High-grade Brain Cancer Research Articles

Patterning Networks of Grade IV Glioblastoma on Silicon Chip.

Glioblastoma (GBM) is the most aggressive high-grade brain cancer with a median survival time of <15 months. Due to GBMs fast and infiltrative growth patient prognosis is poor with recurrence after treatment common. Investigating GBMs ability to communicate, specifically via Ca2+ signaling, within its functional tumour networks may unlock new therapeutics to reduce the rapid infiltration and growth which currently makes treatment ineffective. This work aims to produce patterned networks of GBM cells such that the Ca2+ communication at a network level can be repeatedly and reliably investigated.

Evaluating nanoparticle localisation in glioblastoma multicellular tumour spheroids by surface enhanced Raman scattering.

Glioblastoma multiforme (GBM) is a particularly aggressive and high-grade brain cancer, with poor prognosis and life expectancy, in urgent need of novel therapies. These severe outcomes are compounded by the difficulty in distinguishing between cancerous and non-cancerous tissues using conventional imaging techniques. Metallic nanoparticles (NPs) are advantageous due to their diverse optical and physical properties, such as their targeting and imaging potential. In this work, the uptake, distribution, and location of silica coated gold nanoparticles (AuNP-SHINs) within multicellular tumour spheroids (MTS) derived from U87-MG glioblastoma cells was investigated by surface enhanced Raman scattering (SERS) optical mapping. MTS are three-dimensional in vitro tumour mimics that represent a tumour in vivo much more closely than that of a two-dimensional cell culture. By using AuNP-SHIN nanotags, it is possible to readily functionalise the inner gold surface with a Raman reporter, and the outer silica surface with an antibody for tumour specific targeting. The nanotags were designed to target the biomarker tenascin-C overexpressed in U87-MG glioblastoma cells. Immunochemistry indicated that tenascin-C was upregulated within the core of the MTS, however limitations such as NP size, quiescence, and hypoxia, restricted the penetration of the nanotags to the core and they remained in the outer proliferating cells of the spheroids. Previous examples of MTS studies using SERS demonstrated the incubation of NPs on a 2D monolayer of cells, with the subsequent formation of the MTS from these pre-incubated cells. Here, we focus on the localisation of the NPs after incubation into pre-formed MTS to establish a better understanding of targeting and NP uptake. Therefore, this work highlights the importance for the investigation and translation of NP uptake into these 3D in vitro models.

MODL-22. Establishment of a novel system to specifically trace and ablate quiescent/slow cycling cells in high-grade glioma

Abstract Pediatric and adult high-grade gliomas are the most common malignant brain tumors, with poor prognosis due to recurrence and tumor infiltration after surgical removal and chemotherapy. Quiescent/slow cycling stem cells have been proposed to be one of the main players of tumor relapse but their involvement in in the infiltration remain unclear. Despite they have been described in mouse models or after transcriptional profiling of human tumor samples, their direct visualization, targeting and ablation remains a challenge. Here, we identified a population of malignant cells expressing Prominin-1 in a non-proliferating state in pediatric high-grade glioma patients. We next used a fluorescent cell cycle sensor to visualize quiescence tumor cells in mouse brain cancer and human cancer organoids. In particular, we characterized them within the tumor revealing the invasiveness capacity of slow cycling tumor cells. Furthermore, we generated a new system to specifically trace and ablate such cells. Indeed, lineage tracing experiments allowed to trace quiescent Prom1 progeny in the tumors after temozolomide treatment. In addition, the selective ablation of quiescent Prom1+ cells in mouse brain cancer reduced tumor infiltration and improved survival. Furthermore, time-lapse experiments showed that slow cycling cells are also able to infiltrate co-cultured human brain cancer organoids. Finally, using our new cancer organoid- and GBM spheroids-based models we identified a drug acting on quiescent cells leading to a reduction in cell invasion. Overall, our data show that quiescence/slow cycling cells are key driver of tumor invasiveness, the major malignant feature of high-grade brain cancer.

Determining KLF14 tertiary structure and diagnostic significance in brain cancer progression

Expression analysis of new protein targets may play a crucial role in the early detection and diagnosis of brain tumor progression. The study aimed to investigate the possible relation of KLF14, TPD52, miR-124, and PKCε in the development and progression of brain cancer and space occupying lesion (SOL) of the brain. One hundred human blood samples comprising varying diagnostic groups (SOL brain, grade I, II, III, IV) were analyzed by real-time quantitative PCR to determine the expression level of KLF14, TPD52, miR-124, and PKCε. TPD52 and PKCε were upregulated in brain cancer by 2.5- and 1.6-fold, respectively, whereas, KLF14 and miR-124 were downregulated in brain cancer. In metastatic and high-grade brain cancer, TPD52 and PKCε expression were up-regulated and KLF14 and miR-124 expression were down-regulated. Further, these genes were found to be differentially expressed in the blood of patients with SOL. Upregulation of TPD52 and PKCε, however, reduced expression of KLF14 and miR-124 in SOL of the brain as compared to healthy controls. Expression analysis of TPD52, KLF14, miR-124, and PKCε provided useful information on the differences existing between the normal brain and SOL, in addition to gliomas; thus, might prove to be useful having diagnostic or prognostic value.

Urgent elective pathway service reconfiguration facilitates increased use of surgical adjuncts, improvement in survival trends, and reduced hospital stay for glioblastoma patients

Abstract Aims Glioblastomas (GB) are the most common and aggressive of intrinsic brain tumours. Median survival with maximal therapy is reported to be 14.6 months. Service reconfiguration at the Queen Elizabeth Hospital Birmingham (QEHB) has transformed the service for high grade brain cancer patients, including GB, from a predominantly emergency pathway based system to one of planned urgent-elective admissions consisting of: A. Patient-focused, consultant-led, research orientated “one stop shop” model of integrated outpatient neurosurgical oncology clinic B. Standardisation of urgent elective pathways C. Incorporation of neuro-surgical intra-operative adjuncts (neuro-monitoring, 5-ALA) into routine surgical practice for oncology. Using this model, we have reduced hospital length of stay (with associated financial savings), improved extent of resection and achieved a trend towards increased survival. Method We retrospectively identified patients with primary histological diagnoses of GB (WHO grade IV), who underwent surgery over a six year period, from 01/01/2014 to 31/12/2019, from the QEHB pathology database. Data was collected for demographics, surgical and oncological therapy, use of intra-operative adjuncts, emergency and elective admission status, year of admission, length of stay (LOS), and extent of resection (EOR) on first post-operative MRI scan from hospital databases. Survival was analysed using the Kaplan-Meier method and independent-samples median testing for survival. Proportion of patients undergoing resective surgery and admission status was calculated by year. Overall median survival was calculated and subgroup comparisons made of patients by: age, admission status, year of admission, biopsy or resection, oncology treatment. Hospital length of stay was calculated for patients by surgical procedure, admission pathways and compared across the year. Financial data taken from averages of inpatient episode costs were used to estimate cost savings. Results 610 patients underwent primary procedures for GB, of which 64 were still alive at time of analysis (02/02/2021). Median overall survival time was 9.53 months, this was greatest in patients who underwent resection with completion of Stupp protocol: 28.67 months (n=114). From 2014 to 2019, there has been an increase in elective admission rates (28.1% to 90.3%, p&amp;lt;0.001) and increased proportion of resective surgery (68.4% to 81.9%, p&amp;lt;0.001). There is a trend of improved survival from 2014 to 2019 (median 7.95 and 11.08 months, χ2=9.249, p=0.002). Increasing use of intra-operative adjuvants improved EOR (χ2 =31.064, p&amp;lt;0.001). Through improved urgent-elective admission rates, hospital length of stay has decreased by five days for craniotomies and six days for biopsies. Cost analysis of three cases demonstrated that reducing the LOS by one night alone result in an average cost saving of approximately £750 per patient per night. Conclusion Switching to a system of planned and urgent elective based admission, with standardisation of neuro-oncology patient pathways, increased use of intra-operative adjuncts, earlier oncology multidisciplinary input and outpatient review, has improved the extent of GB resection, led to shorter length of hospital stay associated with significant financial savings and achieved a trend towards increased overall survival.

OS13.3.A Establishment of a novel system to specifically trace and ablate quiescent/slow cycling cells in high-grade glioma

Abstract BACKGROUND High-grade gliomas are the most common malignant brain tumors, with poor prognosis due to recurrence and tumor infiltration after surgical removal and chemotherapy. Quiescent/slow cycling stem cells have been proposed to be one of the main players of tumor relapse but their involvement in in the infiltration remain unclear. Despite they have been described in mouse models or after transcriptional profiling of human tumor samples, their direct visualization, targeting and ablation remains a challenge. MATERIAL AND METHODS Tumors were induced over-expressing oncogenic forms of MET and p53 in the subventricular zone (SVZ) of P2 mouse brain as well as human forebrain organoids. The co-expression with specific cell cycle sensors as well as lineage specific CreERT2 under control of stem cells promoters allowed to visualize and target glioma stem cells. RESULTS Here, we used a fluorescent cell cycle sensor to visualize quiescent tumor cells in mouse brain cancer and human cancer organoids. In particular, we characterized them within the tumor revealing the invasiveness capacity of slow cycling tumor cells. Furthermore, we generated a new system to specifically trace and ablate such cells. Indeed, lineage tracing experiments allowed to trace quiescent Prom1 progeny in the tumors after temozolomide treatment. In addition, the selective ablation of qProm1 in mouse brain cancer reduced tumor infiltration. Finally, time-lapse experiments showed that slow cycling cells are also able to infiltrate co-cultured human brain cancer organoids. CONCLUSION Overall, our data show that quiescent/slow cycling cells are key driver of tumor invasiveness, the major malignant feature of high-grade brain cancer.

Targeting Orphan G Protein-Coupled Receptor 17 with T0 Ligand Impairs Glioblastoma Growth.

Simple SummaryGlioblastoma multiforme (GBM), or glioblastoma chemotherapy, has one of the poorest improvements across all types of cancers. Despite the different rationales explored in targeted therapy for taming the GBM aggressiveness, its phenotypic plasticity, drug toxicity, and adaptive resistance mechanisms pose many challenges in finding an effective cure. Our manuscript reports the expression and prognostic role of orphan receptor GPR17 in glioma, the molecular mechanism of action of the novel ligand of GPR17, and provides evidence how the T0 agonist promotes glioblastoma cell death through modulation of the MAPK/ERK, PI3K–Akt, STAT, and NF-κB pathways. The highlights are as follows: GPR17 expression is associated with greater survival for both low-grade glioma (LGG) and GBM; GA-T0, a potent GPR17 receptor agonist, causes significant GBM cell death and apoptosis; GPR17 signaling promotes cell cycle arrest at the G1 phase in GBM cells; key genes are modulated in the signaling pathways that inhibit GBM cell proliferation; and GA-T0 crosses the blood–brain barrier and reduces tumor volume.Glioblastoma, an invasive high-grade brain cancer, exhibits numerous treatment challenges. Amongst the current therapies, targeting functional receptors and active signaling pathways were found to be a potential approach for treating GBM. We exploited the role of endogenous expression of GPR17, a G protein-coupled receptor (GPCR), with agonist GA-T0 in the survival and treatment of GBM. RNA sequencing was performed to understand the association of GPR17 expression with LGG and GBM. RT-PCR and immunoblotting were performed to confirm the endogenous expression of GPR17 mRNA and its encoded protein. Biological functions of GPR17 in the GBM cells was assessed by in vitro analysis. HPLC and histopathology in wild mice and an acute-toxicity analysis in a patient-derived xenograft model were performed to understand the clinical implication of GA-T0 targeting GPR17. We observed the upregulation of GPR17 in association with improved survival of LGG and GBM, confirming it as a predictive biomarker. GA-T0-stimulated GPR17 leads to the inhibition of cyclic AMP and calcium flux. GPR17 signaling activation enhances cytotoxicity against GBM cells and, in patient tissue-derived mesenchymal subtype GBM cells, induces apoptosis and prevents proliferation by stoppage of the cell cycle at the G1 phase. Modulation of the key genes involved in DNA damage, cell cycle arrest, and in several signaling pathways, including MAPK/ERK, PI3K–Akt, STAT, and NF-κB, prevents tumor regression. In vivo activation of GPR17 by GA-T0 reduces the tumor volume, uncovering the potential of GA-T0–GPR17 as a targeted therapy for GBM treatment. Conclusion: Our analysis suggests that GA-T0 targeting the GPR17 receptor presents a novel therapy for treating glioblastoma.

A Flux-Limited Model for Glioma Patterning with Hypoxia-Induced Angiogenesis

We propose a model for glioma patterns in a microlocal tumor environment under the influence of acidity, angiogenesis, and tissue anisotropy. The bottom-up model deduction eventually leads to a system of reaction–diffusion–taxis equations for glioma and endothelial cell population densities, of which the former infers flux limitation both in the self-diffusion and taxis terms. The model extends a recently introduced (Kumar, Li and Surulescu, 2020) description of glioma pseudopalisade formation with the aim of studying the effect of hypoxia-induced tumor vascularization on the establishment and maintenance of these histological patterns which are typical for high-grade brain cancer. Numerical simulations of the population level dynamics are performed to investigate several model scenarios containing this and further effects.

A Phase 2 Randomised, Double Blind Clinical Trial Assessing the Tolerability of Two Different Ratios of Medicinal Cannabis in Patients with Glioblastoma Multiforme (GBM)

Background: Glioblastoma (GBM) remains incurable. Patients seek complementary treatments when standard treatments are ineffective, or when cancer symptoms are uncontrolled. Cannabis has intense interest as both therapy and palliation for GBM, but little prospective controlled data exists. We studied the safety and tolerability of two different quality-assured medicinal cannabis preparations in patients with GBM, taking standard treatments. Patients and Methods: Patients with recurrent high-grade glioma (anaplastic astrocytoma or GBM) with no history of substance abuse enrolled in a single-centre phase II double-blind randomized clinical trial comparing two preparations of medicinal cannabis (tetrahydrocannabinol (THC) to cannabidiol (CBD), THC 1:1 CBD and THC 4:1 CBD) taken once daily at night. Retrospective historically matched controls were audited from clinical records as comparators. Results: Patient self-referral for screening was overwhelming (n=921). Both preparations were well tolerated. Side effects included dry mouth, tiredness at night, dizziness, drowsiness, and impaired motor coordination, but no serious adverse events were attributable to the intervention. Sleep was reported as improved from baseline in both groups (95% CI 2.4-2.77; p=0.009). Overall quality of life (FACT-Br) was improved from baseline in both groups with the 1:1 preparation favouring both physical (95% CI 6.49-7.92; p=0.025) and functional (95% CI 15-16.9; p=0.014) capacity. Disease control appeared no worse than historical controls; only 10% of patients experienced progressive disease during the study period, and median overall survival was 11 months. Conclusion: A single nightly dose of medicinal cannabis was safe, well tolerated and appeared to improve sleep, functional wellbeing and quality of life (QOL) compared to baseline, with no negative impact on disease control. The 1:1 preparation is favoured for future studies in people with high-grade brain cancer. Trial Registration: The trial protocol was registered with the Australian New Zealand Clinical Trial Registry (ANZCTR): ACTRN12617001287325. Funding Statement: The funding body, Bioceuticals Pty Ltd, had no involvement with the protocol development, implementation of the trial, collection nor analysis of the trial. The trial was conducted by an independent research institution, Endeavour College of Natural Health which was the initial sponsor for the trial which has now been transferred to Southern Cross University (SCU) due to the lead researcher transferring universities. The funding body paid fees directly to Endeavour and SCU for the trial and supplied the investigational products for the trial in kind Declaration of Interests: All investigators declare no conflict of interest with this study. Prof Charlie Teo and Dr Mike Sughrue are shareholders and founders of Omniscient neurotechnologies for brain tumours but does not conflict with this study. Ethics Approval Statement: The trial was approved by human research ethical committees (HREC), South Eastern Sydney Local Health District HREC: 18/028, University of Technology Sydney HREC: ETH 18-2761 and Endeavour College of Natural Medicine HREC: 20180821.

Vascular targeting of LIGHT normalizes blood vessels in primary brain cancer and induces intratumoural high endothelial venules.

High-grade brain cancer such as glioblastoma (GBM) remains an incurable disease. A common feature of GBM is the angiogenic vasculature, which can be targeted with selected peptides for payload delivery. We assessed the ability of micelle-tagged, vascular homing peptides RGR, CGKRK and NGR to specifically bind to blood vessels in syngeneic orthotopic GBM models. By using the peptide CGKRK to deliver the tumour necrosis factor (TNF) superfamily member LIGHT (also known as TNF superfamily member 14; TNFSF14) to angiogenic tumour vessels, we have generated a reagent that normalizes the brain cancer vasculature by inducing pericyte contractility and re-establishing endothelial barrier integrity. LIGHT-mediated vascular remodelling also activates endothelia and induces intratumoural high endothelial venules (HEVs), which are specialized blood vessels for lymphocyte infiltration. Combining CGKRK-LIGHT with anti-vascular endothelial growth factor and checkpoint blockade amplified HEV frequency and T-cell accumulation in GBM, which is often sparsely infiltrated by immune effector cells, and reduced tumour burden. Furthermore, CGKRK and RGR peptides strongly bound to blood vessels in freshly resected human GBM, demonstrating shared peptide-binding activities in mouse and human primary brain tumour vessels. Thus, peptide-mediated LIGHT targeting is a highly translatable approach in primary brain cancer to reduce vascular leakiness and enhance immunotherapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Trajectories of Symptom Clusters, Performance Status, and Quality of Life During Concurrent Chemoradiotherapy in Patients With High-Grade Brain Cancers.

Symptom clusters must be identified in patients with high-grade brain cancers for effective symptom management during cancer-related therapy. The aims of this study were to identify symptom clusters in patients with high-grade brain cancers and to determine the relationship of each cluster with the performance status and quality of life (QOL) during concurrent chemoradiotherapy (CCRT). Symptoms were assessed using the Memorial Symptom Assessment Scale, and the performance status was evaluated using the Karnofsky Performance Scale. Quality of life was assessed using the Functional Assessment of Cancer Therapy-General. This prospective longitudinal survey was conducted before CCRT and at 2 to 3 weeks and 4 to 6 weeks after the initiation of CCRT. A total of 51 patients with newly diagnosed primary malignant brain cancer were included. Six symptom clusters were identified, and 2 symptom clusters were present at each time point (ie, "negative emotion" and "neurocognitive" clusters before CCRT, "negative emotion and decreased vitality" and "gastrointestinal and decreased sensory" clusters at 2-3 weeks, and "body image and decreased vitality" and "gastrointestinal" clusters at 4-6 weeks). The symptom clusters at each time point demonstrated a significant relationship with the performance status or QOL. Differences were observed in symptom clusters in patients with high-grade brain cancers during CCRT. In addition, the symptom clusters were correlated with the performance status and QOL of patients, and these effects could change during CCRT. The results of this study will provide suggestions for interventions to treat or prevent symptom clusters in patients with high-grade brain cancer during CCRT.

Diagnostic significance of arterial spin labeling in the assessment of tumor grade in brain.

The objective of the current meta.analysis was to assess the arterial spin labeling. (ASL) perfusion imaging measurement of cerebral blood flow. (CBF) in patients with brain tumors, and assessing preoperative tumor grade in brain. PubMed, Web of Science, Embase, China BioMedicine (CBM), CINAHL, Cochrane Library, and China National Knowledge Infrastructure (CNKI) databases were chosen to evaluate the associations between ASL and brain cancer. Two reviewers separately evaluated the quality of the included trials. Standardized mean difference (SMD) at 95% confidence interval (95% CI) was also calculated. Finally, 475 patients were enrolled into this meta-analysis from 12 eligible studies and were selected for statistical analysis. Results showed that relative tumor blood flow (rTBF) and relative cerebral blood flow (rCBF) in high-grade brain cancer patients were faster than those in low-grade brain cancer patients. Subgroup analysis stratified by country implied that ASL may be the main prediction of increased rTBF in high-grade brain cancer patients among USA, Korea and China; and rCBF was faster in high-grade brain cancer using ASL in USA and China. Further reference by tissue-stratified analysis revealed a positive association of rTBF with high-grade brain cancer by utilization of ASL in all the experimental subgroups, while rCBF was only correlated in white subgroups. These results showed that rTBF and rCBF were faster in high-grade brain cancer patients, suggesting that ASL may provide suitable measurement for the differential diagnosis of tumor grade in brain.

OP19 * SERUM SPECTROSCOPY OF BRIAN TUMOURS: A RAPID AND ACCURATE DIAGNOSTIC TOOL

INTRODUCTION: The ability to diagnose brain tumours rapidly from serum would allow for rapid testing and decreased time to results providing a responsive diagnostic environment. ATR-FTIR (Attenuated Total Reflection - Fourier Transform Infrared Spectroscopy) generates a serum sample - fingerprint - since biomolecules exhibit different responses to different wavelengths of light. We have previously used ATR-FTIR to discriminate high grade brain cancer, low-grade brain cancer and normal serum to sensitivities and specificities on average of 93.75 and 96.53 % respectively. This paper reports the analysis of 429 patients. METHOD: 1 µl of serum was analysed in triplicate from 429 patients via ATR-FTIR. This dataset was analysed using pattern recognition algorithms for the following models 1) Cancer (n = 314) vs Non-cancer (n = 122), 2) Metastatic Brain Cancer (n = 192) vs Brain Cancer (n = 124) vs Non-Cancer (n = 122), 3) Organ of tumour origin, 4)High grade glioma (n = 52) vs low grade glioma (n = 24) vs meningioma (n = 47) 5) Subtype of Brain Cancer. RESULTS: ATR-FTIR spectroscopy was able to discriminate different groupings with high accuracy. The predicted range for each diagnostic layer was between 78.26 - 100.00 % sensitivity and specificity respectively. Interrogation of the loadings plots showed differences in Amide and lipid content of the serum. CONCLUSION: A robust reproducible method has been developed for the diagnosis of small volumes of serum. In addition multiple layers of diagnostic information can be provided from the same spectral dataset. ATR-FTIR has shown to be an excellent clinical tool for rapid diagnosis enabling a dynamic and responsive clinical environment. Future work will investigate spectroscopic changes during treatment.

Hypoxia induced CA9 inhibitory targeting by two different sulfonamide derivatives including Acetazolamide in human Glioblastoma

HIF-1α regulated genes are mainly responsible for tumour resistance to radiation- and chemo-therapy. Among these genes, carbonic anhydrase isoform IX (CA9) is highly over expressed in many types of cancer especially in high grade brain cancer like Glioblastoma (GBM). Inhibition of the enzymatic activity by application of specific chemical CA9 inhibitor sulphonamides (CAI) like Acetazolamide (Aza.), the new sulfonamide derivative carbonic anhydrase inhibitor (SU.D2) or indirect inhibitors like the HIF-1α inhibitor Chetomin or molecular inhibitors like CA9-siRNA are leading to an inhibition of the functional role of CA9 during tumorigenesis. Human GBM cells were treated with in vitro hypoxia (1, 6, or 24h at 0.1%, O2). Aza. application was at a range between 250 and 8000nM and the HIF-1α inhibitor Chetomin at a concentration range of 150–500nM. Cell culture plates were incubated for 24h under hypoxia (0.1% O2). Further, CA9-siRNA constructs were transiently transfected into GBM cells exposed to extreme hypoxic aeration conditions. CA9 protein expression level was detectable in a cell-type specific manner under normoxic conditions. Whereas U87-MG exhibited a strong aerobic expression, U251 and U373 displayed moderate and GaMG very weak normoxic CA9 protein bands. Aza. as well as SU.D2 displayed inhibitory characteristics to hypoxia induced CA9 expression in the four GBM cell lines for 24h of hypoxia (0.1% O2) at concentrations between 3500 and 8000nM, on both the protein and mRNA level. Parallel experiments using CA9-siRNA confirmed these results. Application of 150–500nM of the glycolysis inhibitor Chetomin under similar oxygenation conditions led to a sharply reduced expression of both CA IX protein and CA9 mRNA levels, indicating a clear glucose availability involvement for the hypoxic HIF-1α and CA9 expression in GBM cells. Hypoxia significantly influences the behaviour of human tumour cells by activation of genes involved in the adaptation to hypoxic stress. The main objective in malignant GBM therapy is either to eradicate the tumour or to convert it into a controlled, quiescent chronic disease. Aza., SU.D2, Chetomin or CA9-siRNA possesses functional CA9 inhibitory characteristics when applied against human cancers with hypoxic regions like GBM. They may be used as alternative or in conjunction with other direct inhibitors possessing similar functionality, thereby rendering them as potential optimal tools for the development of an optimized therapy in human brain cancer treatment.

Modulation of Carbonic Anhydrase 9 (CA9) in Human Brain Cancer

Hypoxia is a crucial factor in tumour aggressiveness and its treatment resistance, particularly in human brain cancer. Tumour resistance against radiation- and chemo- therapy is facilitated by oxygenation reduction at tumour areas. HIF-1α regulated genes are mostly responsible for this type of resistance. Among these genes, carbonic anhydrase isoform 9 (CA9) is highly overexpressed in many types of cancer especially in high grade brain cancer like GBM. CA IX contributes to tumour environment acidification by catalyzing the carbon dioxide hydration to bicarbonate and protons, leading to the acquisition of metastasic phenotypes and chemoresistance to weakly basic anticancer drugs and therefore to inadequate application of radio-therapeutic or chemotherapeutic anti-cancer treatment strategies. Inhibition of this enzymatic activity by application of specific chemical CA9 inhibitors (sulphonamide derivative compounds) or indirect inhibitors like HIF-1α inhibitors (chetomin) or molecular inhibitors like CA9-siRNA leads to reversion of these processes, leading to the CA9 functional role inhibition during tumourigenesis. Hypoxia significantly influences the tumour microenvironment behaviour via activation of genes involved in the adaptation to the hypoxic stress. It also represents an important cancer prognosis indicator and is associated with aggressive growth, malignant progression, metastasis and poor treatment response. The main objective in malignant GBM therapy is either to eradicate the tumour or to convert it into a controlled, quiescent chronic disease. Sulfonamide derivative compounds with CA9 inhibitory characteristics represent one of the optimal treatment options beside other CA9 inhibitory agents or chemical inhibitory compounds against its main regulating transcription factor which is the hypoxia induced HIF-1α when applied against human cancers with hypoxic regions like GBM, bearing potential for an effective role in human brain tumour therapeutic strategies. Glycolytic inhibitors, when added in controlled doses under hypoxia, lead to a reduced accumulation of HIF-1α and can function as indirect hypoxia regulated genes inhibitors like CA9. These may be used as alternative or in conjunction with other direct inhibitors like the sulphonamide derivate compounds, chetomin or specific siRNAs, or other different chemical compounds possessing similar functionality making them as optimal tools for optimized therapy development in cancer treatment, especially against human brain cancer. Further experimental analysis towards the tumour stage specific inhibitory CA9 characteristics determination are necessary to find the optimal therapeutic solutions among the different available modalities; whether they are direct or indirect chemical, molecular or natural inhibitors to be able to set up successful treatment approaches against the different human tumour diseases.