Glioblastoma multiforme (GBM), classified by the World Health Organization (WHO) as a grade IV glioma, represents the most aggressive and lethal primary brain tumor. Despite the current therapeutic standard, which includes surgical resection followed by radiotherapy and chemotherapy with Temozolomide (TMZ), patient prognosis remains extremely poor, with an average survival of only 14/16 months. This lack of a resolutive therapeutic effect is primarily due to the pronounced intratumoral heterogeneity and the intrinsic resistance of GBM to treatment, features strongly associated with the presence of glioblastoma stem cells (GBCSCs) and particularly accentuated in the mesenchymal (MES) molecular subtype. Resistance is further strengthened by tumour-specific molecular programs and by the highly immunosuppressive tumour microenvironment. Over the years, several experimental studies have highlighted the crucial role of ion channels in tumour progression. In this context, these channels display unique molecular properties and are referred to as oncogenic channels, or “oncochannels.” Among them, the Na⁺/Ca²⁺ exchanger (NCX), a key mediator of intracellular calcium homeostasis, has been implicated in promoting invasiveness in immortalized GBM cell lines such as U251. The main goal of this thesis was to consolidate and expand this knowledge by investigating the functional role of NCX in a more clinically relevant, highly aggressive GBM model. To this end, a primary mesenchymal GBM cell line (GBM19) was used, providing an in vitro system that more faithfully reflects the biological complexity observed in vivo than immortalized cell lines. Furthermore, this thesis aimed to explore the relationship between NCX involvement in GBM19 cell migration and its potential role in the spontaneous formation of structures associated with vasculogenic mimicry (VM). A combination of complementary experimental approaches was employed, including wound healing assays, time-lapse microscopy, and immunofluorescence. GBM19 cells exhibited distinctive growth features, characterized by the presence of well-defined circular structures. Time-lapse microscopy revealed two cellular subpopulations: leading-edge cells, primarily involved in scratch closure, and loop cells, positioned along the circular structures. Immunofluorescence analyses revealed significantly higher VE-cadherin expression in loop cells than in leading-edge cells, supporting the hypothesis that VM contributes to the migratory behaviour of this line. Pharmacological inhibition of NCX profoundly impaired migratory capacity: at 40 hours, scratch closure in bepridil-treated cultures stalled at 81.15%, whereas control cultures achieved complete closure. Moreover, NCX inhibition disrupted loop formation, which remained significantly wider and unclosed even at 40 hours. The results identify NCX activity as a determining factor for GBM19 cell motility and migration. The data also reveal a clear association between NCX function and vasculogenic mimicry, suggesting that this transporter may represent a dual-purpose therapeutic target that simultaneously impairs both migratory pathways and VM-related processes that support tumour expansion. Notably, the effective bepridil concentration in GBM19 (6.25 μM) is approximately eight times lower than previously reported doses in immortalized cell lines, indicating increased sensitivity in primary mesenchymal GBM cells and offering encouraging prospects for the development of pharmacological strategies with reduced systemic toxicity. Overall, this work provides new insights into the electrophysiological and morphological heterogeneity of mesenchymal GBM and lays essential groundwork for future research aimed at defining the molecular mechanisms through which NCX governs invasion and the formation of VM-associated structures.
Il glioblastoma multiforme (GBM), classificato dall'Organizzazione Mondiale della Sanità (OMS) come glioma di grado IV, rappresenta il tumore cerebrale primario più aggressivo e letale. Nonostante l'attuale standard terapeutico, che include la resezione chirurgica seguita da radioterapia e chemioterapia con Temozolomide (TMZ), la prognosi dei pazienti rimane estremamente sfavorevole, con una sopravvivenza media limitata a 14/16 mesi. Questo mancato effetto terapeutico risolutivo è in gran parte dovuto alla pronunciata eterogeneità intratumorale e alla resistenza intrinseca del GBM al trattamento, caratteristiche fortemente associate alla presenza di cellule staminali di glioblastoma (GBCSC) e particolarmente accentuate nel sottotipo molecolare mesenchimale (MES). Nel corso degli anni, diversi studi sperimentali hanno dimostrato il ruolo cruciale di canali ionici nella progressione dei tumori che, in questo contesto, possiedono delle caratteristiche molecolari uniche e prendono il nome di oncocanali. Tra questi, lo scambiatore Na⁺/Ca²⁺ (NCX), un mediatore cruciale dell'omeostasi del calcio intracellulare, è implicato nella promozione dell'invasività in linee cellulari di GBM immortalizzate come U251. L'obiettivo principale di questa tesi è stato quello di consolidare queste conoscenze studiando il contributo funzionale di NCX in un modello di GBM clinicamente più rilevante e altamente aggressivo. A tal fine, è stata utilizzata una linea cellulare primaria di GBM di sottotipo mesenchimale (GBM19), fornendo un sistema in vitro che riflette più fedelmente la complessità biologica osservata in vivo rispetto alle linee di cellule immortalizzate. Inoltre, questo lavoro di tesi ha permesso di porre in relazione il coinvolgimento di NCX nella migrazione delle cellule GBM19 e il suo potenziale ruolo nella formazione spontanea di strutture associate al mimetismo vasculogenico (VM). Per raggiungere questo obiettivo, è stata utilizzata una combinazione di approcci sperimentali complementari, quali il wound healing assay, la microscopia time-lapse e l’immunofluorescenza. Le cellule GBM19 hanno mostrato caratteristiche di crescita distintive in cui si riscontra la presenza di strutture circolari ben definite. I risultati ottenuti attraverso la microscopia time lapse hanno permesso di identificare due sottopopolazioni cellulari: le leading edge cells, primariamente coinvolte nella chiusura dello scratch e le loop cells, affacciate sulle strutture circolari. Le analisi di immunofluorescenza hanno rivelato un'espressione significativamente più elevata del marcatore endoteliale VE-caderina associato alla VM sulle loop cells rispetto alle leading edge cells, supportando l'ipotesi che la VM contribuisca al comportamento migratorio di questa linea. L'inibizione farmacologica di NCX ha profondamente compromesso la capacità migratoria: a 40 ore, la chiusura dello scratch nella condizione trattata con bepridil si è arrestata all'81,15%, mentre le colture di controllo hanno raggiunto una chiusura completa. Inoltre, l'inibizione di NCX ha alterato la formazione dei loop, che rimanevano significativamente più ampi e non chiusi chiusi anche a 40 ore. I risultati ottenuti, identificano l'attività di NCX come un fattore determinante per la motilità e la migrazione delle cellule GBM19. I dati rivelano anche una chiara associazione tra la funzione di NCX e il mimetismo vasculogenico, suggerendo che il trasportatore possa rappresentare un bersaglio terapeutico a duplice scopo, in grado di compromettere simultaneamente le vie di migrazione e quelle correlate alle VM che facilitano l'espansione tumorale. Nel complesso, questo lavoro offre nuove informazioni sull'eterogeneità elettrofisiologica e morfologica del GBM mesenchimale e pone le basi essenziali per future ricerche volte a delineare i meccanismi molecolari attraverso i quali NCX governa l'invasione e la formazione di strutture associate alle VM.
Ruolo dello scambiatore NCX nella migrazione di cellule di glioblastoma e nel mimetismo vasculogenico
DMELLO, NATHAN GREGORY
2024/2025
Abstract
Glioblastoma multiforme (GBM), classified by the World Health Organization (WHO) as a grade IV glioma, represents the most aggressive and lethal primary brain tumor. Despite the current therapeutic standard, which includes surgical resection followed by radiotherapy and chemotherapy with Temozolomide (TMZ), patient prognosis remains extremely poor, with an average survival of only 14/16 months. This lack of a resolutive therapeutic effect is primarily due to the pronounced intratumoral heterogeneity and the intrinsic resistance of GBM to treatment, features strongly associated with the presence of glioblastoma stem cells (GBCSCs) and particularly accentuated in the mesenchymal (MES) molecular subtype. Resistance is further strengthened by tumour-specific molecular programs and by the highly immunosuppressive tumour microenvironment. Over the years, several experimental studies have highlighted the crucial role of ion channels in tumour progression. In this context, these channels display unique molecular properties and are referred to as oncogenic channels, or “oncochannels.” Among them, the Na⁺/Ca²⁺ exchanger (NCX), a key mediator of intracellular calcium homeostasis, has been implicated in promoting invasiveness in immortalized GBM cell lines such as U251. The main goal of this thesis was to consolidate and expand this knowledge by investigating the functional role of NCX in a more clinically relevant, highly aggressive GBM model. To this end, a primary mesenchymal GBM cell line (GBM19) was used, providing an in vitro system that more faithfully reflects the biological complexity observed in vivo than immortalized cell lines. Furthermore, this thesis aimed to explore the relationship between NCX involvement in GBM19 cell migration and its potential role in the spontaneous formation of structures associated with vasculogenic mimicry (VM). A combination of complementary experimental approaches was employed, including wound healing assays, time-lapse microscopy, and immunofluorescence. GBM19 cells exhibited distinctive growth features, characterized by the presence of well-defined circular structures. Time-lapse microscopy revealed two cellular subpopulations: leading-edge cells, primarily involved in scratch closure, and loop cells, positioned along the circular structures. Immunofluorescence analyses revealed significantly higher VE-cadherin expression in loop cells than in leading-edge cells, supporting the hypothesis that VM contributes to the migratory behaviour of this line. Pharmacological inhibition of NCX profoundly impaired migratory capacity: at 40 hours, scratch closure in bepridil-treated cultures stalled at 81.15%, whereas control cultures achieved complete closure. Moreover, NCX inhibition disrupted loop formation, which remained significantly wider and unclosed even at 40 hours. The results identify NCX activity as a determining factor for GBM19 cell motility and migration. The data also reveal a clear association between NCX function and vasculogenic mimicry, suggesting that this transporter may represent a dual-purpose therapeutic target that simultaneously impairs both migratory pathways and VM-related processes that support tumour expansion. Notably, the effective bepridil concentration in GBM19 (6.25 μM) is approximately eight times lower than previously reported doses in immortalized cell lines, indicating increased sensitivity in primary mesenchymal GBM cells and offering encouraging prospects for the development of pharmacological strategies with reduced systemic toxicity. Overall, this work provides new insights into the electrophysiological and morphological heterogeneity of mesenchymal GBM and lays essential groundwork for future research aimed at defining the molecular mechanisms through which NCX governs invasion and the formation of VM-associated structures.| File | Dimensione | Formato | |
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Descrizione: This experimental thesis was written by Mr. Nathan Gregory D’Mello and conducted at the Laboratory of Neurobiology and Integrated Physiology, under the supervision of Prof.ssa Paola Rossi and Dott.ssa Erica Cecilia Pariori.
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https://hdl.handle.net/20.500.14239/32663