Spatial transcriptomics in glioblastoma: is knowing the right zip code the key to the next therapeutic breakthrough?

Spatial transcriptomics, the technology of visualizing cellular gene expression landscape in a cells native tissue location, has emerged as a powerful tool that allows us to address scientific questions that were elusive just a few years ago. This technological advance is a decisive jump in the technological evolution that is revolutionizing studies of tissue structure and function in health and disease through the introduction of an entirely new dimension of data, spatial context. Perhaps the organ within the body that relies most on spatial organization is the brain. The central nervous system's complex microenvironmental and spatial architecture is tightly regulated during development, is maintained in health, and is detrimental when disturbed by pathologies. This inherent spatial complexity of the central nervous system makes it an exciting organ to study using spatial transcriptomics for pathologies primarily affecting the brain, of which Glioblastoma is one of the worst. Glioblastoma is a hyper-aggressive, incurable, neoplasm and has been hypothesized to not only integrate into the spatial architecture of the surrounding brain, but also possess an architecture of its own that might be actively remodeling the surrounding brain. In this review we will examine the current landscape of spatial transcriptomics in glioblastoma, outline novel findings emerging from the rising use of spatial transcriptomics, and discuss future directions and ultimate clinical/translational avenues.

Autophagy-dependent ferroptosis as a potential treatment for glioblastoma.

Glioblastoma (GBM) is the most common malignant primary brain tumor with a poor 5-year survival rate. Autophagy is a conserved intracellular degradation system that plays a dual role in GBM pathogenesis and therapy. On one hand, stress can lead to unlimited autophagy to promote GBM cell death. On the other hand, elevated autophagy promotes the survival of glioblastoma stem cells against chemotherapy and radiation therapy. Ferroptosis is a type of lipid peroxidation-mediated regulated necrosis that initially differs from autophagy and other types of cell death in terms of cell morphology, biochemical characteristics, and the gene regulators involved. However, recent studies have challenged this view and demonstrated that the occurrence of ferroptosis is dependent on autophagy, and that many regulators of ferroptosis are involved in the control of autophagy machinery. Functionally, autophagy-dependent ferroptosis plays a unique role in tumorigenesis and therapeutic sensitivity. This mini-review will focus on the mechanisms and principles of autophagy-dependent ferroptosis and its emerging implications in GBM.

Diffusing alpha-emitters radiation therapy in combination with temozolomide or bevacizumab in human glioblastoma multiforme xenografts.

Glioblastoma multiforme (GBM) is at present an incurable disease with a 5-year survival rate of 5.5%, despite improvements in treatment modalities such as surgery, radiation therapy, chemotherapy [e.g., temozolomide (TMZ)], and targeted therapy [e.g., the antiangiogenic agent bevacizumab (BEV)]. Diffusing alpha-emitters radiation therapy (DaRT) is a new modality that employs radium-224-loaded seeds that disperse alpha-emitting atoms inside the tumor. This treatment was shown to be effective in mice bearing human-derived GBM tumors. Here, the effect of DaRT in combination with standard-of-care therapies such as TMZ or BEV was investigated. In a viability assay, the combination of alpha radiation with TMZ doubled the cytotoxic effect of each of the treatments alone in U87 cultured cells. A colony formation assay demonstrated that the surviving fraction of U87 cells treated by TMZ in combination with alpha irradiation was lower than was achieved by alpha- or x-ray irradiation as monotherapies, or by x-ray combined with TMZ. The treatment of U87-bearing mice with DaRT and TMZ delayed tumor development more than the monotherapies. Unlike other radiation types, alpha radiation did not increase VEGF secretion from U87 cells in culture. BEV treatment introduced several days after DaRT implantation improved tumor control, compared to BEV or DaRT as monotherapies. The combination was also shown to be superior when starting BEV administration prior to DaRT implantation in large tumors relative to the seed size. BEV induced a decrease in CD31 staining under DaRT treatment, increased the diffusive spread of 224Ra progeny atoms in the tumor tissue, and decreased their clearance from the tumor through the blood. Taken together, the combinations of DaRT with standard-of-care chemotherapy or antiangiogenic therapy are promising approaches, which may improve the treatment of GBM patients.

Luteolin enhances erlotinib's cell proliferation inhibitory and apoptotic effects in glioblastoma cell lines.

The epidermal growth factor (EGFR) receptor is frequently overexpressed in glioblastoma multiforme IV (GBM). Increased expression of EGFR leads to increased proliferation, decreased apoptosis, and increased resistance to chemotherapeutic agents. A small molecule called erlotinib inhibits EGFR receptors by binding to their adenosine triphosphate (ATP) binding sites. It is FDA approved to treat a variety of EGFR-mediated cancers. Several clinical trials have explored a combination of erlotinib with other agents to treat glioblastoma since it is believed that erlotinib would benefit patients with GBM with EGFR mutations or expression. Luteolin, a natural flavonoid, inhibits cell growth and induces apoptosis in cancer cells. We investigated the combined effects of erlotinib and luteolin on proliferation and apoptosis on glioblastoma cell lines overexpressing EGFR or glioma cells expressing truncated EGFR (ΔEGFR). In a concentration-dependent fashion, the combination of luteolin and erlotinib reduced cell proliferation (p < 0.05) and induced apoptosis by cleaving PARP and increasing caspase expression. In addition, the combination of luteolin and erlotinib reduced the phosphorylation of downstream EGFR cell signaling molecules such as Akt, NF kappa B, and STAT3 in a concentration-dependent manner. These findings suggest that combining luteolin with erlotinib offers a potential treatment strategy for glioblastoma multiforme IV.

"Aerobic glycolytic imaging" of human gliomas using combined pH-, oxygen-, and perfusion-weighted magnetic resonance imaging.

PURPOSE: To quantify abnormal metabolism of diffuse gliomas using "aerobic glycolytic imaging" and investigate its biological correlation. METHODS: All subjects underwent a pH-weighted amine chemical exchange saturation transfer spin-and-gradient-echo echoplanar imaging (CEST-SAGE-EPI) and dynamic susceptibility contrast perfusion MRI. Relative oxygen extraction fraction (rOEF) was estimated as the ratio of reversible transverse relaxation rate R2' to normalized relative cerebral blood volume. An aerobic glycolytic index (AGI) was derived by the ratio of pH-weighted image contrast (MTRasym at 3.0 ppm) to rOEF. AGI was compared between different tumor types (N = 51, 30 IDH mutant and 21 IDH wild type). Metabolic MR parameters were correlated with 18F-FDG uptake (N = 8, IDH wild-type glioblastoma), expression of key glycolytic proteins using immunohistochemistry (N = 38 samples, 21 from IDH mutant and 17 from IDH wild type), and bioenergetics analysis on purified tumor cells (N = 7, IDH wild-type high grade). RESULTS: AGI was significantly lower in IDH mutant than wild-type gliomas (0.48 ± 0.48 vs. 0.70 ± 0.48; P = 0.03). AGI was strongly correlated with 18F-FDG uptake both in non-enhancing tumor (Spearman, ρ = 0.81; P = 0.01) and enhancing tumor (ρ = 0.81; P = 0.01). AGI was significantly correlated with glucose transporter 3 (ρ = 0.71; P = 0.004) and hexokinase 2 (ρ = 0.73; P = 0.003) in IDH wild-type glioma, and monocarboxylate transporter 1 (ρ = 0.59; P = 0.009) in IDH mutant glioma. Additionally, a significant correlation was found between AGI derived from bioenergetics analysis and that estimated from MRI (ρ = 0.79; P = 0.04). CONCLUSION: AGI derived from molecular MRI was correlated with glucose uptake (18F-FDG and glucose transporter 3/hexokinase 2) and cellular AGI in IDH wild-type gliomas, whereas AGI in IDH mutant gliomas appeared associated with monocarboxylate transporter density.

Long Non-Coding RNAs in Diagnosis, Treatment, Prognosis, and Progression of Glioma: A State-of-the-Art Review.

Glioma is the most common malignant central nervous system tumor with significant mortality and morbidity. Despite considerable advances, the exact molecular pathways involved in tumor progression are not fully elucidated, and patients commonly face a poor prognosis. Long non-coding RNAs (lncRNAs) have recently drawn extra attention for their potential roles in different types of cancer as well as non-malignant diseases. More than 200 lncRNAs have been reported to be associated with glioma. We aimed to assess the roles of the most investigated lncRNAs in different stages of tumor progression and the mediating molecular pathways in addition to their clinical applications. lncRNAs are involved in different stages of tumor formation, invasion, and progression, including regulating the cell cycle, apoptosis, autophagy, epithelial-to-mesenchymal transition, tumor stemness, angiogenesis, the integrity of the blood-tumor-brain barrier, tumor metabolism, and immunological responses. The well-known oncogenic lncRNAs, which are upregulated in glioma, are H19, HOTAIR, PVT1, UCA1, XIST, CRNDE, FOXD2-AS1, ANRIL, HOXA11-AS, TP73-AS1, and DANCR. On the other hand, MEG3, GAS5, CCASC2, and TUSC7 are tumor suppressor lncRNAs, which are downregulated. While most studies reported oncogenic effects for MALAT1, TUG1, and NEAT1, there are some controversies regarding these lncRNAs. Expression levels of lncRNAs can be associated with tumor grade, survival, treatment response (chemotherapy drugs or radiotherapy), and overall prognosis. Moreover, circulatory levels of lncRNAs, such as MALAT1, H19, HOTAIR, NEAT1, TUG1, GAS5, LINK-A, and TUSC7, can provide non-invasive diagnostic and prognostic tools. Modulation of expression of lncRNAs using antisense oligonucleotides can lead to novel therapeutics. Notably, a profound understanding of the underlying molecular pathways involved in the function of lncRNAs is required to develop novel therapeutic targets. More investigations with large sample sizes and increased focus on in-vivo models are required to expand our understanding of the potential roles and application of lncRNAs in glioma.

Macromolecular Drug Carriers for Targeted Glioblastoma Therapy: Preclinical Studies, Challenges, and Future Perspectives.

Glioblastoma, the most common, aggressive brain tumor, ranks among the least curable cancers-owing to its strong tendency for intracranial dissemination, high proliferation potential, and inherent tumor resistance to radiation and chemotherapy. Current glioblastoma treatment strategies are further hampered by a critical challenge: adverse, non-specific treatment effects in normal tissue combined with the inability of drugs to penetrate the blood brain barrier and reach the tumor microenvironment. Thus, the creation of effective therapies for glioblastoma requires development of targeted drug-delivery systems that increase accumulation of the drug in the tumor tissue while minimizing systemic toxicity in healthy tissues. As demonstrated in various preclinical glioblastoma models, macromolecular drug carriers have the potential to improve delivery of small molecule drugs, therapeutic peptides, proteins, and genes to brain tumors. Currently used macromolecular drug delivery systems, such as liposomes and polymers, passively target solid tumors, including glioblastoma, by capitalizing on abnormalities of the tumor vasculature, its lack of lymphatic drainage, and the enhanced permeation and retention (EPR) effect. In addition to passive targeting, active targeting approaches include the incorporation of various ligands on the surface of macromolecules that bind to cell surface receptors expressed on specific cancer cells. Active targeting approaches also utilize stimulus responsive macromolecules which further improve tumor accumulation by triggering changes in the physical properties of the macromolecular carrier. The stimulus can be an intrinsic property of the tumor tissue, such as low pH, or extrinsic, such as local application of ultrasound or heat. This review article explores current preclinical studies and future perspectives of targeted drug delivery to glioblastoma by macromolecular carrier systems, including polymeric micelles, nanoparticles, and biopolymers. We highlight key aspects of the design of diverse macromolecular drug delivery systems through a review of their preclinical applications in various glioblastoma animal models. We also review the principles and advantages of passive and active targeting based on various macromolecular carriers. Additionally, we discuss the potential disadvantages that may prevent clinical application of these carriers in targeting glioblastoma, as well as approaches to overcoming these obstacles.

Drug delivery approaches for the treatment of glioblastoma multiforme.

CONTEXT: Glioblastoma multiforme (GBM) is by far the most common and aggressive form of glial tumor. It is characterized by a highly proliferative population of cells that invade surrounding tissue and that frequently recur after surgical resection and chemotherapy. Over the last decades, a number of promising novel pharmacological approaches have been investigated, but most of them have failed clinical trials due to some side-effects such as toxicity and poor drug delivery to the brain. The major obstacle in the treatment of GBM is the presence of the blood-brain barrier (BBB). Due to their relatively high molecular weight, most therapeutic drugs fail to cross the BBB from the blood circulation. OBJECTIVE: This paper sheds light on the characteristics of GBM and the challenges of current pharmacological treatments. A closer look is given to the role of nanotechnology in the field of drug delivery, and its application in the treatment of brain tumors such as GBM. METHOD: For this purpose, effort was made to select the most recent studies using predefined search criteria that included at least one of the following keywords in the PubMed and Medline databases: glioblastoma, drug delivery, blood-brain barrier, nanotechnology, and nanoparticle. CONCLUSION: Breakthrough in nanotechnology offers promising applications in cancer therapy and targeted drug delivery. However, more efforts need to be devoted to the development of novel therapeutic strategies that enable the delivery of drugs to desired areas of the brain with limited side-effects and higher therapeutic efficiency.

Treatment of glioma by cisplatin-loaded nanogels conjugated with monoclonal antibodies against Cx43 and BSAT1.

Targeted drug delivery for brain tumor treatment is one of the important objectives in nanomedicine. Human glioblastoma is the most frequent and aggressive type of brain tumors. The preferential expression of membrane protein connexin 43 (Cx43) and brain-specific anion transporter (BSAT1) in the tumor and peritumoral area is a key component for targeted drug delivery. The purpose of this study was to design cisplatin-loaded nanogels conjugated with monoclonal antibodies to Cx43 and BSAT1 for treatment of intracranial gliomas 101/8. MRI volumetric analysis of tumor-bearing rats indicated significantly reduced tumor volume with cisplatin-loaded targeted-nanogel treatment compared to other formulations. The median survival of rats treated with targeted nanogels conjugated with specific mAbs against extracellular loops of Cx43 and BSAT1 were 27 and 26.6 days higher than that in control group, respectively. For the first time we demonstrated the efficiency of mAb-targeted cisplatin-loaded nanogels in the experimental model of glioma 101/8. This approach could facilitate the development of new drug delivery systems for the treatment of gliomas.