| Abstract |
Glioblastoma (GBM) is the most malignant brain cancer among adults, according to the World Health Organization (WHO). It is characterized by excessive proliferation and infiltration, along with extensive inter- and intra-tumoral heterogeneity. Current standard therapy includes maximal safe surgical resection, followed by concurrent radiation and/or adjuvant chemotherapy. However, in spite of intensive treatment, patients have a poor prognosis, due to the high recurrence potential of GBM. Identifying GBM biomarkers could help in earlier disease diagnosis, patient classification and stratification, as well as in the characterization of specific features regarding the tumor physiology and aggressiveness, paving the way to new therapeutic strategies.
Growing evidence indicate that GBM exploits mechanisms of neurogenesis and neurodevelopment to further facilitate tumor progression. There is recent evidence suggesting, high expression of the Promyelocytic Leukemia Protein (PML), a tumor suppressor and cell regulator, in primary GBM samples. PML is expressed in all tissues and implicated in various ways to cancer biology. In brain, PML participates in the physiological migration of the neural progenitor cells, which have been also hypothesized to serve as the cell of origin of GBM. Recent studies in PML knocked-down mice indicate a common PML-mediated migratory pathway in both adult neurogenesis and GBM invasion within the central nervous system. Furthermore, recent findings in the biology of malignant gliomas have revealed that glioma cells extend long membranous protrusions called tumor microtubes (TMs), which share certain characteristics with the axonal and dendritic growth of developing neurons. TMs are utilized as pathways for the proliferation and invasion of brain tumors and serve as points of intersection for the formation of the GBΜ network, enabling rapid and long-distance multicellular communication. Intercellular calcium waves propagate through this network via gap junctions and synchronize GBM cells, orchestrating various cellular activities that promote tumor progression and resistance to therapeutic interventions.
In this work, we aim to explore more thoroughly the role of PML in GBM growth, invasion, glioma network formation, and treatment by utilizing a variety of models including 2D/3D in vitro and ex vivo cultures. In our experiments, we utilize the established U87MG cell line, which has been genetically modified for conditional overexpression or silencing of the PML protein. Specifically, we explore the role of the PML protein in the proliferation of GBM cells using 2D in vitro biological models. Additionally, we examine whether the PML protein can modulate the TM-network and we investigate the effect of TM-network on GBM proliferation and response to treatment by employing the gap junction channel blocker carbenoxolone (CBX). Moving beyond 2D cultures, we employ advanced 3D biological models, such as spheroids and organotypic brain slices (ex vivo), to explore the PML-mediated effects on tumor growth and invasion. To visualize and quantify these effects, we utilize conventional optical microscopy techniques along with confocal microscopy.
Our overall findings indicate that PML overexpression suppresses cell proliferation, while it induces the invasive capacity of the U87MG cells. Furthermore, the organotypic brain slices seemed to facilitate further the invasive capacity of GBM cells and the maintenance of the invasive patterns in both in vitro and ex vivo conditions affirm the consistency of our observations in a more physiologic realistic environment. Moreover, our findings suggest that the PML protein can influence the connectivity of the GBM network. Finally, our results indicate that GBM network plays a role in tumor growth, as evidenced by the observed effect of inhibiting gap junctions with CBX. Unravelling further the various factors affecting GBM proliferation, invasion and treatment response, as well as the various evolutional trajectories brought on by clonal alterations in gliomas, could pave the way into new therapeutic strategies and the identification of potential biomarkers for GBM progression
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