Ex-vivo drug screening of surgically resected glioma stem cells to replace murine avatars and provide personalise cancer therapy for glioblastoma patients [version 1; peer review: 2 approved]

With diminishing returns and high clinical failure rates from traditional preclinical and animal-based drug discovery strategies, more emphasis is being placed on alternative drug discovery platforms. Ex vivo approaches represent a departure from both more traditional preclinical animal-based models and clinical-based strategies and aim to address intra-tumoural and inter-patient variability at an earlier stage of drug discovery. Additionally, these approaches could also offer precise treatment stratification for patients within a week of tumour resection in order to direct tailored therapy. One tumour group that could significantly benefit from such ex vivo approaches are high-grade gliomas, which exhibit extensive heterogeneity, cellular plasticity and therapy-resistant glioma stem cell (GSC) niches. Historic use of murine-based preclinical models for these tumours has largely failed to generate new therapies, resulting in relatively stagnant and unacceptable survival rates of around 12-15 months post-diagnosis over the last 50 years. The near universal use of DNA damaging chemoradiotherapy after surgical resection within standard-of-care (SoC) therapy regimens provides an opportunity to improve current treatments if we can identify efficient drug combinations in preclinical models that better reflect the complex inter-/intra-tumour heterogeneity, GSC plasticity and inherent DNA damage resistance mechanisms. We have therefore developed and optimised a high-throughput ex vivo drug screening platform; GliExP, which maintains GSC populations using immediately dissociated fresh surgical tissue. As a proof-of-concept for GliExP, we have optimised SoC therapy responses and screened 30+ small molecule therapeutics and preclinical compounds against tumours from 18 different patients, including multi-region spatial heterogeneity sampling from several individual tumours. Our data therefore provides a strong basis to build upon GliExP to incorporate combination-based oncology therapeutics in tandem with SoC therapies as an important preclinical alternative to murine models (reduction and replacement) to triage experimental therapeutics for clinical translation and deliver rapid identification of effective treatment strategies for individual gliomas

Inhibition of ATR prevents macropinocytosis driven retraction of neurites and opposes invasion in GBM

Glioblastoma (GBM) is the most common and aggressive type of primary brain tumour and remains incurable despite decades of research. GBM are characterised by highly infiltrative growth patterns that contribute to the profound cognitive and neurological symptoms experienced by patients, and to inevitable recurrence following treatment. Novel treatments that reduce infiltration of the healthy brain have potential to ameliorate clinical symptoms and improve survival. Here, we report a novel role of the Ataxia telangiectasia and Rad 3 related kinase (ATR) in supporting the invasive properties of GBM cells through the regulation of macropinocytosis-driven internalisation of integrin adhesion receptors. We demonstrate that inhibition of ATR opposes GBM migration in vitro, and correspondingly reduces infiltrative behaviour in orthotopic mouse models. These results indicate that ATR inhibition, in addition to its use as a radiosensitiser, may be effective in reducing GBM infiltration and its associated symptoms

Generation and characterisation of the Sheffield Living Biobank (SLB) of post-surgical residual Glioblastoma to identify novel therapeutic targets.

Glioblastoma remains the most common and aggressive CNS malignancy diagnosed accounting for ~5,300 deaths in the UK annually, and around 200,000 globally each year. Additionally, there has been no significant universal improvement to disease outcome or survival since the implementation of the chemotherapeutic Temozolomide (TMZ) in 2005. Numerous biological blockades perturbing the development of novel effective therapies have been identified which include extensive inter-patient heterogeneity, the presence of the blood-brain barrier (BBB) hindering the bioavailability of novel therapeutics, the highly invasive nature of the disease hindering extent of resection (EOR) safely possible, an immune suppressive tumour microenvironment (TME) and the presence of a treatment resistant subpopulations of cells, termed glioma stem cells (GSCs), which have been indicated as pivotal contributors to the currently inevitable recurrence of the disease post-“Stupp” regimen treatment and the death of the patient (median survival of 12-15 months). Recent reports have also now highlighted extensive intratumoural heterogeneity between spatially divergent populations of the same tumour (to a similar degree as inter-patient variation) and a lack of pre-clinical models capable of recapitulating this heterogeneity ultimately allowing for full characterisation of the disease. Here we display a wealth of data characterising a novel biobank of GSC populations isolated from the necrotic core and distal invasive edge of large en-bloc specimens resected within the Royal Hallamshire Hospital, Sheffield. These samples contain large portions of infiltrated brain (not possible in all resections) which allow for direct characterisation of the typically residual GSC populations left behind after surgery. This PhD study focussed on the characterisation of the DNA damage response (DDR) within these populations as these repair cascades directly reverse the damage induced by both standard- of-care (SoC) therapies (radiotherapy and TMZ) employed in the treatment of GBM. Additionally, therapies targeting these cascades have displayed unparalleled efficacy in a range of other cancers. This led us to pose the hypothesis that “GSCs typically left behind after surgery are inherently treatment resistant due to increased expression of both GSC stem markers and the DDR”. Initial biomolecular characterisation of these Core (resected) and Edge (residual) GSC populations (propagated in clinically relevant 3D AlvetexTM architecture) revealed differential expression and activation of several DDR mediators both endogenously and in response to SoC therapies. Additionally, we also recorded differential expression of several GSC markers suggestive of differential cell composition within these populations reflective of the previously established “Verhaak” GSC cell states. Further investigation also revealed differential DNA repair kinetics and chromatin order between Core and Edge GSC populations. We therefore believe that the differences identified within this study contribute to the differential TMZ sensitivity identified within these heterogeneity models. Interestingly, we did not record substantial differences in IR sensitivity between Core and Edge populations, however, we did observe differences in radio-sensitisation capacity of the clinically relevant ATR inhibitor (AZD6738) and ATM inhibitor (AZD1390), the latter of which is currently under investigation in clinical trials for GBM (NCT03523628). Finally, bulk RNA-seq datasets generated from these Core and Edge GSC populations revealed stark differences in the expression of all DNA repair cascades and stem markers again suggestive of differential GSC cell states between resected and residual tumour cells. This data also highlighted potential distinct preferences for different DNA repair pathways between these stem cell states therefore highlighting multimodal DDR interruptions as a viable treatment strategy for targeting entire tumour GSC populations. It is imperative to remember, all of the differences identified within this study largely followed a locational signature, however, there was significant exceptions to this ideology even within our small sample size leading us to reject our initial hypothesis. In conclusion, the unprecedented insight these models have provided suggests differential DNA repair capacity resulting in differential treatment sensitivity to SoC therapies, with further characterisation of these models employing single-cell technologies and the strategies outlined within the future work aiding in elucidating the viability of novel treatment strategies ideally leading to improved disease outcome. This has led us to pose a revised hypothesis which states: “Individual “Verhaak” GSC subtypes (Classical, Proneural and Mesenchymal) display distinct DDR expression and activation signatures, which with further characterisation, presents an exciting potential therapeutic window for the development of novel therapeutics capable of targeting the entire GBM tumour.

Ex-vivo drug screening of surgically resected glioma stem cells to replace murine avatars and provide personalise cancer therapy for glioblastoma patients [version 2; peer review: 2 approved]

With diminishing returns and high clinical failure rates from traditional preclinical and animal-based drug discovery strategies, more emphasis is being placed on alternative drug discovery platforms. Ex vivo approaches represent a departure from both more traditional preclinical animal-based models and clinical-based strategies and aim to address intra-tumoural and inter-patient variability at an earlier stage of drug discovery. Additionally, these approaches could also offer precise treatment stratification for patients within a week of tumour resection in order to direct tailored therapy. One tumour group that could significantly benefit from such ex vivo approaches are high-grade gliomas, which exhibit extensive heterogeneity, cellular plasticity and therapy-resistant glioma stem cell (GSC) niches. Historic use of murine-based preclinical models for these tumours has largely failed to generate new therapies, resulting in relatively stagnant and unacceptable survival rates of around 12-15 months post-diagnosis over the last 50 years. The near universal use of DNA damaging chemoradiotherapy after surgical resection within standard-of-care (SoC) therapy regimens provides an opportunity to improve current treatments if we can identify efficient drug combinations in preclinical models that better reflect the complex inter-/intra-tumour heterogeneity, GSC plasticity and inherent DNA damage resistance mechanisms. We have therefore developed and optimised a high-throughput ex vivo drug screening platform; GliExP, which maintains GSC populations using immediately dissociated fresh surgical tissue. As a proof-of-concept for GliExP, we have optimised SoC therapy responses and screened 30+ small molecule therapeutics and preclinical compounds against tumours from 18 different patients, including multi-region spatial heterogeneity sampling from several individual tumours. Our data therefore provides a strong basis to build upon GliExP to incorporate combination-based oncology therapeutics in tandem with SoC therapies as an important preclinical alternative to murine models (reduction and replacement) to triage experimental therapeutics for clinical translation and deliver rapid identification of effective treatment strategies for individual gliomas