group. Postgraduate projects in this area have analysed the cellular response to tumour-targeted drugs such as polyphenols in collaboration with Dr Nikki Jordan-Mahy and TRAIL and death receptor agonists with Dr Neil Cross (SHU). A £700 studentship grant was obtained from the BSI to support this work, and this research was presented at the Yorkshire Society for Immunology meeting in May 2018. A grant to support this work was submitted to the BMRC project funding round in January 2019.

(vii) Role of the nuclear lamina proteins in cell nucleus dynamics in blood cancer models. (Dr Tanya Klymenko)

The aim of this study is to explore LMNB1 dynamics and its involvement in the pathogenesis of B-cell malignancies through several objectives: Investigation of the ratio of nuclear lamins in soft and stiff tissue using cell lines; Examination of the ratio and phosphorylation of nuclear lamins upon kinase inhibition in mice models of CLL; Investigation of changes in nuclear lamin ratio; Investigation of the phosphorylation patterns and changes in genome expression upon kinase inhibition; Investigation of the impact of increased LMNB1 expression in mice on formation of CLL malignant lymphocytes; Examination of the changes in LMNB1-genome interactions that translate into characteristic changes in gene expression.

(b) Application of biophysical techniques to the study of cancer biomarkers in fingermarks, solid tumours and 3D models of solid tumours

(i) Biomarkers of Breast Cancer in Fingermarks (Dr Laura Cole, Professor Simona Francese)

Non-invasive detection of cancer using fingertip smears. This project is funded by the MRC CiC scheme. The project intends to bridge the gap between screening, diagnosis and monitoring of breast cancer by employing fingertip exocrine secretion smears and use oncological molecular markers contained within to enable the discrimination of healthy versus breast cancer patients. Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) will be employed to recover cancer signatures, assisted by statistical modelling to discriminate oncological versus healthy patients and thus determining the specificity and sensitivity of this technique. The method is not initially intended to replace breast biopsies, rather to offer a preliminary specific and sensitive, non-invasive and pain-free molecular screening opportunity in order to: 1. Reduce the occurrences of patient discomfort and embarrassment (and so enhance screening uptake rates); 2. Reduce false positives/negatives 3. Indicate appropriateness of further and more in depth tests which. will reduce costs to the public health sector, by decreasing the number of requests for advanced diagnostic tests, for first contact and follow up patients. These significant advantages hold the promise to break new ground in screening, diagnosis and monitoring of breast cancer.

(ii) Use of Mass Spectrometry Imaging to Study Solid Tumours (Professor Malcolm Clench and Dr Laura Cole)

Mass spectrometry imaging presents a powerful label free multiplexed methodology to study disease process, in solid tumours. Projects in this area have studied pancreatic cancer and uveal melanoma. The technique can be used to study the metabolome and proteome of the tumour enabling "molecular maps" of processes occurring in the tumour to be generated.

(iii) Use of Mass Spectrometry Imaging to Study Drug Distribution and Response in Solid Tumours and 3-D Models of Solid Tumours. (Dr Neil Cross, Dr David Smith, Dr Laura Cole and Professor Malcolm Clench)

Mass spectrometry imaging presents a powerful label free multiplexed methodology to study drugs, drug metabolites and responses to treatment in solid tumours and 3D models of solid tumours. Projects in this area have studied the drugs AQ4N, doxorubicin, docetaxel and bevacizumab. The technique can be used to study the drug and responses to treatment in both the metabolome and proteome enabling "molecular maps" of processes occurring in the tumour to be generated.