Developing stem-cell containing organoids as a pre-clinical model for colorectal cancer therapeutics

Colorectal cancer (CRC) is the second most common cause of cancer related deaths in the UK. Whilst identification of molecular events that contribute to the initiation and progression of CRC have facilitated the development of predictive biomarker-driven therapeutics, their success in the clinic has been restricted by the lack of anticipated responses. Furthermore, not all genetic signatures of a tumour have been linked to related drug targets, highlighting the need for novel development of compounds and better therapeutic rationales for the treatment of patient subsets. The limited success of targeted therapies, both within the clinic and drug discovery pipeline, has been attributed to a lack of effective preclinical models that are capable of capturing the complexity of deregulated signalling networks. The development of functional readouts that better represent tumour complexity, is therefore imperative to confirm the effects of hypothesis-driven therapeutics. This thesis therefore aimed to investigate whether 3D CRC organoids , which show a degree of greater complexity compared to preceding in vitro models, could be applied as suitable readouts for stratified medicine programmes and novel compounds within the drug discovery setting. To achieve this, a panel of 3D patient-derived CRC organoids cultures were generated. Suitable methodologies were established to facilitate organoids towards quantifiable, robust assay formats. This platform enabled the study of organoids within an in vitro clinical trial setting, based upon treatments administered within the ongoing FOCUS 4 stratified medicine trial, exploring organoids’ capacity to predict responses to targeted therapeutics. Organoids were differentially sensitive to therapies, irrespective of their genotypic background. It will be interesting to see whether prediction-response correlations observed in this study are typical of those seen in patients and whether functional readouts will be required to support stratified medicine approaches. Quantitative image-based analysis was also found to identify signatures of organoid responses against novel Wnt signalling inhibitors, suggesting that organoids may constitute a platform that can be used to study the effects of targeting a prospective cancer stem cell (CSC) population. Taken together, the findings in this thesis highlight the utility of patient derived organoid models as a functional model to evaluate novel therapeutic strategies, potentially generating clinically relevant hypotheses

Improving and accelerating the differentiation and functional maturation of human stem cell-derived neurons: role of extracellular calcium and GABA

Neurons differentiated from pluripotent stem cells using established neural culture conditions often exhibit functional deficits. Recently, we have developed enhanced media which both synchronize the neurogenesis of pluripotent stem cell-derived neural progenitors and accelerate their functional maturation; together these media are termed SynaptoJuice. This pair of media are pro-synaptogenic and generate authentic, mature synaptic networks of connected forebrain neurons from a variety of induced pluripotent and embryonic stem cell lines. Such enhanced rate and extent of synchronized maturation of pluripotent stem cell-derived neural progenitor cells generates neurons which are characterized by a relatively hyperpolarized resting membrane potential, higher spontaneous and induced action potential activity, enhanced synaptic activity, more complete development of a mature inhibitory GABAA receptor phenotype and faster production of electrical network activity when compared to standard differentiation media. This entire process – from pre-patterned neural progenitor to active neuron – takes 3 weeks or less, making it an ideal platform for drug discovery and disease modelling in the fields of human neurodegenerative and neuropsychiatric disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease and Schizophrenia

3D imaging of colorectal cancer organoids identifies responses to Tankyrase inhibitors

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model

Efficient intravenous tumor targeting using the αvβ6 integrin-selective precision virotherapy Ad5NULL-A20

We previously developed a refined, tumor-selective adenovirus, Ad5NULL-A20, harboring tropism ablating mutations in each major capsid protein, to ablate all native means of infection. We incorporated a 20-mer peptide (A20) in the fiber knob for selective infection via αvβ6 integrin, a marker of aggressive epithelial cancers. Methods: To ascertain the selectivity of Ad5NULL-A20 for αvβ6-positive tumor cell lines of pancreatic and breast cancer origin, we performed reporter gene and cell viability assays. Biodistribution of viral vectors in mice harboring xenografts with low, medium, and high αvβ6 levels was quantified by qPCR for viral genomes 48 h post intravenous administration. Results: Ad5NULL-A20 vector transduced cells in an αvβ6-selective manner, whilst cell killing mediated by oncolytic Ad5NULL-A20 was αvβ6-selective. Biodistribution analysis following intravenous administration into mice bearing breast cancer xenografts demonstrated that Ad5NULL-A20 resulted in significantly reduced liver accumulation coupled with increased tumor accumulation compared to Ad5 in all three models, with tumor-to-liver ratios improved as a function of αvβ6 expression. Conclusions: Ad5NULL-A20-based virotherapies efficiently target αvβ6-integrin-positive tumors following intravenous administration, validating the potential of Ad5NULL-A20 for systemic applications, enabling tumor-selective overexpression of virally encoded therapeutic transgenes

Monitoring intracellular oxygen concentration: Implications for hypoxia studies and real-time oxygen monitoring

The metabolic properties of cancer cells have been widely accepted as a hallmark of cancer for a number of years and have shown to be of critical importance in tumour development. It is generally accepted that tumour cells exhibit a more glycolytic phenotype than normal cells. In this study, we investigate the bioenergetic phenotype of two widely used cancer cell lines, RD and U87MG, by monitoring intracellular oxygen concentrations using phosphorescent Pt-porphyrin based intracellular probes. Our study demonstrates that cancer cell lines do not always exhibit an exclusively glycolytic phenotype. RD demonstrates a reliance on oxidative phosphorylation whilst U87MG display a more glycolytic phenotype. Using the intracellular oxygen sensing probe we generate an immediate readout of intracellular oxygen levels, with the glycolytic lines reflecting the oxygen concentration of the environment, and cells with an oxidative phenotype having significantly lower levels of intracellular oxygen. Inhibition of oxygen consumption in lines with high oxygen consumption increases intracellular oxygen levels towards environmental levels. We conclude that the use of intracellular oxygen probes provides a quantitative assessment of intracellular oxygen levels, allowing the manipulation of cellular bioenergetics to be studied in real time

Anti-Folate Receptor alpha-directed Antibody Therapies Restrict the Growth of Triple Negative Breast Cancer

PURPOSE: Highly-aggressive triple negative breast cancers (TNBCs) lack validated therapeutic targets and have high risk of metastatic disease. Folate Receptor alpha (FRα) is a central mediator of cell growth regulation that could serve as an important target for cancer therapy. EXPERIMENTAL DESIGN: We evaluated FRα expression in breast cancers by genomic (N = 3414) and immunohistochemical (N = 323) analyses and its association with clinical parameters and outcomes. We measured the functional contributions of FRα in TNBC biology by RNA interference and the anti-tumor functions of an antibody recognizing FRα (MOv18-IgG1), in vitro and in human TNBC xenograft models. RESULTS: FRα is overexpressed in significant proportions of aggressive basal like/TNBC tumors, and in post-neoadjuvant chemotherapy-residual disease associated with a high risk of relapse. Expression is associated with worse overall survival. TNBCs show dysregulated expression of thymidylate synthase, folate hydrolase 1 and methylenetetrahydrofolate reductase, involved in folate metabolism. RNA interference to deplete FRα decreased Src and ERK signaling and resulted in reduction of cell growth. An anti-FRα antibody (MOv18-IgG1) conjugated with a Src inhibitor significantly restricted TNBC xenograft growth. Moreover, MOv18-IgG1 triggered immune-dependent cancer cell death in vitro by human volunteer and breast cancer patient immune cells, and significantly restricted orthotopic and patient-derived xenograft growth. CONCLUSIONS: FRα is overexpressed in high-grade TNBC and post-chemotherapy residual tumors. It participates in cancer cell signaling and presents a promising target for therapeutic strategies such as antibody-drug conjugates, or passive immunotherapy priming Fc-mediated anti-tumor immune cell responses

Forced cell-cycle exit and modulation of GABAA, CREB and GSK3β signaling promote functional maturation of induced pluripotent stem cell-derived neurons

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal sub-types and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time-courses of functional maturation which are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to post-synaptic receptor maturation. Here, we describe a simple protocol which employs the sequential addition of just two supplemented media which have been formulated to separate the two key phases of neural differentiation - the neurogenesis and synaptogenesis - each characterized by different signaling requirements. Employing these media, this new protocol synchronised neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials, moreover they exhibited augmented: i) spontaneous electrical activity; ii) regenerative induced action potential train activity; iii) Na+ current availability, and; iv) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAA receptor phenotype which was demonstrated by Ca2+ imaging and the ability of GABAA receptor blockers to evoke seizurogenic network activity in multi-electro array recordings. Furthermore, since this protocol can exploit expanded and frozen pre-patterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens

Targeting TRIM37-driven centrosome dysfunction in 17q23-amplified breast cancer

Genomic instability is a hallmark of cancer, and has a central role in the initiation and development of breast cancer1,2. The success of poly-ADP ribose polymerase inhibitors in the treatment of breast cancers that are deficient in homologous recombination exemplifies the utility of synthetically lethal genetic interactions in the treatment of breast cancers that are driven by genomic instability3. Given that defects in homologous recombination are present in only a subset of breast cancers, there is a need to identify additional driver mechanisms for genomic instability and targeted strategies to exploit these defects in the treatment of cancer. Here we show that centrosome depletion induces synthetic lethality in cancer cells that contain the 17q23 amplicon, a recurrent copy number aberration that defines about 9% of all primary breast cancer tumours and is associated with high levels of genomic instability4-6. Specifically, inhibition of polo-like kinase 4 (PLK4) using small molecules leads to centrosome depletion, which triggers mitotic catastrophe in cells that exhibit amplicon-directed overexpression of TRIM37. To explain this effect, we identify TRIM37 as a negative regulator of centrosomal pericentriolar material. In 17q23-amplified cells that lack centrosomes, increased levels of TRIM37 block the formation of foci that comprise pericentriolar material-these foci are structures with a microtubule-nucleating capacity that are required for successful cell division in the absence of centrosomes. Finally, we find that the overexpression of TRIM37 causes genomic instability by delaying centrosome maturation and separation at mitotic entry, and thereby increases the frequency of mitotic errors. Collectively, these findings highlight TRIM37-dependent genomic instability as a putative driver event in 17q23-amplified breast cancer and provide a rationale for the use of centrosome-targeting therapeutic agents in treating these cancers