Search

Bioelectrochemistry journal paper entitled "High-voltage 10ns delayed paired or bipolar pulses for in vitro bioelectric experiments" published online in September 2020 and to appear in Volume 137February 2021, 107648.

The effect of nsPEF on glioblastoma cancer-like stem cell transcription and protein expression paper accepted to presentation to the 2020 BIOEM conference

International audience

International audience; Glioblastoma multiforme (GBM) is a common and aggressive tumour of the glia affecting the central nervous system. It is highly invasive and resistant to all current treatments, with the median survival time for patients being 14.6 months. Current standard treatment includes surgical resection, chemotherapy and radiotherapy 1,2. Cancer stem-like cells (CSC) have been identified in GBM tumour masses 3 and their multipotentiality, ability to self-renew and resistance to chemotherapy make them a key target for novel GBM treatments.

International audience

International audience

Conference Paper entitled "Rhodamine B Temperature Dosimetry of Biological Samples Interacting with Electromagnetic Fields in Macrosystems" for the IEEE IMS symposium 11-15 June 2019, Boston, USA

International audience; This study looks at the application of genetically encoded calcium indicators (GECIs), to investigate the spatiotemporal nature of nsPEF-induced calcium signals, using fluorescent live cell imaging. GECIs can be targeted to different cellular locations and, by using a plasma membrane expressed GECI, we demonstrate calcium entry into the cell as a wave, initiated at the cathode side, following nsPEF treatment. This wave was masked by commercially available chemical, cytosolic, calcium indicators.

International audience; Carcinoma of the colon and rectum known as colorectal cancer (CRC) is a common cancer that causes around 700 000 deaths from CRC annually and more than 3.5 millions of patients living with it [1]. In addition to the liver, which is the most commonly affected organ, the peritoneum (the membrane that covers the entire abdominal cavity and viscera) is another common metastatic site. Indeed, peritoneal metastases are found in 10 to 25% of cases of CRC [2,3]. The localization remains only peritoneal in about 25% of these cases. Until recently, oncologists and digestive surgeons have considered peritoneal carcinomatosis (CPc, the implantation of tumor cells throughout the peritoneal cavity) as an incurable disease [4]. Currently, the only treatment for validated CPc is based on a complete cytoreduction associated with intraperitoneal hyperthermia chemotherapy (CHIP), supervised by systemic chemotherapy [5]. This treatment has improved the long-term survival of these highly selected patients with a median survival of between 22 and 60 months [6,7]. However, this curative surgical treatment is only available to patients with resectable CPc. The majority of patients with CPc is considered unresectable at the time of diagnosis [8] and will not be able to access this curative treatment despite systemic neoadjuvant chemotherapy. In addition, this surgical treatment is associated with a morbidity of 30% in these already fragile patients [9] and any complication may delay the introduction of post-operative systemic chemotherapy which is equally important in their management.It is for these reasons that it is imperative to work on therapeutic alternatives to treat these many patients with unresectable peritoneal carcinomatosis. High-intensity pulsed electric fields with nanosecond durations (3-300ns; nsPEFs) have emerged as a promising tool for tumor ablation [10-12]. The physical mechanisms and specific type of cell death that occurs following the application of nsPEFs is under investigation by several groups and initial studies have shown effects on cell signaling through calcium and cell death pathways [13-14], mitochondria [15] and cytoskeleton [17-19].In order to develop a new model of peritoneal colorectal cancer in mouse suitable for studies on nsPEF treatment, we grafted murine colon carcinoma CT-26 cells expressing luciferase in immunocompetent BALB-c mice by intravenous, subcutaneous, intraperitoneal or laparotomy injection and followed the tumoral growth by bioluminescence over 15 days post-grafting. In vitro CT-26 cells were analyzed by flow cytometry for their sensitivity to nsPEFs (survival, mitochondrial potential and permeabilization). In parallel, human and mice peritoneal tumoral tissue were observed by multiphoton microscopy in order to evaluate the tumor microenvironment.

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.; 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.

The effect of nsPEF on glioblastoma cancer-like stem cell transcription and protein expression paper accepted to presentation to the 2020 BIOEM conference