Pauline BREGIGEON defends her PhD on October 10th, 2023.
Place : amphitheatre 201 in W1 building at Ecole Centrale Lyon, in Ecully
Jury :
Rapporteurs :
Marie-Pierre ROLS, Directrice de Recherche CNRS, IPBS Université de Toulouse
Vincent SENEZ, Directeur de Recherche CNRS, CANTHER Université de Lille
Examinateurs :
Lluis MIR, Directeur de Recherche CNRS, METSY Université Paris-Saclay
Tomás GARCIA-SANCHEZ, Docteur, BERG Universitat Pompeu Fabra
Invités:
Laure FRANQUEVILLE, Ingénieure de Recherche CNRS, Ampère
Frédéric PRAT, Professeur, AP-HP
Encadrement :
Christian VOLLAIRE, Professeur, Ampère, Directeur de thèse
Marie FRENEA-ROBIN, Professeur, Ampère, Co-encadrante de thèse
Charlotte RIVIERE, Maître de Conférences, ILM, Co-encadrante de thèse
Julien MARCHALOT, Maître de Conférences, Ampère, Co-encadrant de thèse
Abstract :
In the search for new cancer treatments, electrochemotherapy (ECT) has emerged as a solid alternative to conventional chemotherapy, reducing doses and hence side effects. This method is based on the phenomenon of reversible electroporation (EPN), which occurs when pulsed electric fields of controlled parameters are applied to cells, causing a temporary increase of their permeability. The growing interest in this type of treatment requires the development of reliable in vitro tumor models. Cell aggregates called spheroids have been identified as relevant, as they more closely mimic the structure of a micro-tissue, in which cells interact with each other, than cells grown in 2D. The most commonly used techniques to electroporate spheroids are first to fabricate them, using methods that do not always allow to obtain regular size, and then to introduce them into an EPN cuvette or electroporate them one by one with hand-held electrodes. However, these techniques can damage them and lead to inhomogeneous field distribution. Our approach, based on the use of a microsystem containing a microstructured hydrogel in which several hundred of spheroids are cultured, aims to overcome these drawbacks, since to our knowledge, there is no solution enabling culture of spheroids of controlled size, shape and location, their monitoring and their EPN in a single device.
After characterizing the microsystem from an electrical and fluidic point of view, a proof of concept for its use as an ECT treatment test platform was carried out, using bleomycin. The results, obtained by fluorescent labeling revealed by microscopy, are consistent with the literature, showing efficacy on cell mortality in just a few days only induced when combined with EPN.
In a second phase, research work was focused on the complexification of the tumor environment (TME) of spheroids with the addition of fibroblast cells, as part of the Inter-Carnot Impulse project aimed at improving the treatment of pancreatic cancer using physical methods. Indeed, this TME has a major impact in vivo on the resistance to cancer treatment, especially in the case of pancreatic cancer. Two anticancer drugs were compared: bleomycin and gemcitabine, a classic chemotherapy agent. The results show that the efficacy of the latter is increased when combined with EPN, although it is also weakly effective in the absence of EPN. Moreover, preliminary results seem to show a greater effect of the treatment on spheroids without fibroblasts, reflecting the reproduction of the impact of the TME, although further developments are still needed to make it even denser.
At the same time, the microsystem has been adapted for impedance measurement, enabling the monitoring of spheroids without observation, while characterizing their electrical properties. This should eventually enable the study of other applications such as electro-gene therapy, which requires the use of electrodes made of non-transparent materials. The results obtained show the possibility to monitor growth and the effect of chemical molecules permeabilizing cells with bio-impedance, as a first step towards monitoring the effect of EPN, using a low-conductivity medium, in order to improve measurement sensitivity. Future work will tackle the structuration of the electrodes with microelectrode arrays (MEA), in order to move from global impedance measurement to more precise individual monitoring.
Keywords: microsystem – electroporation – spheroid – electrochemotherapy – bio-impedance