NIHRDataInsights/HRCSData huggingface dataset:
Title: Predictive biomarkers of response to DNA repair targeting agents in sporadic prostate cancer
Funder: Medical Research Council

Abstract: Prostate cancer (PC) is the commonest cancer in men and is highly heterogeneous; despite this, molecular stratification to guide treatment is not standard of care. Defects in DNA repair genes in sporadic PC present a therapeutic opportunity. Preclinical studies indicate that cell lines defective in genes involved in homologous recombination, including BRCA1/2, ATM, ATR, CHEK2 and RAD51, are sensitive to PARP inhibitors (PARPi). We have now shown that PARPi have antitumour activity in sporadic PC in clinical trials of niraparib (Sandhu et al, 2013) and olaparib (unpublished). AIMS I. To determine the frequency of DNA repair defects in sporadic PC and the impact of these on prognosis and response to endocrine treatment. II. To determine the prevalence of DNA repair defects in patients responding or resistant to PARPi. III. To evaluate the functional relevance, in preclinical models, of genomic aberrations identified in these tumor biopsies studied in Aims I and II. IV. To clinically qualify a biomarker suite that will serve as a predictive biomarker of sensitivity to PARPi in sporadic CRPC. METHODOLOGY 1. Next generation sequencing (NGS) of CRPC samples using a customized panel to identify DNA repair defects. These data will be correlated with clinical data. 2. Use of whole genome sequencing data of sporadic CRPC responding or resistant to PARPi to identify defects in DNA repair that associate with response. 3. Knockout and knock-in functional studies of identified genes, in in vitro studies, to determine their impact on PARPi sensitivity and DNA repair. 4. Customized, high throughput, targeted and high coverage NGS (>1000 fold coverage) using the MiSeq Illumina platform of all the identified genes to prospectively select patients for planned large randomized studies. IMPLICATIONS: It is envisioned that these studies will generate the first biomarker suite to guide systemic treatment selection for prostate cancer.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS
Title: Recapitulating the adult stem cell and cancer stem cell niche in vitro using 3D engineered matrices
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Breast cancer is a prevalent heterogeneous disease that may have multiple cells-of-origin requiring specific therapeutic strategies. Experimentation to uncover the cell signaling and molecular deregulation in cancer, together with pharmacological drug testing remain highly dependent on the generation of large numbers of specific in vivo animal models. Stem cells, characterised by their proliferative and self-renewal capacity, have been implicated as key players in cancer development, however cancer stem cells and the cell-of-origin remain distinct concepts. Stem cells reside within a niche, which provides instructive signals in the form of cell-derived soluble factors, cell-cell contacts and extracellular matrix within a 3-dimensional environment. To recapitulate this niche in vitro, we envisage a tissue engineering approach to be necessary, seeding multiple cell types of murine and animal origin into biomaterial scaffolds with controlled biochemical and physical parameters. This project encompasses the development, characterization and validation of a fully humanized tissue analogue of breast. The development of such a system could greatly enhance research into normal and cancer stem cells and studies of tumour initiation and progression, as well as providing potent therapeutic research tools. Furthermore, the availability of robust, replicable 3-dimensional niche culture models could result in the replacement of animals for stem cell research and ultimately reduce the numbers of animals used for preliminary drug screening, as efficacy on cancer stem cells could be readily assessed.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY)
Health categories: CANCER AND NEOPLASMS
Title: Developing an integrated “in vitro carcinogenicity predictive tool” utilising in vitro cell signalling and cell behaviour assessment coupled with in vitro genotoxicity data
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Cancer arises due to the accumulation of multiple disparate abnormalities governing how cells control their growth. Some of these abnormalities involve the accumulation of DNA mutations, hence DNA mutagens (agents that alter DNA) are nearly always capable of inducing cancer. Therefore a traditional way to assess cancer risk of new chemical agents is merely to focus on DNA mutability. Unfortunately, this approach does not detect chemicals that cause cancer in other (non-DNA) ways. It is also not 100% accurate in detecting chemicals that do mutate DNA, and even throws up numerous false positives (wrongly classifies safe chemicals as mutagens), which require animal experiments for clarification. In this proposal we aim to develop a better approach to detecting carcinogens, by focussing on induced abnormalities in cell signals controlling cellular growth and on how cells behave by monitoring multiple cellular parameters after exposure to the test chemicals. We hypothesise that this more holistic approach will yield a better prediction of which chemicals are potential carcinogens. As this apporach is entirely in vitro (cultured cells), this could have a major impact on the use of animals in safety testing and reduce the numbers of animals needed by tens of thousands per year.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: CANCER AND NEOPLASMS
Title: Development of human epithelial cell transformation models for carcinogen screening, employing defined phenotypic endpoints mechanistically representative of rate-limiting events in human carcinogenesis.
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: The use of laboratory animals to screen chemicals for carcinogenic activity is set to decline because of new EC regulations and continuing public pressure. This has revived interest in cell culture models for this purpose. Such cell transformation assays (CTAs) should: (i) include those based on metabolically competent normal human cells of epithelial origin (the source of most human cancers), (ii) employ objective transformation criteria that reflect key molecular events driving human cancer development, and (iii) be responsive to non-genotoxic and genotoxic carcinogens. This project has been formulated to develop human CTAs meeting all these requirements. New models will be based on human mammary epithelial cells and squamous epithelial cells. Assays will employ transformation endpoints directly linked to two key events responsible for the immortality of human cancer cells (senescence-bypass). The applicants have already demonstrated the feasibility of developing exploitable CTAs using these approaches. Novel human CTAs will be exhaustively validated against panels of genotoxic and non-genotoxic carcinogens. New assays will be available for further evaluation by industry and regulatory bodies by the end of the project. Human CTAs emerging from the project will have a major impact on reducing animal usage in carcinogenicity assessment, and therefore on the 3Rs.


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: CANCER AND NEOPLASMS
Title: Using induced pluripotent stem cells (iPSC) as a replacement for in vivo models to screen novel therapies which target self-renewal pathways in chronic myeloid leukaemia
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Chronic myeloid leukaemia (CML) is often described as a paradigm for the cancer stem cell hypothesis. CML develops when a multipotent haemopoietic stem cell (HSC) acquires the Philadelphia chromosome (Ph), a reciprocal translocation between the long arms of chromosomes 9 and 22, designated t(9;22)(q34;q11). The result is the expression of a constitutively active tyrosine kinase called BCR-ABL, the putative cause of CML. Emerging evidence indicates that CML is sustained by a small pool of highly quiescent cancer stem cells (CSCs), with self-renewing capacity, which reside within the bone marrow niche. These primitive CSCs are insensitive to treatment including specific BCR-ABL tyrosine kinase inhibitor (TKI) therapy (imatinib, dasatinib, nilotinib, bosutinib), making them difficult to eradicate. Recent interest has focused on dual targeting methods inhibiting BCR-ABL tyrosine kinase activity in combination with specific small-molecule inhibitors, that target conserved self-renewal pathways (Wnt, Hedgehog [Hh], bone morphogenic protein [BMP], transforming growth factor-β [TGFβ] and Notch pathways). However obtaining sufficient numbers of these CSCs to perform the detailed biochemistry analysis necessary to determine efficacy of these potential treatments is proving difficult. Induced pluripotent stem cells (iPSC) have the potential to solve this problem. Generating disease-specific iPSC from patient cells will allow us to produce both primitive CSCs and more differentiated progeny for drug discovery in CML. This ideal model will enable the efficacy of individual drug treatments versus combination treatments to be evaluated at multiple levels and more importantly their ability to eradicate the most primitive CSC populations responsible for minimal residual disease, relapse and disease progression. Studies suggest that identifying agents that specifically target the CSCs is likely to be essential for the elimination (cure) of leukaemia.


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: CANCER AND NEOPLASMS
Title: Development of a non-animal model for characterizing drug-resistant tumours.
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: A small molecule inhibitor of histone deacetylases (HDACi) is currently in use for the treatment of refractory cutaneous T-cell lymphoma and more than eight HDACis are in clinical trials for use against a range of solid and haematological malignancies, either singly or in combination therapies. However, this approach is limited as cancerous cells develop resistance to HDACi treatment, through unknown mechanisms No biomarkers are available to distinguish HDACi sensitive and resistant tumour cells. The route to generating resistant tumour cells to identify biomarkers and mechanisms is to induce tumours in mice and induce resistance in order to isolate cells. This uses many mice and involves protocols rated as moderate severity. We will develop a non-animal model for HDACi resistance by exploiting the easy genetic manipulation of Dictyostelium discoideum to identify mutations conferring HDACi-resistance. Dictyostelium is an ideal model for studying histone modifications, as modifications are conserved between Dictyostelium and human cells, including a number not found in yeast. HDACis inhibit Dictyostelium development facilitating genetic screens for resistant mutants. A pilot screen successfully identified a candidate gene and we will extend this analysis. We have two aims 1. Characterising the histone modifications in resistant strains will identify bio-marker profiles associated with resistance. 2. Elucidating the mechanism of action of the proteins/RNAs encoded by target genes will increase our understanding of the mechanisms by which HDACis target tumour cells. This will facilitate targeting HDACis to responsive cancers and developing strategies (combination therapies or inhibitors with altered specificity) to overcome resistance, minimising use of animals


Research activity codes: 5.1 PHARMACEUTICALS;5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: CANCER AND NEOPLASMS
Title: The validation of defined genotoxic thresholds, leading to better in vitro risk assessments of carcinogenic potential
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: As part of the safety assessment of new chemicals (pharmaceuticals, pesticides etc), a tiered approach to genotoxicity (DNA damaging) testing is currently recommended. This approach is designed to prevent human exposures to agents that can damage DNA, as DNA damage is closely linked to cancer development. The tiered approach involves all chemicals entering stage 1 tests where DNA damage induction is assessed in cells cultured in the laboratory. Negative results in these tests reassure the manufacturer that DNA damage is unlikely to be induced by that agent. However, any chemicals testing positive in these tests are usually then put through stage 2 tests which are carried out in animals (mice usually). This can involve thousands of such animal tests being carried out in the UK per year. The stage 2 tests are designed to overcome problems with false positive results which can occur in stage 1 tests and also to more rigorously assess the risks to human health. The false positive results in stage 1 tests can often arise due to high doses of chemicals being used. High doses of chemical have traditionally been used to ensure that DNA damaging effects are identified and it has widely been assumed that the effects are induced in a linear manner. That is, if a high dose is positive, then a low dose will also be positive. We have recently shown how important dose can be in terms of DNA damage. We uniquely demonstrated that DNA damaging agents whilst giving positive results at high doses, are not DNA damaging at low doses. This confirms that “safe” doses exist, even for potent DNA damaging agents which are known to induce cancer. Hence, identifying these critical dose ranges is vital if we are to assess the effects of dose on DNA damage. If chemicals in stage 1 tests are only positive at high doses and not at low doses, and these positive effects were seen at doses estimated to be well above human exposures, then this would suggest that these chemicals would not be a human health risk. Importantly, the progression of these chemicals into stage 2 (animal) tests might not be necessary. Hence, by changing the testing strategy to consider the effects of dose, we could reduce the numbers of animal tests in toxicology. To add weight to the argument that DNA damaging thresholds exist and can be fully explained, we need high quality, cell-based studies of DNA damage induction for a wide range of chemicals. This application is aimed at supplying new knowledge in this field to impact on the testing strategies for DNA damage assessment with the ultimate aim of reducing the number of animal tests.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS
Title: In vitro methods for replacement of current in vivo assays for development of drugs against heterogeneous cancer stem cell populations.
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: The development of resistance to therapeutic regimens is a major problem for cancer therapy, as it precludes complete ablation of the tumour and leaves highly malignant and drug resistant cells remaining at the tumour site. These then drive local and distant tumour recurrence, the main cause of cancer mortality. Recent evidence has demonstrated the important role that cancer stem cells play in this process, as they express heightened resistance to current therapies compared to the majority non-stem cell population. They thus survive and drive tumour recurrence. In the course of my current project, I have identified two cancer stem cell populations in squamous cell carcinoma (SCC). One has an epithelial phenotype and a CD44highESAhigh marker profile, and the other has a more mesenchymal phenotype and a CD44highESAlow marker profile. Cells are able to switch in both directions between these two phenotypes through the processes of epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET). The epithelial phenotype exhibits much faster growth and the mesenchymal phenotype has greatly enhanced invasive capacity. Importantly, the results obtained in vitro were reproducible using an in vivo transplantation system, with the only difference being that the in vivo murine system is inferior in terms of the amount of useful information generated about the cell types under investigation. I also now have evidence to demonstrate that both the epithelial and mesenchymal cancer stem cell phenotypes exhibit therapeutic resistance, but differences in their responses to individual drugs suggests differing underlying mechanisms of therapeutic resistance. Therefore, new methods of targeting both phenotypes are required in order to develop therapies that clear all malignant cells and thus prevent tumour recurrence. I aim to develop an in vitro cell line model that enables high-throughput testing of compounds for their ability to target both cancer stem cell thenotypes. I will also use this model to assess the mechanisms of therapeutic resistance. In addition to work using cell lines, I will develop in vitro assays on fresh tumour specimens to enable comparison with data gained using cell lines and thus provide evidence that cell lines are a relevant model for therapeutic development.


Research activity codes: 5.1 PHARMACEUTICALS;5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: CANCER AND NEOPLASMS
Title: Replacement of murine transplantation assays by 3-dimensional in vitro substitutes for the assessment of therapeutic responses of cancer stem cells.
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Recent research points to important roles of sub-populations of cancer stem cells (CSCs) in the growth and spread of cancers. Of particular interest, CSCs are several-fold more resistant to therapeutic killing, a property likely to be related to tumour recurrence. This raises the need to discover drugs with effective actions in killing them. This effort has now been rendered more complex by the demonstration that epithelial-mesenchymal transition (EMT) reversibly switches CSCs between a sessile epithelial phenotype and a motile EMT phenotype. In these manifestations CSCs differ in their drug resistance patterns. It is further apparent that rates of phenotypic switching, and hence therapeutic responses, are influenced both by cell intrinsic factors and by stimuli from the tumour microenvironment. Currently, tumour transplantation to mice is considered the "gold standard" for studies of the properties and therapeutic responses of CSCs. This is based in the assumption that environmental requirements for stem cell survival are too complex to be adequately modelled in vitro. However, mouse models are far from perfect and basic CSC properties, including therapeutic resistance and entry into EMT, are retained even in standard 2 dimensional cultures on plastic. 3-dimensional co-culture of human tumour cells with fibroblasts results in interactions and cellular changes that even more closely mimic those occurring in the in vivo situation. To replace animal transplantation for stem cell assays, we therefore propose to (a) generate a heterotypic 3D culture system, containing both tumour and stromal cells, in which the cellular responses associated with EMT mimic those present in vivo, (b) develop EMT reporter systems enabling rapid analysis of cellular responses to known inducers/blockers of EMT, (c) assess the utility of this system for high throughput assays of agents suitable for the control of EMT and for killing CSCs in their various phenotypic manifestations.


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: CANCER AND NEOPLASMS
Title: Targeted, radiolabelled near-infrared quantum dots for high sensitivity and resolution, dual modality imaging of human tumours in mice
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: This proposal will use near infrared quantum dots (NIR-QDs), to improve sensitivity and resolution of imaging in advanced pre-clinical cancer models. Successful development of NIR-QDs will have 3Rs impact, enabling use of more refined, patient-relevant cancer models and reducing numbers of animals used. Academia and Pharma are focussed on developing new cancer drugs resulting in the use of >430,000 mice/year. We estimate that since more advanced patient-relevant cancer models will predict drug failure earlier in preclinical testing, numbers of animals used for continued evaluation of such failing drugs would be reduced by half (~170,000 animals/year). Bioluminescent/fluorescent imaging despite advantages has drawbacks, especially for use in advanced cancer models. Bioluminescent imaging requires administration of substrate (an additional in vivo procedure); organic fluorophores photo-bleach and have limited sensitivity due to light absorption by tissues. For application in advanced models using close-to-patient tissues, standard labelling methods cause loss of cellular heterogeneity resulting in models unrepresentative of the original tumour. The NIR-QDs we will develop do not photo-bleach, will be water-soluble and optically active in the NIR wavelength, providing improved tissue penetration and sensitivity, and will incorporate targeting ligands for uptake by specific populations of cells within complex xenografts, and a positron-emitting radionuclide for translation into the PET/CT imaging platform. The knowledge gained will have scientific as well as 3Rs impact, leading to development of probes that can be more broadly applied to imaging complex tissues in diseases other than cancer, and in a non-disease context e.g. bioengineered tissues. The probes also have potential for economic impact, through improving the drug discovery cascade by refinement of patient-relevant pre-clinical cancer models that are better able to predict clinical outcomes.


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: CANCER AND NEOPLASMS