<p dir="ltr">Live cell imaging is a method to optically investigate living cells using microscopy images. It plays an increasingly important role in biomedical research as well as drug development. In this thesis, we focus on label-free mammalian cell tracking and label-free abnormally shaped nuclei segmentation of microscopy images. We propose a method to use a precomputed velocity field to enhance cell tracking performance. Additionally, we propose an ensemble method, Weighted Mask Fusion (WMF), combining the results of multiple segmentation models with shape analysis, to improve the final nuclei segmentation mask. We also propose an edge-aware Mask RCNN and introduce a hybrid architecture, an ensemble of CNNs and Swin-Transformer Edge Mask R-CNNs (HER-CNN), to accurately segment irregularly shaped nuclei of microscopy images. Our experiments indicate that our proposed method outperforms other existing methods for cell tracking and abnormally shaped nuclei segmentation.</p><p dir="ltr">While image-based dietary assessment methods reduce the time and labor required for nutrient analysis, the major challenge with deep learning-based approaches is that the performance is heavily dependent on the quality of the datasets. Challenges with food data include suffering from high intra-class variance and class imbalance. In this thesis, we present an effective clustering-based training framework named ClusDiff for generating high-quality and representative food images. From experiments, we showcase our method’s effectiveness in enhancing food image generation. Additionally, we conduct a study on the utilization of synthetic food images to address the class imbalance issue in long-tailed food classification.</p>

Les maladies cardiovasculaires (MCV) sont la principale cause de mortalité dans les pays industrialisés. L'hypercholestérolémie constitue un facteur de risque majeur pour les MCV. Elle est caractérisée par des niveaux élevés de lipoprotéines de faible densité (LDL, aussi appelé “mauvais cholestérol”). La présence prolongée de haut niveaux de LDL dans la circulation augmente le risque de formation de plaques athérosclérotiques, ce qui peut conduire à l'obstruction des artères et l'infarctus du myocarde. Le LDL est normalement extrait du sang par sa liaison au récepteur du LDL (LDLR) qui est responsable de son endocytose dans les hépatocytes. Des études génétiques humaines ont identifié PCSK9 (proprotein convertase subtilisin/kexin type 9) comme le troisième locus responsable de l'hypercholestérolémie autosomique dominante après le LDLR et son ligand l’apolipoprotéine B-100. PCSK9 interagit avec le LDLR et induit sa dégradation, augmentant ainsi les niveaux plasmatiques de LDL. Les mutations gain de fonction (GF) de PCSK9 sont associées à des niveaux plasmatiques élevés de LDL et à l'apparition précoce des MCV, alors que les mutations perte de fonction (PF) de PCSK9 diminuent le risque de MCV jusqu’à ~ 88% grâce à une réduction du LDL circulant. De ce fait, PCSK9 constitue une cible pharmacologique importante pour réduire le risque de MCV. PCSK9 lie le LDLR à la surface cellulaire et/ou dans l'appareil de Golgi des hépatocytes et provoque sa dégradation dans les lysosomes par un mécanisme encore mal compris. Le but de cette étude est de déterminer pourquoi certaines mutations humaines de PCSK9 sont incapables de dégrader le LDLR tandis que d'autres augmentent sa dégradation dans les lysosomes. Plusieurs mutations GF et PF de PCSK9 ont été fusionnées à la protéine fluorecente mCherry dans le but d'étudier leur mobilité moléculaire dans les cellules hépatiques vivantes. Nos analyses quantitatives de recouvrement de fluorescence après photoblanchiment (FRAP) ont montré que les mutations GF (S127R et D129G) avaient une mobilité protéique plus élevée (> 35% par rapport au WT) dans le réseau trans- Golgien. En outre, nos analyses quantitatives de recouvrement de fluorescence inverse après photoblanchiment (iFRAP) ont montré que les mutations PF de PCSK9 (R46L) avaient une mobilité protéique plus lente (<22% par rapport au WT) et une fraction mobile beaucoup plus petite (<40% par rapport au WT). Par ailleurs, nos analyses de microscopie confocale et électronique démontrent pour la toute première fois que PCSK9 est localisée et concentrée dans le TGN des hépatocytes humains via son domaine Cterminal (CHRD) qui est essentiel à la dégradation du LDLR. De plus, nos analyses sur des cellules vivantes démontrent pour la première fois que le CHRD n'est pas nécessaire à l'internalisation de PCSK9. Ces résultats apportent de nouveaux éléments importants sur le mécanisme d'action de PCSK9 et pourront contribuer ultimement au développement d'inhibiteurs de la dégradation du LDLR induite par PCSK9. / Coronary heart diseases (CHD) are a leading cause of death in Western societies. Hypercholesterolemia is a major risk factor for CHD. It is characterized by high levels of circulating low-density lipoprotein cholesterol (LDL, also called "bad cholesterol"). The prolonged presence of elevated levels of LDL in the circulation increases the risk of formation of atherosclerotic plaques, which can lead to obstruction of arteries and myocardial infarction. LDL is normally cleared from the blood through the binding of its sole protein constituent apolipoprotein B100 to hepatic LDL receptor (LDLR), which mediates its endocytosis in the liver. Human genetic studies have identified PCSK9 as the third gene responsible of autosomal dominant hypercholesterolemia after LDLR and its ligand apolipoprotein B100. PCSK9 interacts with the LDLR and induces its degradation thereby causing plasma LDL levels to rise. PCSK9 gain-of-function (GOF) mutations are associated with elevated plasma LDL levels and premature CHD while PCSK9 loss-offunction (LOF) mutations reduce the risk of CHD up to ~88% owing to reduction of circulating LDL. Accordingly, PCSK9 is recognized as a major pharmacological target to lower the risk of CHD. PCSK9 binds the LDLR at the cell surface and/or in the Golgi apparatus of hepatocytes and causes its degradation in lysosomes by a mechanism not yet clearly understood. The goal of this study was to determine why some human PCSK9 mutations fail to induce LDLR degradation while others increase it in lysosomes. Several PCSK9 LOF and GOF mutations were fused to the fluorescent protein mCherry to study their molecular mobility in living human liver cells. Our quantitative analysis of fluorescence recovery after photobleaching (FRAP) showed that PCSK9 GOF mutations S127R and D129G have a higher protein mobility (>35% compared to WT) at the trans- Golgi network (TGN). Our quantitative analysis of inverse fluorescence recovery after photobleaching (iFRAP) showed that PCSK9 LOF mutation R46L presented a much slower protein mobility (<22% compared to WT) and a much slower mobile fraction (<40% compared to WT). In addition, our confocal and electron microscopy analyses demonstrate for the first time that PCSK9 is localized and concentrated at the TGN of human hepatocytes. Furthermore, our results demonstrate that PCSK9 localization in the TGN is mediated through its C-terminal cysteine and histidine-rich domain (CHRD), which is essential for LDLR degradation. Also, our live-cell analyses demonstrate for the first time that the CHRD is not required for internalization of PCSK9. These results provide important new information on the mechanism of action of PCSK9 and may ultimately help in the development of inhibitors of the PCSK9-induced LDLR degradation.

Le virus de l'hépatite C (HCV) infecte environ 180 millions de personnes à travers le monde. De nouveaux antiviraux ont récemment été mis sur le marché mais ils présentent des effets indésirables. La recherche de nouvelles cibles thérapeutiques reste donc d'actualité. Mes principaux travaux ont consisté à développer des approches de biochimie et d'imagerie sur cellules vivantes pour étudier les mécanismes moléculaires d'action des antiviraux silibinine (SbN) et arbidol (ARB) sur HCV. Nous avons montré que SbN et ARB inhibent des vésicules entourées de clathrine et ne sont pas délivrés aux endosomes précoces. SbN et ARB inhibent également l'infection d'autres virus entrant par endocytose clathrine-dépendante, ce qui expliquerait leur activité à large spectre. J'ai également contribué à un projet initié depuis quelques mois au sein de l'équipe. L'hypothèse était qu'un élément présent dans le microenvironnement hépatique (MEH) jouerait un rôle essentiel dans l'infection par HCV. Nous nous sommes intéressés au syndécan-1 (SDC1) car il est fortement exprimé à la surface des hépatocytes. Nos travaux montrent que la déplétion de SDC1 diminue fortement l'infection. SDC1 colocalise à la surface des hépatocytes non infectés avec CD81, un récepteur connu de HCV. Dans les jours suivant l'infection, cette colocalisation est perturbée. Ces données suggèrent que SDC1 serait un co-facteur d'entrée de HCV, agissant en combinaison avec CD81, et que l'infection réorganiserait les molécules du MEH, ce qui pourrait à long terme contribuer à la persistance de l'infection / Hepatitis C virus (HCV) is a global health concern infecting 170 million people worldwide. New antivirals recently received the approval for the treatment against HCV infection but they display many side effects. Research for new therapeutic targets therefore remains an important topic. My main work was to develop approaches in biochemistry and live cell imaging to study the molecular mechanisms of action of antivirals silibinin (SbN) and arbidol (ARB) on HCV infection. We show that SbN and ARB alter clathrin-mediated endocytosis. Viral particles are trapped in clathrin-positive structures and cannot be delivered to the early endosomal compartment, thereby preventing infection. SbN and ARB also prevent cell infection by viruses that enter through clathrin-mediated endocytosis, which could explain their broad spectrum activity. I also contribute to a project initiated for a few months in the lab. We hypothsized that a molecule present in the immediate surrouding of the hepatocyte microenvironment could play a role in HCV infection. We focused on the syndecan-1 (SDC1) because it is essentially anchored on the surface of hepatocytes. We show that SDC1 depletion leads to a drastic decrease of the viral infectivity. SDC1 colocalizes on the unfected hepatocyte surface with the already identified HCV recptor CD81. This colocalization vanished within days in infected cells. These data suggest that SDC1 could act as a cellular co-factor for HCV entry, in combination with CD81; thus infection could reorganized molecules of the hepatocyte microenvironment and contribute to HCV hepatotropism and the peristence of infection

L'utilisation de champs électriques pulsés (PEF) dans les secteurs de la médecine et de la biotechnologie est devenue de plus en plus courante au cours des dernières décennies. La recherche a montré qu'en ajustant la durée du PEF, nous pouvons prédire quels effets seront observés. Alors que les PEF dans la gamme micro - milliseconde ont été utilisés pour perméabiliser la membrane cellulaire et améliorer l'absorption de médicament ou de protéine, le PEF nanoseconde (nsPEF) a démontré des effets uniques sur les organites intracellulaires. Les deux PEF et nsPEF ont démontré un potentiel thérapeutique pour une variété de pathologies humaines, y compris le traitement du cancer. Utilisant l'imagerie des cellules vivantes, cette thèse a étudié in vitro les effets de champs pulsés d'une durée de 10 ns à 10 ms sur des lignées cancéreuses (U87 glioblastome multiforme) et non cancéreuses (neurones hippocampes de souris (HT22) et cellules ovariennes du hamster chinois (CHO)). Des résultats publiés antérieurement ont démontré que les cellules cancéreuses sont plus sensibles aux champs électriques que les cellules saines. Nos résultats sont en accord avec ces résultats, dans la mesure où les cellules U87 ont subi une dépolarisation significativement plus importante de leur potentiel transmembranaire après une seule impulsion électrique à toutes les durées. Dans un ensemble d'expériences parallèles, malgré des seuils de champ électrique similaires pour la perméabilisation membranaire, les cellules U87 ont démontré une absorption significativement améliorée de YO-PRO par rapport aux autres lignées cellulaires. Bien que les cellules U87 aient subi le plus grand changement dans la dépolarisation membranaire et la perméabilisation membranaire, elles ont également montré la constante de rescellement de la membrane la plus rapide, qui était environ 30 secondes plus rapide que les autres lignées cellulaires. Pour élucider certains des mécanismes sous-jacents par lesquels les cellules U87 répondent aux champs électriques, une série d'expériences a examiné le rôle des canaux ioniques transmembranaires. Plusieurs études récentes ont rapporté que les PEF peuvent agir directement sur les canaux ioniques voltage-dépendants. En utilisant divers modulateurs de canaux ioniques pharmacologiques spécifiques et à action large, nous avons démontré que nous pouvions presque entièrement inhiber la dépolarisation membranaire induite par le champ électrique dans les cellules U87 en bloquant certains canaux cationiques. Ces résultats étaient assez spécifiques, tels que le canal de potassium de grande conductance (BK), les canaux calciques de type L et T, et le canal cationique non spécifique, TRPM8, étaient capables d'inhiber la dépolarisation tandis que le blocage d'autres canaux ioniques ne produisait aucun changement significatif. . Les travaux de cette thèse ont montré que la lignée cellulaire maligne U87 présentait une plus grande sensibilité aux champs électriques allant de 10 ns à 10 ms par rapport aux lignées cellulaires non cancéreuses étudiées. Des améliorations potentielles aux protocoles de traitement actuels ont été proposées sur la base des résultats présentés ici. / The use of pulsed electric fields (PEF) in medical and biotechnology sectors has become increasingly prevalent over the last few decades. Research has shown that by adjusting the duration of the PEF we can predict what effects will be observed. Whereas PEF in the micro-to-millisecond range have been used to permeabilize the cell membrane and enhance drug or protein uptake, nanosecond PEF (nsPEF) have demonstrated unique effects on intracellular organelles. Both PEF and nsPEF have demonstrated therapeutic potential for a variety of human pathologies, including the treatment of cancer. Using live-cell imaging, this thesis investigated, in vitro, the effects of pulsed fields ranging in duration from 10 ns to 10 ms on cancerous (U87 glioblastoma multiforme) and non-cancerous cell lines (mouse hippocampal neurons (HT22) and Chinese hamster ovary (CHO) cells). Previously published results have demonstrated that cancerous cells have a greater sensitivity to applied electric fields than healthy cells do. Our results are in agreement with these findings, insofar as the U87 cells underwent a significantly greater depolarization of their transmembrane potential following a single electric pulse at all durations. In a parallel set of experiments, despite having similar electric field thresholds for membrane permeabilization, the U87 cells demonstrated significantly enhanced YO-PRO uptake compared to the other cells lines. Although U87 cells underwent the greatest change in both membrane depolarization and membrane permeabilization, they also showed the fastest membrane resealing constant, which was approximately 30 seconds faster than other cell lines. To elucidate some of the underlying mechanisms by which U87 cells respond to electric fields, a series of experiments looked at the role of transmembrane ion channels. Several recent studies have reported that PEFs can act directly on voltage-gated ion channels. Using a variety of specific and broad acting pharmacological ion channel modulators, we demonstrated that we could almost entirely inhibit the electric field-induced membrane depolarization in U87 cells by blocking certain cationic channels. These results were quite specific, such that the big conductance potassium (BK) channel, L- and T-type calcium channels, and the non-specific cationic channel, TRPM8, were able to inhibit depolarization while blocking other ion channels produced no significant change. The work in this thesis showed that the malignant U87 cell line showed a greater sensitivity to electric fields from ranging from 10 ns – 10 ms when compared to the non-cancerous cell lines that were investigated. Potential improvements to current treatment protocols have been proposed based on the findings presented herein.

Broad distribution and activity limit the utility of anti-cancer compounds by causing unacceptable systemic toxicity and narrow therapeutic indices. To improve tumour accumulation, drug-loaded macromolecular assemblies have been designed to replace conventional surfactant-based formulations. Their nanoscale size enhances tumour accumulation via hyperpermeable vasculature and reduced lymphatic drainage. Incorporating targeting ligands introduces cell specificity through receptor-specific binding and uptake, enabling drugs to reach intracellular targets. In this work, the targeting properties of polymer nanoparticle micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG) were verified using in vitro and in vivo models of breast cancer. To select a relevant mouse model, the vascular and lymphovascular properties of two tumour xenograft models were compared. Greater accumulation of a model nanocarrier was observed in orthotopic mammary fat pad (MFP) tumours than size matched ectopic subcutaneous tumours, suggesting that the organ environment influenced the underlying pathophysiology. Immunostaining revealed greater vascular thickness, density and size, and thinner basement membranes in MFP tumours, likely contributing to greater blood perfusion and vascular permeability. Based on these observations, MFP tumour-bearing mice were used to characterize the pharmacokinetics and biodistribution of a taxol drug, docetaxel, encapsulated in poly(TMCC-co-LA)-g-PEG nanoparticles. The nanoparticle formulation demonstrated longer docetaxel circulation in plasma compared to the conventional surfactant-based formulation. As a result, greater docetaxel retention was uniquely measured in tumour tissue, extending exposure of tumour cells to the active compound and suggesting potential for increased anti-cancer efficacy. Furthermore, active targeting of antibody-modified nanoparticles to live cells was shown to be selective and receptor-specific. Binding isotherms were used to quantify the impact of antibody density on binding strength. The equilibrium binding constant increased linearly with the average number of antibodies per particle, which is consistent with a single antibody-antigen interaction per particle. This mechanistic understanding enables binding behaviour to be adjusted in a predictive manner and guides rational nanoparticle design. These studies validate poly(TMCC-co-LA)-g-PEG nanoparticles as a platform for targeted delivery to cancer on both a tissue and cellular level, forming a compelling justification for further pre-clinical evaluation of this system for safety and efficacy in vivo.

Semiconductor quantum dots (QDs) have emerged as a new class of fluorescent probes and labeling agents for biological samples. QDs are bright, highly photostable and allow simultaneous excitation of multiple emissions. Owing to these properties, QDs hold exceptional promise in enabling intracellular biochemical studies and diagnosis with unprecedented sensitivity and accuracy. However, use of QD probes inside living cells remains a challenge due to difficulties in delivery of nanoparticles without causing aggregation and imaging single nanoparticles inside living cells. In this dissertation, a systematic approach to deliver, image and locate single QDs inside living cells is presented and the properties of molecular motor protein driven QD transport are studied. First, spectroscopic and imaging methods capable of differentiating single nanoparticles from the aggregates were developed. These technologies were validated by differentiating surface protein expression on viral particles and by enabling rapid counting of single biomolecules. Second, controlled delivery of single QDs into living cells is demonstrated. A surprising finding is that single QDs associate non-specifically with the dynein motor protein complex and are transported to the microtubule organizing center. Accurate localization and tracking of QDs inside cell cytoplasm revealed multiple dynein motor protein attachment resulting in increased velocity of the QDs. Further, spectrin molecule which is known to recruit dynein motor protein complex to phospholipid micelles was found to associate with the QDs. These results may serve as a benchmark for developing new QD surface coatings suitable for intracellular applications. Since, nanoparticles are similar in size to viral pathogens; better understanding of nanoparticle-cell interactions should also help engineer nanoparticle models to study virus-host cell interactions. (Contains AVI format multimedia files)

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Broad distribution and activity limit the utility of anti-cancer compounds by causing unacceptable systemic toxicity and narrow therapeutic indices. To improve tumour accumulation, drug-loaded macromolecular assemblies have been designed to replace conventional surfactant-based formulations. Their nanoscale size enhances tumour accumulation via hyperpermeable vasculature and reduced lymphatic drainage. Incorporating targeting ligands introduces cell specificity through receptor-specific binding and uptake, enabling drugs to reach intracellular targets. In this work, the targeting properties of polymer nanoparticle micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG) were verified using in vitro and in vivo models of breast cancer. To select a relevant mouse model, the vascular and lymphovascular properties of two tumour xenograft models were compared. Greater accumulation of a model nanocarrier was observed in orthotopic mammary fat pad (MFP) tumours than size matched ectopic subcutaneous tumours, suggesting that the organ environment influenced the underlying pathophysiology. Immunostaining revealed greater vascular thickness, density and size, and thinner basement membranes in MFP tumours, likely contributing to greater blood perfusion and vascular permeability. Based on these observations, MFP tumour-bearing mice were used to characterize the pharmacokinetics and biodistribution of a taxol drug, docetaxel, encapsulated in poly(TMCC-co-LA)-g-PEG nanoparticles. The nanoparticle formulation demonstrated longer docetaxel circulation in plasma compared to the conventional surfactant-based formulation. As a result, greater docetaxel retention was uniquely measured in tumour tissue, extending exposure of tumour cells to the active compound and suggesting potential for increased anti-cancer efficacy. Furthermore, active targeting of antibody-modified nanoparticles to live cells was shown to be selective and receptor-specific. Binding isotherms were used to quantify the impact of antibody density on binding strength. The equilibrium binding constant increased linearly with the average number of antibodies per particle, which is consistent with a single antibody-antigen interaction per particle. This mechanistic understanding enables binding behaviour to be adjusted in a predictive manner and guides rational nanoparticle design. These studies validate poly(TMCC-co-LA)-g-PEG nanoparticles as a platform for targeted delivery to cancer on both a tissue and cellular level, forming a compelling justification for further pre-clinical evaluation of this system for safety and efficacy in vivo.

Les impulsions de champ électrique nanoseconde de forte intensité (nsPEF) ont été proposées pour le traitement du cancer avec des effets secondaires minimes et peu susceptibles de conduire à une résistance de la tumeur au traitement. Le glioblastome multiforme (GBM) est un cancer du cerveau incurable montrant une résistance aux traitements actuels tels que la chirurgie, la radiothérapie et la chimiothérapie. Dans cette thèse, l'imagerie des cellules vivantes est utilisée pour étudier in vitro les effets de nsPEF sur une lignée de cellules de glioblastome humain (U87-MG), et pour évaluer la pertinence de l'utilisation des nsPEF en tant que nouveau traitement pour le GBM. En accord avec les résultats publiés précédemment, nous montrons que les cellules U87-MG répondent aux nsPEF avec une poration de la membrane plasmique, une augmentation rapide du calcium intracellulaire et une perte progressive du potentiel de membrane mitochondriale. De nouveaux résultats montrent que 100 impulsions de 10 ns délivrées à 44 kV/cm perturbent la dynamique de croissance des microtubules indépendamment du calcium et du gonflement, ces derniers étant connus pour provoquer la dépolymérisation des microtubules. La microscopie à super-résolution nous a permis de visualiser les flexions et ruptures de microtubules après l'application nsPEF suggérant un effet plus direct des impulsions. L'étude des nsPEF sur le calcium a également été menée via des indicateurs de calcium génétiquement encodés (GECIs) qui permettent une comparaison entre les GECIs et les indicateurs chimiques couramment utilisés. En utilisant le GECI GCaMP, le potentiel d'expression des GECIs dans des endroits subcellulaires spécifiques a permis de mettre en évidence une onde de calcium induite par l'application des nsPEF, grâce à une forme de GCaMP fixée à la membrane plasmatique. Ce phénomène, qui n'est pas habituel avec des indicateurs chimiques cytosoliques classiques en raison de la diffusion, permet de confirmer l'origine extracellulaire des pics de calcium post nsPEF. Cette thèse démontre que les nsPEF appliqués à des cellules U87-MG induisent plusieurs effets cellulaires majeurs et potentiellement destructeurs. La perturbation du réseau de microtubules par les nsPEF pourrait éventuellement être exploitée comme un antimitotique, administré localement, pour le traitement de GBM, avec des effets secondaires systémiques réduits et une faible résistance au traitement. / High powered, nanosecond duration pulsed electric fields (nsPEF) have been proposed as a minimal side-effect, electrical cancer therapy that is unlikely to result in tumour resistance. Glioblastoma multiforme (GBM) is an incurable brain cancer showing resistance to current treatments such as surgery, radiotherapy and chemotherapy. This thesis uses live-cell imaging to look in vitro at the effects of nsPEF on a human glioblastoma cell line (U87-MG) in a first step towards assessing its suitability as a novel treatment for GBM. In agreement with previously published results we show that U87-MG cells respond to nsPEF with plasma membrane poration, a rapid increase in intracellular calcium and a gradual loss of mitochondrial membrane potential. We present novel results showing that 100, 10 ns pulses delivered at 44 kV/cm disrupt microtubule growth dynamics in a way that is independent of calcium and swelling, both of which are known to cause microtubule depolymerisation. Super-resolution microscopy allowed us to visualise microtubules bending and breaking following nsPEF application suggesting a more direct effect of the pulse. We look also at the application of genetically encoded calcium indicators (GECIs) to nsPEF calcium studies making a comparison between GECIs and commonly used chemical indicators. Using the GECI GCaMP, we show the advantages of being able to express GECIs in specific subcellular locations by visualising an nsPEF induced calcium wave with a plasma membrane bound form of GCaMP. This event, which is not evident with classic cytosolic chemical indicators due to diffusion, helps confirm the extracellular origin of the post-nsPEF calcium spike. The work in this thesis demonstrates that nsPEF causes several major, and possibly destructive, cellular events when applied to U87-MG cells. The disruption of the microtubule network by nsPEF could potentially be exploited as a locally administered antimitotic, for GBM treatment, with reduced systemic side effects and lower occurrences of resistance.