REPOSITORY > RESULTS

Abstract

Glioblastoma (GB) is the most common and aggressive primary tumour of the central nervous system. Despite maximal possible surgical resection of the tumours and aggressive treatment regimens with radiotherapy and chemotherapy, patient overall survival is less than 2 years, as patients eventually develop resistance to therapy, resulting in recurrent tumours. The main challenges of therapeutic failure are based on the infiltrative growth of the tumour into the brain parenchyma, the presence of a small population of therapy-resistant GB stem cells (GSCs), and extensive inter- and intra-tumour heterogeneity. Cancer models that efficiently represent the complexity of the tumour are therefore crucial in the era of precision medicine.
The establishment of a standardized tissue bank of high-quality biospecimens annotated with clinical information is central for reliable translational research. We contributed glioma samples to the Slovenian GlioBank, containing glioma tissue samples, cell models, and corresponding clinical data for use in cancer research. We used GB differentiated cells and GSCs as models to screen for the efficacy of the cannabinoids delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabigerol (CBG) on cell viability and invasion. CBG effectively impaired the relevant hallmarks of GB progression, with comparable cytotoxic effects to THC, but with the additional ability to inhibit GB cell invasion. Moreover, CBG could target therapy-resistant GSCs, which are the root of cancer development and extremely resistant to various other treatments. Thus, CBG could represent a new yet unexplored adjuvant treatment strategy for GB.
We established a novel 3D in vitro model of GB organoids (GBOs) prepared from patient-derived tumours that represents a more clinically relevant tumour model. The GBOs, unlike the previously used 3D GB cells and GSCs, capture the entire tumour microenvironment. The intra- and inter-tumour heterogeneity of in vivo tumours (including variable transcriptional profiles and cellular compositions) was thus preserved in GBOs. We demonstrated that GBOs from most of the patients were resistant to irradiation and chemotherapy with temozolomide, as no significant effects on GBO viability or invasion were observed. Further analyses revealed that certain target genes were differentially expressed in the treated GBOs, such as E3 ubiquitin-protein ligase (MDM2) and cyclin-dependent kinase inhibitor 1A (CDKN1A). Both genes are involved in the DNA damage response and cell cycle signalling pathways. Our results implicate on activation of p53-related pathways in the tumour microenvironment (TME) of GB tumours and shed light on possible mechanisms underlying the resistance of GB to therapy. GBOs can represent a novel, clinically-relevant culture system for evaluating specific responses of GB patients to therapy and have the potential to change drug and novel biomarker discoveries.
Discovering novel therapeutic targets is critical for developing efficient treatments. We explored the role of the novel potential biomarker cathepsin X in GB progression. Cathepsin X expression and activity were found to be upregulated in human GB tissues compared to low-grade gliomas and nontumour brain tissues. Cathepsin X was localised in GB cells and tumour-associated macrophages and microglia. Subsequently, potent, selective, irreversible (AMS36) and reversible (Z7) cathepsin X inhibitors were tested in vitro. These inhibitors decreased the viability of patient-derived GB cells. Furthermore, there was a correlation between the high proteolytic activity of cathepsin X and C-terminal cleavage of neuron-specific enzyme γ-enolase, a target of cathepsin X. Cathepsin X and γ-enolase were colocalised in GB tissues, preferentially in GB-associated macrophages and microglia. Taken together, our results on patient-derived tissues and cells suggest that cathepsin X plays a role in GB progression and as such is a potential target for therapeutic approaches against GB.
Overall, this work contributes to the establishment of a platform for basic and translational research in the field of neuro-oncology and to a deeper understanding of the pathobiology and therapeutic resistance of GB.