Specific genetic mutations in glioma can predict prognosis as well as predict the potential benefits of particular treatments. There is an important and urgent need to establish whether current reporting of race and ethnicity data in contemporary studies generating new genome-scale sequencing data for gliomas is adequate. We aimed to provide a snapshot describing the current state of racial and ethnic representation in recently published studies which, have generated de novo genome-scale sequencing data from glioma samples. We searched PubMed, Embase, Web of Science, and Scopus and systematically reviewed articles published be- tween January and November 2023 reporting de novo genome-scale sequencing data generated using samples from patients diagnosed with a glioma (according to WHO 2021 criteria) to characterise the reporting and com- position of race and ethnicity data. Thirty-five studies involving 5,601 patients were analysed. Race or ethnicity data was reported in only 3 studies (8.6%), of which none provided omic data in a format that could be stratified by race or ethnicity. Reporting varied by continent with all 3 studies including race or ethnicity data based in North America. Where racial data was available, we found that samples used for genome-scale characterisation came from patients reported as being White in 91.1% cases (41 patients), with 6.7% (3 patients) reported as Black and 2.2% (1 patient) as Hispanic. Race and ethnicity data is exquisitely underreported in contemporary glioma studies generating genome-scale omic data, and where it is reported, the data is typically not shared in a format that makes stratification of genetic variants and their impact by ethnicity possible. Improved reporting of race and ethnicity data, along- side initiatives to broaden representation, are required to enhance our understanding of glioma biology across different populations and ultimately, provide more equitable advancements in healthcar

Background:The expanding repertoire of studies generating genome-scale omic datasets from glioma samples provides a generational opportunity to uncover mechanisms driving aggressive biology and develop new treatments. However, ensuring such studies reflect the breadth of racial groups and ethnicities affected by gliomas is critical to support equity in future therapeutic advances. We therefore report a contemporary snapshot of the representation of race and ethnicity in omic glioma studies. Methods:We searched PubMed, Embase, Web of Science, and Scopus and systematically reviewed articles published between January and November 2023 reporting de novo genome-scale sequencing data generated using samples from patients diagnosed with glioma (according to World Health Organization 2021 criteria) to characterize the reporting and composition of race and ethnicity data. Results:Thirty-five studies involving 5601 patients were analyzed. Race or ethnicity data was reported in only 3 studies (8.6%), of which none provided omic data in a format that could be stratified by race or ethnicity. Reporting varied by continent with all 3 studies including race or ethnicity data based in North America. Where racial data was available, we found that samples used for genome-scale characterization came from patients reported as being White in 91.1% cases (41 patients), with 6.7% (3 patients) reported as Black and 2.2% (1 patient) as Hispanic. Conclusions:These studies underscore an urgent need for improved reporting and representation to enhance our understanding of glioma biology across different populations and guide targeted initiatives from policymakers and funders to support equitable improvements in healthcare.

Prostate cancer bone metastases are commonly treated with radium-223 (Ra-223); however, patients ultimately experience relapse. These metastases are currently incurable and there is an unmet need to improve the efficacy of Ra-223 treatment regimens. Ra-223 causes DNA strand breaks within tumor cells that are in close proximity to bone. We hypothesized that relapse is partly due to Ra-223-induced activation of DNA damage-response pathways; therefore, inhibiting DNA repair pathways with ATM inhibitors (ATMi), currently in clinical trials for other cancers, would radiosensitize bone metastases, increasing anti-tumor efficacy of Ra-223. To test this hypothesis, 2 mouse models of prostate cancer bone metastasis were administered 20 mg/kg/d ATMi on day 2, 7 or 10 and Ra-223 treatment (50 kBq/kg/wk or 300 kBq/kg/wk) commenced 24 h after first ATMi treatment. The 300 kBq/kg Ra-223 reduced PC3 prostate cancer bone metastases by 60.9%-87.4% compared with placebo. Radiosensitization with either the ATMi AZD0156 or AZD1390 prior to 300 kBq/kg Ra-223 treatment further reduced bone metastasis by 94.1% and 88.7%, respectively, whereas combining AZD1390 with 50 kBq/kg synergistically reduced tumor size and the number of mice with tumors in bone by 50% compared with Ra-223 alone. Treating early-stage RM1 prostate cancer bone metastasis with a combination of AZD1390 and 50 kBq/kg Ra-223 had no additional benefits compared with Ra-223 alone. However, delaying treatment to mimic overt bone metastases resulted in a 50% reduction in bone metastasis when ATMi was added prior to Ra-223 compared with Ra-223 alone. Notably, adding ATMi prior to Ra-223 did not exacerbate Ra-223-induced adverse effects on the bone. Instead, this treatment combination increased subsets of anti-tumor immune cells. Taken together, our data suggest that ATMi may be an effective radiosensitizer for increasing efficacy of Ra-223 in prostate cancer bone metastases, reducing Ra-223-induced adverse effects on bone.

Cancer cells undergo morphological changes and phenotype switching to promote invasion into healthy tissues. Manipulating the transitional morphological states in cancer cells to prevent tumor dissemination may enhance survival and improve treatment response. We describe two members of the RhoGTPase activating protein (ARHGAP) family, ARHGAP12 and ARHGAP29, as regulators of transitional morphological states in glioma via Src kinase signaling events, leading to morphological changes that correspond to phenotype switching. Moreover, we establish a link between glycogen synthase kinase 3 (GSK-3) inhibition and β-catenin translocation in altering transcription of ARHGAP12 and ARHGAP29. Silencing ARHGAP12 causes loss of N-cadherin and adoption of mesenchymal morphology, a characteristic feature of aggressive cellular behavior. In patients with glioblastoma (GBM), we identify a link between ARHGAP12 and ARHGAP29 co-expression and recurrence after treatment. Consequently, we propose that further investigation of how ARHGAPs regulate transitional morphological events to drive cancer dissemination is warranted.

SUMOylation, the covalent attachment of the small ubiquitin-like modifier (SUMO) to target proteins, and its reversal, deSUMOylation by SUMO proteases like Sentrin-specific proteases (SENPs), are crucial for initiating cellular responses to hypoxia. However, their roles in subsequent adaptation processes to hypoxia such as mitochondrial autophagy (mitophagy) remain unexplored. Here, we show that general SUMOylation, particularly SUMO2/3 modification, suppresses mitophagy under both normoxia and hypoxia. Furthermore, we identify deSUMO2/3-ylation enzyme SENP3 and mitochondrial Fission protein 1 (FIS1) as key players in hypoxia-induced mitophagy (HIM), with SUMOylatable FIS1 acting as a crucial regulator for SENP3-mediated HIM regulation. Interestingly, we find that hypoxia promotes FIS1 SUMO2/3-ylation and triggers an interaction between SUMOylatable FIS1 and Rab GTPase-activating protein Tre-2/Bub2/Cdc16 domain 1 family member 17 (TBC1D17), which in turn suppresses HIM. Therefore, we propose a novel SUMOylation-dependent pathway where the SENP3-FIS1 axis promotes HIM, with TBC1D17 acting as a fine-tuning regulator. Importantly, the SENP3-FIS1 axis plays a protective role against hypoxia-induced cell death, highlighting its physiological significance, and hypoxia-inducible FIS1-TBC1D17 interaction is detectable in primary glioma stem cell-like (GSC) cultures derived from glioblastoma patients, suggesting its disease relevance. Our findings not only provide new insights into SUMOylation/deSUMOylation regulation of HIM but also suggest the potential of targeting this pathway to enhance cellular resilience under hypoxic stress.

Background Glioblastoma is a highly infiltrative, currently incurable brain cancer. To date, translation of novel therapies for glioblastoma from the laboratory into clinical trials has relied heavily on in vitro cell culture and murine (subcutaneous and orthotopic) xenograft models using cells derived from the main bulk of patient tumours. However, it is the residual cells left-behind after surgery that are responsible for disease progression and death in the clinic. A lack of substantial improvements in patient survival for decades suggests commonly used murine xenograft models, a key step before clinical trials, do not reflect the biology of residual disease in patients. Methods To address this, we have developed the ‘Sheffield Protocol’ to generate ex vivo models that reflect both resected, and post-surgical residual disease from the same patient. The protocol leverages parallel derivation of inherently treatment-resistant glioblastoma stem cells (GSCs) from ‘core’ and distant ‘edge’ regions through careful macrodissection of a large en bloc specimen, such as from a partial lobectomy for tumour, followed by tissue dissociation and propagation in serum-free media. Opportunistic en bloc specimen use can liberate the most distant infiltrative cells feasibly accessible from living patients. Results We provide an example illustrating that resected and residual disease models represent spatially divergent tumour subpopulations harbouring distinct transcriptomic and cancer stem cell marker expression profiles. We also introduce the ‘Sheffield Living Biobank’ of glioma models (SLB) that incorporates over 150 GSC lines from 60+ patients, including 44+ resected and residual models, which are available for academic use via MTA. Conclusions These models provide a novel tool to reduce animal xenograft usage by improving candidate drug triage in early preclinical studies and directly replacing animal studies for some therapies that are post-Phase 1+ clinical trial for other cancers/conditions to, ultimately, deliver more effective treatments for post-surgical residual disease in glioblastoma.

Glioblastoma is an aggressive, incurable brain cancer with poor five-year survival rates of around 13% despite multimodal treatment with surgery, DNA-damaging chemoradiotherapy and the recent addition of Tumour Treating Fields (TTFields). As such, there is an urgent need to improve our current understanding of cellular responses to TTFields using more clinically and surgically relevant models, which reflect the profound spatial heterogeneity within glioblastoma, and leverage these biological insights to inform the rational design of more effective therapeutic strategies incorporating TTFields. We have recently reported the use of preclinical TTFields using the inovitroTM system within 2D glioma stem-like cell (GSC) models and demonstrated significant cytotoxicity enhancement when co-applied with a range of therapeutically approved and preclinical DNA damage response inhibitors (DDRi) and chemoradiotherapy. Here we report the development and optimisation of preclinical TTFields delivery within more clinically relevant 3D scaffold-based primary GSC models of spatial heterogeneity, and highlight some initial enhancement of TTFields potency with temozolomide and clinically approved PARP inhibitors (PARPi). These studies, therefore, represent an important platform for further preclinical assessment of TTFields-based therapeutic strategies within clinically relevant 3D GSC models, aimed towards accelerating clinical trial implementation and the ultimate goal of improving the persistently dire survival rates for these patients.

BACKGROUND:Glioblastomas have highly infiltrative growth patterns that contribute to recurrence and poor survival. Despite infiltration being a critical therapeutic target, no clinically useful therapies exist that counter glioblastoma invasion. Here, we report that inhibition of ataxia telangiectasia and Rad 3 related kinase (ATR) reduces invasion of glioblastoma cells through dysregulation of cytoskeletal networks and subsequent integrin trafficking.METHODS:Glioblastoma motility and invasion were assessed in vitro and in vivo in response to ATR inhibition (ATRi) and ATR overexpression using time-lapse microscopy, two orthotopic glioblastoma models, and intravital imaging. Disruption to cytoskeleton networks and endocytic processing were investigated via high-throughput, super-resolution and intravital imaging.RESULTS:High ATR expression was associated with significantly poorer survival in clinical datasets while histological, protein expression, and spatial transcriptomics using glioblastoma tumor specimens revealed higher ATR expression at infiltrative margins. Pharmacological inhibition with two different compounds and RNAi targeting of ATR opposed the invasion of glioblastoma, whereas overexpression of ATR drove migration. Subsequent investigation revealed that cytoskeletal dysregulation reduced macropinocytotic internalization of integrins at growth-cone-like structures, resulting in a tumor microtube retraction defect. The biological relevance and translational potential of these findings were confirmed using two orthotopic in vivo models of glioblastoma and intravital imaging.CONCLUSIONS:We demonstrate a novel role for ATR in determining invasion in glioblastoma cells and propose that pharmacological targeting of ATR could have far-reaching clinical benefits beyond radiosensitization.

Abstract High grade gliomas are the most common primary brain cancer malignancies with ~300,000 global diagnoses each year with glioblastomas (WHO grade IV) conferring the worst prognoses. This arises from their treatment resistance, rationalised by the presence of glioma stem cell-like (GSC) tumour sub-regions and high intratumoural spatio-heterogeneity. TTFields are clinically approved alternating electric fields of intermediate frequency that exert anti-mitotic effects and invoke downregulation of the DDR. Therefore, TTFields alongside clinically developed, and blood brain barrier (BBB) penetrating DDR inhibitors (DDRi) could boost cell death and overcome treatment resistance through chemo-/radiosensitisation. Primary GSC cell lines were derived from surgically resected and clinically defined tumour core and invasive edge regions and grown in more biologically relevant 3D-printed AlvetexTM scaffolds, offering highly clinically relevant models. Cells were then pre-treated with DDRi, subjected to chemo-/radiotherapy and then incubated under TTFields (200 kHz, 72 hrs) with their survival measured by clonogenic assays after 3 weeks. Immunofluorescence and western blot analysis were used to analyse the combination treatment effects on the DDR. In multiple 3D-grown core/edge GSC models, western blot analyses revealed that the DDR was altered by TTFields and clonogenic survival assays revealed that TTFields alongside DDRi led to significant chemo-/radiosensitisation. TTFields alongside DDRi effectively enhances cell death by significant treatment sensitisation effects in clinically relevant 3D GSCs which exhibit extensive intra and intertumoral spatio-heterogeneity. Given that all the DDRi used are BBB penetrable and are either clinically approved or in clinical trials, TTFields could additionally compliment these treatment regimens in gliomas to boost their efficacy.

Abstract Glioblastoma is a grade IV brain tumour with poor survival rates of 15 months post-diagnosis, and 5-year survival rates of 10%. Limited progress over the past 40 years to improve the current treatment regimen of surgery and chemo-/radio-therapy are mainly due to tumour heterogeneity that drive resistance mechanisms. The persistence of glioma stem-like cells (GSCs) sub-populations, particularly within post-surgical residual tumour tissue. ERK5 is an emerging oncology drug target that we previously highlighted as a potential target for combined therapy with temozolomide (TMZ) in glioblastoma due to heightened ERK5 expression, which is associated with poor survival. This current project aims to expand on our previous findings by establishing whether targeting ERK5 can sensitise glioblastoma cells to TMZ using primary derived ex vivo GSC models of intratumor heterogeneity and residual disease. Primary GSC models were derived from patients at the Royal Hallamshire Hospital. The PROTAC OS11 (CRUK & University of Manchester) was used to degrade ERK5. DNA damage and cell survival were evaluated following treatment of TMZ alone and in combination with OS11. Consistent with our findings in established glioma cell lines, OS11 was able to robustly degrade ERK5 in a 3D GSC model for at least 72hrs. Combining OS11 and TMZ in GSCs led to a significant increase in DNA damage, with MGMT negative cells exhibiting a greater increase in DNA damage compared to MGMT positive cells. However, unlike our previous findings in established cell lines, this did not lead to reduced cell survival in combination with TMZ. These findings further support the preclinical use of ex vivo models that better represent intratumoural heterogeneity and post-surgical GCS niches. Overall, these data highlight the value of preclinical validation in more clinically relevant models to identify the best possible novel therapeutic approaches to pursue further towards clinical delivery.

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.

Delivering therapies to deeply seated brain tumours (BT) is a major clinical challenge.Magnetic drug targeting (MDT) could overcome this by rapidly transporting magnetised drugs directly into BT.We have developed a magnetic device for application in murine BT models using an array of neodymium magnets with a combined strength of 0.7T.In a closed fluidic system, the magnetic device trapped magnetic nanoparticles (MNP) up to distances of 0.8cm.In mice, the magnetic device guided intravenously administered MNP (<50nm) from the circulation into the brain where they localised within mouse BT.Furthermore, MDT of magnetised Temozolomide (TMZ mag+ ) significantly reduced tumour growth and extended mouse survival to 48 days compared to the other treatment groups.Using the same principles, we built a proof of principle scalable magnetic device for human use with a strength of 1.1T.This magnetic device demonstrated trapping of MNP undergoing flow at distances up to 5cm.MDT using our magnetic device provides an opportunity for targeted delivery of magnetised drugs to human BT.

Background High-grade gliomas are primary brain cancers with unacceptably low and persistent survival rates of 10–16 months for WHO grade 4 gliomas over the last 40 years, despite surgical resection and DNA-damaging chemo-radiotherapy. More recently, tumour-treating fields therapy (TTFields) has demonstrated modest survival benefit and been clinically approved in several countries. TTFields is thought to mediate anti-cancer activity by primarily disrupting mitosis. However, recent data suggest that TTFields may also attenuate DNA damage repair and replication fork dynamics, providing a potential platform for therapeutic combinations incorporating standard-of-care treatments and targeted DNA damage response inhibitors (DDRi). Methods We have used patient-derived, typically resistant, glioma stem-like cells (GSCs) in combination with the previously validated preclinical Inovitro™ TTFields system together with a number of therapeutic DDRi. Results We show that TTFields robustly activates PARP- and ATR-mediated DNA repair (including PARylation and CHK1 phosphorylation, respectively), whilst combining TTFields with PARP1 or ATR inhibitor treatment leads to significantly reduced clonogenic survival. The potency of each of these strategies is further enhanced by radiation treatment, leading to increased amounts of DNA damage with profound delay in DNA damage resolution. Conclusion To our knowledge, our findings represent the first report of TTFields applied with clinically approved or in-trial DDRi in GSC models and provides a basis for translational studies toward multimodal DDRi/TTFields-based therapeutic strategies for patients with these currently incurable tumours.

Abstract AIMS Glioblastoma is the most common cancer arising within the brain accounting for >2,000 deaths/year in the UK. Current standard-of-care therapy consists of maximal safe surgical resection followed by radiochemotherapy which activates the cellular DNA damage response (DDR). However, disease prognosis remains poor (median OS ~12-15 months). This stagnation in the development and clinical translation of novel therapeutics is largely attributed to extensive inter- and intra-tumoral heterogeneity and resistant glioma stem cell (GSC) niches. Traditionally, primary ex vivo and in vivo models of glioblastoma have been generated from the resected tumour mass potentially omitting the post-surgical residual tumour cells that give rise to disease recurrence and morbidity. METHOD Multiple parallel primary ex vivo GSC cell models were generated from anatomically discrete regions of en-bloc partial lobectomy glioblastoma specimens to produce 3D models of resected (tumour “core”) and typically residual (distal invasive tumour “edge”) GSC niches. These models were characterised using an array of biomolecular techniques and 3D based assays. RESULTS Preliminary RNA-seq analyses revealed largely divergent transcriptional landscapes for resected vs residual GSCs and protein expression/immunofluorescence analyses also identified key differences in GSC and DDR ex- pression, and subsequent cellular responses to current standard-of-care radio-chemotherapy regimens. CONCLUSIONS These data reveal phenotypic relationships between GSC “stemness” and DDR activity, therefore highlighting the potential clinical relevance of our novel parallel resected and residual ex vivo glioblastoma models. Characterisation of further models from within our 3D GSC biobank will also aid evaluation of novel therapeutics capable of targeting post-surgical GSC subpopulations leading to improved disease outcomes.

Abstract AIMS High grade gliomas are the most common CNS malignancies and cause ~200,000 deaths/year globally. Glioblastoma (WHO grade IV) confer the worst prognoses with their treatment being hindered by glioma stem cell-like (GSC) tumour sub-regions and high intratumoural spatio-heterogeneity. Tumour Treating Fields (TTFields) exert clinically approved alternating electric fields of intermediate frequency which induces anti-mitotic effects within concomitant deleterious effects on cellular responses to DNA damage (DDR). Therefore, treatment resistance could be overcome with TTFields with clinically developed DDR inhibitors (DDRi) to cause chemo-/radio- sensitisation. METHOD Surgically resected samples derived from the tumour core and invasive edge were processed to create GSC cell lines, offering highly clinically relevant models. These were grown in either 2D or in 3D-printed AlvetexTM scaffolds, treated with DDRi, subjected to chemo-/radio-therapy and then placed under TTFields (200 kHz, 48- 72 hrs). Survival was measured by clonogenic assays after 3 weeks. Immunofluorescence and western blot analysis were used to analyse the combination treatment effects on the DDR. RESULTS TTFields with DDRi led to significant chemo/radio-sensitisation in both 2D and 3D intratumoral core/edge GSC models. Western blot and immunofluorescence analyses revealed that DNA damage was heightened by the combination treatments with lesion resolution being impaired. CONCLUSIONS DDRi with concurrent TTFields effectively leads to significant chemo-/radio-sensitisation in clinically relevant 3D GSCs which model spatio-heterogeneity which is thought to impede treatment effcacy. Given that all the DDRi used are BBB penetrable and are either clinically approved or in clinical trials, TTFields could additionally compliment these treatment regimens in gliomas to boost their effcacy.

Abstract AIMS With diminishing returns and common clinical failure rates from traditional preclinical and animal-based drug discovery strategies, emphasis is being placed on alternative drug discovery platforms, such as ex vivo approaches. Such approaches represent a departure from both preclinical animal-based models and more traditional clinical-based strategies and aim to address intra/inter-patient variability at an earlier stage of drug discovery. Additionally, they could also offer precise treatment stratification for patients within a week of surgery to direct a tailored treatment course. 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, and where historic preclinical models have failed to generate new therapies leading to 50-year stagnant dire survival rates of around 15 months post-diagnosis. METHOD We have developed and optimized a high-throughput ex vivo drug screening platform; GliExP, which maintains GSC populations using immediately dissociated fresh surgical tissue. RESULTS As a proof-of-concept for GliExP, we have optimized standard-of-care chemoradiotherapy responses which can correctly predict MGMT status based on Temozolomide sensitivity. We have screened 30+ small molecule therapeutics and preclinical compounds against 18 different patient’s tumours, including multi-region spatial het- erogeneity sampling from several individual tumours. Drug responses are further correlated to transcriptomic and genetic profiles. CONCLUSIONS Our data provides a strong basis to build upon GliExP to incorporate combination-based oncology therapeutics in tandem with standard-of-care therapies as an important preclinical model to triage experimental therapeutics for clinical translation, delivering rapid identification of effective treatment strategies for individual gliomas.