Glioblastoma multiforme (GBM) is a rare tumor and one of the most challenging malignancies to treat in all of oncology field. GBM is an aggressive solid tumor that mainly affects white matter and the corpus callosum. Typically, the cellular morphology is predominantly astrocytic, but in some cases, a subset of tumor cells may have oligodendroglial or primitive neuroectodermal tumor features.For a majority of patients with GBM, there is no known cause of the disease and no early detection of GBM is available.Despite advances in cancer therapies, nanomedicine approaches included, the treatment of GBM remains inefficient. These failures are likely attributable to the complex, and not yet completely know, biology of this tumour, which is responsible for its strong invasiveness, high degree of metastasis, high proliferation potential and resistance to radiation and chemotherapy, and to the presence of the blood-brain barrier (BBB) that limit the drugs entrance into the brain. Moreover, the intimate connection through which the cells communicate between them plays an important role in these biological processes. In this scenario, tunneling nanotubes (TnTs) are recently gaining importance as a key features in tumor progression and in particular in the re-growth of GBM after surgery.GBM Stem-like Cells (GSCs) represent a subpopulation of cells characterized by increased resistance to chemo- and radiotherapy (RT).Due to their intrinsic tumor-initiating potential and invasiveness, residual resistant GSCs lead to GBM recurrence and progression and represent a crucial target for effective therapies. However targeting GSCs is hardly difficult and complex, due to the presence of the BBB and for GSCs infiltrative nature arousing their dispersion within the brain parenchyma. In this context, we proposed doxorubicin-loaded multifunctionalized liposomes (LIPs) to enable BBB crossing, selective GSCs targeting and anti-tumor immune response activation.LIPs were dually functionalized with mApoE, a modified fragment of the human apolipoprotein E, which binds the LDL receptor (overexpressed on BBB and GBM cells) and chlorotoxin (ClTx) to improve their BBB crossing and tumour targeting respectively. Our results demonstrated the synergistic activity of ClTx-mApoE in boosting doxorubicin-loaded liposomes across the BBB, keeping the anti-tumour activity of the drug loaded: mApoE acts promoting cellular uptake, while ClTx promotes exocytosis of liposomes.Moreover, the encapsulation into mApoE-LIPs prevents DOX toxicity on BBB cells and enhances its accumulation within mouse brain in vivo. mApoE confers GSCs specificity through the engagement of the LDL receptor.When administered to patient-derived GSC NOD/SCID mouse xenograft mApoE-DOXO-LIPs, but not DOX-LIPs, triggered GSC apoptosis resulting in a remarkable reduction of tumor growth and invasion of the contralateral hemisphere through commissural fibers.Apoptotic GSCs prompted microglia/macrophage phagocytic activity coupled to the activation of the antigen-presenting machinery propaedeutic to T cell priming.Importantly, the concomitant administration of radiation enhanced the anti-tumor effects by altering BBB permeability and promoting the expression of LDLr on both BBB and GSCs.RT and adjuvant administration of drug-loaded targeted LIPs represent an effective strategy to deliver cytotoxic molecules, immune cell death inducers particularly, circumventing BBB hurdles and targeting GSCs at the tumor burden, the forefront of GBM recurrence.Moreover, our results demonstrate that TnTs are potentially useful as drug-delivery channels for cancer therapy, facilitating the intercellular redistribution of this drug in close and far away cells, thus reaching isolated tumour niches that are hardly targeted by simple drug diffusion in the brain parenchyma. The differences identified in TnTs formed by GBM cells and normal human astrocytes can be exploited to increase treatments precision and specificity.

Il glioblastoma multiforme (GBM) è un tumore raro del sistema nervoso centrale (SNC), che colpisce principalmente la sostanza bianca e il corpo calloso. Per la maggior parte dei pazienti con GBM, non esiste una causa nota della malattia e non è disponibile alcuna diagnosi precoce. Nonostante i progressi nelle terapie contro il cancro, inclusi gli approcci di nanomedicina, il trattamento del GBM rimane inefficace. Questi fallimenti sono probabilmente attribuibili alla complessa, e non ancora completamente nota, biologia di questo tumore, che è responsabile della sua forte invasività, dell’alto grado di metastasi, dell’alto potenziale di proliferazione e della resistenza alle radiazioni e alla chemioterapia, e alla presenza di della barriera emato-encefalica (BEE) che limita l'ingresso di farmaci nel cervello. Inoltre, l'intima connessione attraverso la quale le cellule comunicano tra loro svolge un ruolo importante in questi processi biologici. In questo scenario, i nanotubi (TnT) stanno recentemente ricevendo attenzione per il loro potenziale ruolo nella progressione del tumore e della sua riformazione dopo l'intervento chirurgico. Le cellule staminali di GBM (GSC) rappresentano una sottopopolazione di cellule caratterizzata da una maggiore resistenza alla chemioterapia e alla radioterapia (RT). A causa del loro potenziale intrinseco di innesco del tumore e dell'invasività, le GSC determinano la ricorrenza del GBM e rappresentano un obiettivo cruciale per lo sviluppo di una terapia efficace. In questo contesto, sono stati sviluppati liposomi(LIP) multifunzionali e caricati con il farmaco antitumorale doxorubicina, per consentire l'attraversamento BEE ed il targeting selettivo delle GSC. I LIP sono stati funzionalizzati con il mApoE, un frammento modificato dell'apolipoproteina umana E, che lega il recettore LDL (sovraespresso su cellule BEE e GBM) e la clorotossina (ClTx) per migliorare rispettivamente l'attraversamento del BBB e il targeting tumorale. I nostri risultati hanno dimostrato l'attività sinergica di ClTx e mApoE nel passaggio del LIP caricati con doxorubicina attraverso il BEE, mantenendo l'attività antitumorale del farmaco caricato: il mApoE agisce promuovendo l'ingresso cellulare, mentre la ClTx promuove l'esocitosi. L’incorporazione della DOX nei LIPs previene la tossicità del farmaco sulle cellule di BEE e ne migliora l'accumulo nel cervello di topo in vivo. I mApoE-DOXO-LIP somministrati a topi con tumori formati da GSC hanno innescato l'apoptosi delle GSC con conseguente notevole riduzione della crescita tumorale e invasione nell'emisfero controlaterale. Le GSC apoptotiche hanno inoltre indotto l'attività di fagocitosi di microglia/macrofagi. È importante sottolineare che la somministrazione concomitante di radiazioni ha migliorato gli effetti anti-tumorali dei mApoE-DOXO LIP, alterando la permeabilità BBB e promuovendo l'espressione del recettore LDL sia sulla BEE che sulle GSC. La RT e la somministrazione adiuvante di LIP caricati con farmaci rappresentano una strategia efficace per fornire molecole citotossiche al SNC, aggirando gli ostacoli della BEE e prendendo di mira le GSC, principali responsabili della recidiva del GBM. Inoltre, i nostri risultati dimostrano che i TnT sono potenzialmente utili come canali di rilascio del farmaco per la terapia del cancro, facilitando la ridistribuzione intercellulare del farmaco in cellule vicine e lontane, raggiungendo così nicchie tumorali isolate che sono difficilmente colpite dalla semplice diffusione del farmaco nel parenchima cerebrale. Le differenze identificate nei TnT formati da cellule GBM e astrociti umani sani possono essere sfruttate per aumentare la precisione e la specificità dei trattamenti.

(2020). A nanomedicine approach for the treatment of glioblastoma multiforme. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2020).

A nanomedicine approach for the treatment of glioblastoma multiforme

FORMICOLA, BEATRICE
2020

Abstract

Glioblastoma multiforme (GBM) is a rare tumor and one of the most challenging malignancies to treat in all of oncology field. GBM is an aggressive solid tumor that mainly affects white matter and the corpus callosum. Typically, the cellular morphology is predominantly astrocytic, but in some cases, a subset of tumor cells may have oligodendroglial or primitive neuroectodermal tumor features.For a majority of patients with GBM, there is no known cause of the disease and no early detection of GBM is available.Despite advances in cancer therapies, nanomedicine approaches included, the treatment of GBM remains inefficient. These failures are likely attributable to the complex, and not yet completely know, biology of this tumour, which is responsible for its strong invasiveness, high degree of metastasis, high proliferation potential and resistance to radiation and chemotherapy, and to the presence of the blood-brain barrier (BBB) that limit the drugs entrance into the brain. Moreover, the intimate connection through which the cells communicate between them plays an important role in these biological processes. In this scenario, tunneling nanotubes (TnTs) are recently gaining importance as a key features in tumor progression and in particular in the re-growth of GBM after surgery.GBM Stem-like Cells (GSCs) represent a subpopulation of cells characterized by increased resistance to chemo- and radiotherapy (RT).Due to their intrinsic tumor-initiating potential and invasiveness, residual resistant GSCs lead to GBM recurrence and progression and represent a crucial target for effective therapies. However targeting GSCs is hardly difficult and complex, due to the presence of the BBB and for GSCs infiltrative nature arousing their dispersion within the brain parenchyma. In this context, we proposed doxorubicin-loaded multifunctionalized liposomes (LIPs) to enable BBB crossing, selective GSCs targeting and anti-tumor immune response activation.LIPs were dually functionalized with mApoE, a modified fragment of the human apolipoprotein E, which binds the LDL receptor (overexpressed on BBB and GBM cells) and chlorotoxin (ClTx) to improve their BBB crossing and tumour targeting respectively. Our results demonstrated the synergistic activity of ClTx-mApoE in boosting doxorubicin-loaded liposomes across the BBB, keeping the anti-tumour activity of the drug loaded: mApoE acts promoting cellular uptake, while ClTx promotes exocytosis of liposomes.Moreover, the encapsulation into mApoE-LIPs prevents DOX toxicity on BBB cells and enhances its accumulation within mouse brain in vivo. mApoE confers GSCs specificity through the engagement of the LDL receptor.When administered to patient-derived GSC NOD/SCID mouse xenograft mApoE-DOXO-LIPs, but not DOX-LIPs, triggered GSC apoptosis resulting in a remarkable reduction of tumor growth and invasion of the contralateral hemisphere through commissural fibers.Apoptotic GSCs prompted microglia/macrophage phagocytic activity coupled to the activation of the antigen-presenting machinery propaedeutic to T cell priming.Importantly, the concomitant administration of radiation enhanced the anti-tumor effects by altering BBB permeability and promoting the expression of LDLr on both BBB and GSCs.RT and adjuvant administration of drug-loaded targeted LIPs represent an effective strategy to deliver cytotoxic molecules, immune cell death inducers particularly, circumventing BBB hurdles and targeting GSCs at the tumor burden, the forefront of GBM recurrence.Moreover, our results demonstrate that TnTs are potentially useful as drug-delivery channels for cancer therapy, facilitating the intercellular redistribution of this drug in close and far away cells, thus reaching isolated tumour niches that are hardly targeted by simple drug diffusion in the brain parenchyma. The differences identified in TnTs formed by GBM cells and normal human astrocytes can be exploited to increase treatments precision and specificity.
RE, FRANCESCA
glioblastoma; nanomedicina; nanoparticelle; cervello; tumore
glioblastoma; nanomedicine; nanoparticles; brain; tumore
BIO/10 - BIOCHIMICA
English
21-gen-2020
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET - 76R
32
2018/2019
open
(2020). A nanomedicine approach for the treatment of glioblastoma multiforme. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2020).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/259340
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