Biomedical research techniques that don’t involve the use of animals are gaining momentum, but those using innovative approaches still face resistance from some quarters. Biomedical research techniques that don’t involve the use of animals are gaining momentum, but those using innovative approaches still face resistance from some quarters.

Abstract Background Glioblastoma multiforme (GBM) is the most prevalent primary brain tumour, with an incidence of 2 per 100,000. The standard clinical treatments do not sufficiently target cell migration and invasion, leading to recurrence after surgical resection and resistance after chemotherapy and radiotherapy. Pre-clinical studies are being conducted to construct artificial substrates that can mimic the tumour microenvironment (TME) to prevent GBM cells from migrating along their primary route through blood vessels and white matter tracts. Alongside, targeted therapies using anti-migratory or ‘migrastatic’ drugs are also being developed. This study aimed to review the therapeutic translational strategies emerging from the study of the GBM microenvironment and anti-migratory drugs. Methods A systematic literature search was carried out using search key terms and synonyms. Full-paper screening was performed based on specific inclusion and exclusion criteria. Results From the systems interrogated, the ‘Nanofibre’ assay is suitable to simulate white matter tracts, while hydrogel-based invasion assays and GBM cerebral organoid (GLICO) mimic the brain extracellular matrix. Inhibitors with anti-migratory activity found in this study are active involving distinct molecular mechanisms and have been tested on cell migration assays. Conclusion Overall, we have analysed therapeutic strategies emerging from an artificial GBM TME approach and from the identification of anti-migratory inhibitors. Both carry potential to improve treatment options to prevent tumour dissemination and spread for GBM.

Temozolomide (TMZ) remains the sole cytotoxic chemotherapeutic option in the management of Glioblastoma (GBM), with tumours eventually developing resistance. We recently reported on members of the ARHGAPs, RhoGTPase-activating proteins, and a role in cell plasticity as potential contributors to therapeutic resistance in GBM with clinical relevance for tumour recurrence (Cheng et al, Cell Reports, 2025). We also demonstrated anti-migratory activity of plant-derived compounds in another study leading to disruption of the GBM cytoskeletal architecture (Thompson et al, JoX, 2024). Here, we explored ARHGAP signaling as a vulnerability in GBM to examine how its modulation could enhance TMZ efficacy, while also highlighting the potential for synergy with plant-derived agents. ARHGAP activity and effect on TMZ potentiation with or without the inclusion of a panel of plant-derived compounds was profiled in previously generated stable GBM knockdown cells targeting ARHGAP 12 and 29. The panel of selected plant-derived compounds included curcumin, senna, black cumin, sulforaphane, hesperidin, EGCG, quercetin and tryptanthrin with selective anti-migratory activity. Established 2D and 3D in vitro methodology allowed investigations of effects on proliferation, migration and subcellular cytoskeletal arrangements. ARHGAP activity correlated with TMZ resistance. Knockdown of ARHGAP 12 or 29 sensitized resistant cells to TMZ, leading to a 10-fold increase in sensitivity. Co-treatment with TMZ and plant-derived compounds in particular curcumin enhanced TMZ activity with distinct cytoskeletal effects observed in 3D spheroids in ARHGAP 12 kd spheroids. ARHGAP inhibition in combination with curcumin disrupted actin dynamics supporting a role for the ARHGAPs in resistance pathways. ARHGAP signaling is a functional vulnerability in GBM that contributes to TMZ resistance. Its inhibition potentiates TMZ efficacy and may synergize with selective plant-derived therapeutics. These findings support the rationale for novel combination strategies integrating selective targeting of the cytoskeleton with RhoGTPase modulators and selective inhibitors to overcome GBM therapy resistance.

Glioblastoma (GMB) treatment remains a substantial unmet need due to its aggressive and highly infiltrative characteristics, facilitated by microstructures within brain tissue such as capillaries and neuronal projections. These key features are seldom represented in pre-clinical models that attempt to evaluate underpinning migratory pathways, leaving drug screening efforts and clinical translation futile. Here, our innovative approach towards modelling biorelevant GBM microenvironments incorporates migratory tracts that are synonymous with migratory pathways observed in GBM patients. This study utilises glioma cell lines U87 and U251 to identify preferential migration strategies in response to physiochemical and mechanical gradients that are fashioned using extracellular matrix-like components. GBM spheroids were printed into collagen conduits of varying density, all encased in a non-cell adhesive hydrogel with stiffness akin to brain matter. Using our newly developed and physiologically advanced bioprinted system and following from our group’s previous work on investigating the role of RhoGTPase-activating proteins (ARHGAPs) as potential contributors to GBM therapeutic resistance (Cheng et al, Cell Reports, 2025) we then introduced stable knockdowns of ARHGAP 12 and 29 to elucidate whether a synergistic effect on migration could be initiated with a panel of antimigratory drugs (CCG-1423, rhosin or combination). Using confocal and light-sheet microscopy, MTT viability assays and physical cytometry, distinct differences were observed in phenotype, adhesion, migration and actin polymerisation between models, with models containing ARHGAP 29 knockdowns and drug cocktail combinations displaying significantly reduced migratory activity, increased adhesion within spheroids, reduced F-actin signalling and reduced cell viability. When compared with classical 2D migration models, striking differences were seen in migratory behaviours, supporting the need for more biorelevant models in predicting in vivo responses. From this, we propose that these sophisticated systems are more capable of tailoring novel, personalised treatments for GBM to subsequently improve patient prognosis.

Human babesiosis is a disease transmitted by the bite of an infected tick or via blood transfusions involving contaminated blood products; in humans, it can lead to severe complications and even death, depending on the clinical history, age and health status of the affected patient. Babesiosis is caused by members of the Babesia spp., protozoan parasites whose life cycle includes sexual reproduction in the arthropod vector and asexual reproduction in the mainly mammalian host. Cases of human babesiosis have been rare, but there are increasing reports of human babesiosis associated with climatic changes affecting the geographical distribution of the parasite and tick vector, enhanced vector-human interactions and improved awareness of the disease in humans. Diagnostics and treatment options for humans are based around discoveries in veterinary research, such as point-of-care testing in cases of bovine babesiosis, and include direct diagnosis by blood smears, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) technologies, and indirect diagnosis by ELISA, immunofluorescence tests (IFAT) and fluorescent in situ hybridisation. Treatment involves a combination of drugs such as azithromycin and atovaquone, or clindamycin and quinine, but more effective options are being investigated, including, but not limited to, trans-chalcones and tafenoquine. Improved surveillance, awareness and diagnosis, as well as advanced technologies to interrupt vector-host interactions, are crucial in managing the increased threat posed by this once-neglected disease in humans.

This review explores the pivotal role of preanalytical variables in bringing liquid biopsy approaches into the clinic for brain tumors. Preanalytical variables encompass a range of critical issues, from blood sample collection and handling to the impact of tumor heterogeneity and patient-specific factors. These variables introduce challenges such as false positives, false negatives, and variability in the analysis of tumor signals, which can hinder the diagnostic and prognostic utility of liquid biopsies. Understanding the nuances of preanalytical variables is essential for the successful implementation of liquid biopsy in clinical settings. This paper delves into strategies aimed at mitigating the influence of preanalytical variables by emphasizing the importance of standardized sample collection protocols, optimized sample processing and storage, quality control measures, and the integration of multiple liquid biopsy modalities.

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.

Glioblastomas (GBMs) are aggressive and invasive cancers of the brain, associated with high rates of tumour recurrence and poor patient outcomes despite initial treatment. Targeting cell migration is therefore of interest in highly invasive cancers such as GBMs, to prevent tumour dissemination and regrowth. One current aim of GBM research focuses on assessing the anti-migratory properties of novel or repurposed inhibitors, including plant-based drugs which display anti-cancer properties. We investigated the potential anti-migratory activity of plant-based products with known cytotoxic effects in cancers, using a range of two-dimensional (2D) and three-dimensional (3D) migration and invasion assays as well as immunofluorescence microscopy to determine the specific anti-migratory and phenotypic effects of three plant-derived compounds, Turmeric, Indigo and Magnolia bark, on established glioma cell lines. Migrastatic activity was observed in all three drugs, with Turmeric exerting the most inhibitory effect on GBM cell migration into scratches and from the spheroid edge at all the timepoints investigated (p < 0.001). We also observed novel cytoskeletal phenotypes affecting actin and the focal adhesion dynamics. As our in vitro results determined that Turmeric, Indigo and Magnolia are promising migrastatic drugs, we suggest additional experimentation at the whole organism level to further validate these novel findings.

Abstract Despite efforts to improve outcomes, patients diagnosed with glioblastoma (GBM) have a median survival of only 12-15 months. Tumors are characterized by rapid growth and invasion. We previously have shown that the broadly selective kinase inhibitor (2'Z,3'E)-6-Bromoindirubin-3'-oxime (BIO) has potent anti-migratory properties in GBM cells. To investigate the mechanisms involved we performed transcriptomic analysis which showed a wide range of alterations in GBM cell lines associated with BIO treatment. This included multiple members of the RhoGTPase activating protein (ARHGAP) family. ARHGAPs are a family of proteins that have been associated with various diseases including cancers but have not been studied in GBM. Using 2D and 3D migration/invasion assays with established and patient derived cell lines as well as in vivo models and human GBM tissue, the role of ARHGAPs 4, 22 and 25 in cell migration and dissemination was investigated. Migration studies after gene knockdown in the patient derived cell line G9 revealed that the three ARHGAPs had pro-or-anti-migratory activity. Preliminary in vivo studies in mice indicated that silencing ARHGAP25 in GBM cells enhances invasiveness. ARHGAP25 negatively regulates RhoA, a key player in cell migration. By decreasing ARHGAP25 activity, RhoA becomes more active, leading to actin polymerization, stress fiber formation, and enhanced invasive behavior in G9-ARHGAP25 knockdown cells. These cells exhibit a rounder, amoeboid phenotype, with reduced protrusions, enabling them to navigate tight spaces and invade surrounding tissue more easily. Conversely, G9 and G9-ARHGAP 22 knockdown cells display a protrusive, elongated phenotype, suggesting a slower form of cell migration, influenced by Rac1 signaling. Knocking down ARHGAP22 promotes RhoA-driven migration, while Rac1 promotes mesenchymal migration through membrane protrusions like lamellipodia. Clinical relevance of the ARHGAPs was determined by an association of ARHGAP expression and survival after treatment in a GBM tissue microarray (TMA) donated by Professor Michel Mittelbronn, which revealed a significant association between high ARHGAP22 expression and longer disease-free survival. Distinct protein localizations were observed for each ARHGAP. ARHGAP22 showed a potential correlation with necrotic regions and blood vessels in GBM tumors. The study's findings enhance our understanding of GBM cell migration, providing potential targets for therapeutic intervention. Overall, the discoveries made represent an important step forward in the identification of new targets for this devastating disease. Citation Format: Philippa C. Vaughn-Beaucaire, Sean Lawler, Anke Bruning-Richardson, William Hawkins, Jorge Jimenez-Macias, Praveen Srinivasan. Stopping brain tumors in their tracks: the role of RhoGTPase activating proteins (ARHGAPs) in glioma cell migration and invasion [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A011.

Polysaccharides such as sodium alginate, pectin and gellan gum are widely used biomaterials, for their ability to easily form hydrogels in the presence of divalent metal ions, such as calcium - a process often cited as a mild crosslinking mechanism. However, when using these materials as substrates for tissue engineering, there is a lack of extensive studies that investigate the impact of elevated calcium concentrations on cell health and behaviour. In this study, we performed an in-depth exploration to understand the potential effects of raising extracellular CaCl2 on cell viability, proliferation, morphology and migration. We used an established glioblastoma (GBM) cell line (U251), human dermal fibroblasts (HDF), and murine osteoblasts (MC3T3) to assess the consequences of using CaCl2 in tissue engineered models to help reevaluate biomaterial suitability and enhance standardisation practices in the field of tissue engineering. Our findings revealed that the addition of CaCl2 induced notable morphological changes in GBM cells when cultured in 3D hydrogels with excess CaCl2 added, leading to a transition from mesenchymal to amoeboid phenotypes, even at a concentration as low as 8 mM. Furthermore, cell viability was reduced in a concentration-dependent manner across all cell types, and migration was also affected. Despite the widespread use of high CaCl2 concentrations to facilitate scaffold gelation, our research unveils that there can be significant risks to cell viability, proliferation, morphology, and migration when such practices are not preceded by cell line-specific experimentation and thorough standardization procedures. This highlights the importance of careful consideration and optimisation of CaCl2 concentration when used as a crosslinking agent for hydrogels intended for use in tissue engineering applications that demand accurate recapitulation of cellular responses and physiological conditions.

Background Glioblastoma (GBM) brain tumors lacking IDH1 mutations (IDHwt) have the worst prognosis of all brain neoplasms. Patients receive surgery and chemoradiotherapy but tumors almost always fatally recur. Results Using RNA sequencing data from 107 pairs of pre- and post-standard treatment locally recurrent IDHwt GBM tumors, we identify two responder subtypes based on longitudinal changes in gene expression. In two thirds of patients, a specific subset of genes is upregulated from primary to recurrence (Up responders), and in one third, the same genes are downregulated (Down responders), specifically in neoplastic cells. Characterization of the responder subtypes indicates subtype-specific adaptive treatment resistance mechanisms that are associated with distinct changes in the tumor microenvironment. In Up responders, recurrent tumors are enriched in quiescent proneural GBM stem cells and differentiated neoplastic cells, with increased interaction with the surrounding normal brain and neurotransmitter signaling, whereas Down responders commonly undergo mesenchymal transition. ChIP-sequencing data from longitudinal GBM tumors suggests that the observed transcriptional reprogramming could be driven by Polycomb-based chromatin remodeling rather than DNA methylation. Conclusions We show that the responder subtype is cancer-cell intrinsic, recapitulated in in vitro GBM cell models, and influenced by the presence of the tumor microenvironment. Stratifying GBM tumors by responder subtype may lead to more effective treatment.

Abstract AIMS The kynurenine (Kyn) pathway plays an important role in the pathogenesis of many cancers including glioblastomas (GBMs). The enzymes, indoleamine-2,3-dioxygenase (IDO1) and tryptophan-2,3-dioxygenase (TDO2), regulate the first and rate-limiting step resulting in the formation of N-formyl-kynurenine, which is then readily converted into Kyn by kynurenine formaldehyde. Cancers expressing the IDO1 and TDO2 enzymes can produce immunosuppressive tryptophan metabolites that compromise the host immune system leading to poor patient prognosis. The inhibitors TD12, TD18 and TD34 were recently developed due to the lack of TDO2 and IDO1/TDO2 inhibitors, of which TD34 can block both IDO1 and TDO2 activity. Here, we investigated the ability of these novel inhibitors to also target cell migration in GBM as part of our ongoing research interest in this area. METHOD Two established brain tumour cell lines, U87 and U251 and one patient-derived cell line, E55, were used to determine IC50 values and anti-migratory concentrations by MTT for application in 2D and 3D migration/invasion assays. RESULTS Cell migration in all cell lines was significantly reduced in the presence of the TD12, TD18 and TD34 inhibitors. E55 appeared to be 100-fold more sensitive to drug activity; immunofluorescence analysis indicates changes in the actin cytoskeleton and cell adhesion CONCLUSIONS Initial results suggest that TDO2 and the IDO1 enzymes play also a role in cell migration in GBM and provide further evidence for the involvement of the Kyn pathway in the pathology of GBMs. This also supports the application of inhibitors targeting TDO2 and IDO1 as novel complementary treatment options in GBM.

Abstract AIMS One of the hallmarks of cancer is cell migration and invasion, a striking feature of glioblastoma (GBM). The highly migratory potential of tumour cells enables recurrence of GBM tumours leading to poor survival and patient death. We recently characterised a panel of migrastatic inhibitors and reported on the ability of glioma cells to overcome drug activity by employing a mesenchymal to amoeboid migratory switch. Here we demonstrate that targeting different signalling pathways involved in cell migration induces synergistic drug activity of single inhibitors. METHOD The previously described inhibitors CCG-1423 and Rhosin Hydrochloride were used singly or in combination to target cell migration in established glioma cell lines. Characterisation of drug activity was achieved by 2D and 3D migration and invasion assays and cytoskeletal and morphological changes were assessed by immunofluorescence assays. RESULTS When used in single applications, the inhibitors did not have a significant effect on cell migration in 2D and 3D. However, combination treatments led to significantly reduced migration in both 2D and 3D with notable effects on the cytoskeleton as evidenced by changes in focal adhesion dynamics and in actin localisation. Cellular features also changed in response to combination treatment as noted by increase in cell size and adhesion to neighbouring cells. CONCLUSIONS The observed effects are in keeping with targeting two distinct signalling pathways driving cells to either adopt amoeboid or mesenchymal cell migration/invasion mechanisms. We propose that a ‘cocktail’ of migrastatic inhibitors should be included in future experimental designs for consideration as novel, complementary treatment strategies in GBM.

Spheroids and organoids are increasingly popular three-dimensional (3D) cell culture models. Spheroid models are more physiologically relevant to a tumor compared to two-dimensional (2D) cultures and organoids are a simplified version of an organ with similar composition. Spheroids are often only formed from a single cell type which does not represent the situation in vivo. However, despite this, both spheroids and organoids can be used in cell migration studies, disease modelling and drug discovery. A drawback of these models is, however, the lack of appropriate analytical tools for high throughput imaging and analysis over a time course. To address this, we have developed an R Shiny app called SpheroidAnalyseR: a simple, fast, effective open-source app that allows the analysis of spheroid or organoid size data generated in a 96-well format. SpheroidAnalyseR processes and analyzes datasets of image measurements that can be obtained via a bespoke software, described herein, that automates spheroid imaging and quantification using the Nikon A1R Confocal Laser Scanning Microscope. However, templates are provided to enable users to input spheroid image measurements obtained by user-preferred methods. SpheroidAnalyseR facilitates outlier identification and removal followed by graphical visualization of spheroid measurements across multiple predefined parameters such as time, cell-type and treatment(s). Spheroid imaging and analysis can, thus, be reduced from hours to minutes, removing the requirement for substantial manual data manipulation in a spreadsheet application. The combination of spheroid generation in 96-well ultra-low attachment microplates, imaging using our bespoke software, and analysis using SpheroidAnalyseR toolkit allows high throughput, longitudinal quantification of 3D spheroid growth whilst minimizing user input and significantly improving the efficiency and reproducibility of data analysis. Our bespoke imaging software is available from https://github.com/GliomaGenomics. SpheroidAnalyseR is available at https://spheroidanalyser.leeds.ac.uk, and the source code found at https://github.com/GliomaGenomics.

Abstract AIMS Glioblastomas (GBMs) are characterised by highly hypoxic regions and the ability to invade into healthy brain tissue promoting tumour dissemination and recurrence; the development of novel anti-migratory inhibitors to target cell invasion and recurrence must consider varying treatment responses in this tumour background. As proof of principle we investigated the anti-migratory activity of two, previously characterised, small molecule inhibitors under normoxic and hypoxic conditions. We hypothesised that anti-migratory drug activity is dependent on hypoxia levels. METHOD Glioma cell line U251 was exposed to BIO-indirubin and Y-27632 alone or in combination and activity was assessed in 2D scratch assays under normoxic (21% O2) and hypoxic (0.1% O2) conditions. Live cell imaging was performed and analysis was carried out using ImageJ. RESULTS Treatment with BIO, Y-27632 or in combination had a statistically significant effect on the inhibition of migration in normoxia and hypoxia (p=0.01); BIO inhibited migration and Y-27632 promoted migration under normoxia, whereas BIO maintained the anti-migratory effect under hypoxia, the effect of Y-27632 was switched towards anti-migratory activity and combination treatment potentiated. The effect of Y-27632 was concomitant with a phenotypic shift in cells under hypoxia from highly elongated to possessing shorter extensions. CONCLUSION An anti-migratory effect of Y-27632 on cell migration was induced under hypoxic conditions suggesting that the adoption of amoeboid migration by the cells allowed targeting migratory pathways under the control of RhoA and ROCK. This confirms that candidate drug activity must be assessed under both conditions to be considered as drugs complementary to chemotherapy as potential novel treatment option.