Yes / We report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it. / This work has been supported by National Research Program of Spain (DPI2011-28262-c04-01) and by the project "MICROANGIOTHECAN" (CIBERBBN, IMIBIC and SEOM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The traditional anticancer drugs are distributed in vivo through systemic blood circulation with a very small portion reaching the tumor site. Targeted drug delivery systems are developed in efforts to concentrate the drug molecules in the tissue of interest while reducing the drug distribution to healthy tissues to reduce the side effects. Liposomes are colloidal systems composed of amphiphilic molecules that assemble into vesicle structures in aqueous media. They are common carriers for targeted drug delivery with the advantages of low toxicity, low immunogenicity and the ability of encapsulating both lipophilic and hydrophilic drugs. Prior research indicated the advantages of triggered release in drug delivery systems. As a specific example, a series of trans-2-aminocyclohexanol based lipids (flipids) have been reported to illustrate a promising strategy to render pH-triggered drug delivery systems: pH-triggered conformational switch. Based on the foregoing, we hypothesize that incorporation of lipids with a pH-sensitive conformational switch and a long-saturated lipid tail can improve the anticancer activities of stealth liposomes. In this study, six new flipids with C-16 saturated hydrocarbon tails were designed. Such lipids were synthesized with high yields by introducing a catalyst (Copper (II) tetrafluoroborate) at a key step of the synthetic scheme. pH-sensitive liposomes (fliposomes) composed of flipids were prepared and loaded with the anticancer drug doxorubicin with high encapsulation efficiency. The physicochemical properties of doxorubicin-loaded fliposomes were characterized and their pH-dependent leakage were investigated. The results showed that among all groups fliposomes containing the C-16 trans-2-morpholylcyclohexanol-based flipid (Mor-C16) exhibited the largest increase of release as the pH dropped form pH 7.4 to 6.0, indicating its good potential of serving as a component in pH-triggered drug delivery systems. Three-dimensional multicellular spheroids (3D MCS) are self-assembled microscale tissue analogs in vitro. They better mimic the native and complex tumor microenvironment than the conventional two-dimensional cell culture systems. In this dissertation study, 3D MCS of six different human cancer cells were successfully cultured and their growing conditions were optimized to obtain 3D MCS of tight structure and reproducible size. The constructed 3D MCS carried heterogeneously distributed live and apoptotic cells as well as acidic inside pH based on confocal microscopic imaging studies. The penetration of doxorubicin-loaded Mor-C16 fliposomes into 3D MCS was imaged by confocal microscopy in comparison to doxorubicin-loaded non pH-sensitive liposomes and free doxorubicin. The anticancer activities of doxorubicin-loaded Mor-C16 fliposomes against 3D MCS of three different cell lines was also evaluated by cell viability. Both the fliposome and the non pH-sensitive liposome formulations more efficiently penetrated into two of the three types of 3D MCS compared to free doxorubicin after 4h drug exposure. However, doxorubicin-loaded Mor-C16 fliposome imposed higher cytotoxicity to all three types of 3D MCS compared to doxorubicin-loaded non pH-sensitive liposome over 72 h drug exposure. Taken together, we propose that fliposomes achieved superior activity against 3D MCS by efficient penetration into 3D MCS, followed by enhanced release of the anticancer drug doxorubicin.

La thérapie photodynamique (PDT) est un traitement alternatif à la chirurgie en oncologie utilisant un photosensibilisateur (PS), la lumière visible et l’oxygène moléculaire. La méta-tétra(hydroxyphényl)chlorine (mTHPC) est l’un des PS de deuxième génération les plus utilisés en clinique en raison de son absorption dans le rouge lointain et d’un rendement quantique en 1O2 élevé. De par sa nature hydrophobe, la mTHPC est partiellement agrégée dans la circulation sanguine diminuant sa biodistribution. Dans le but d’améliorer la sélectivité tumorale de la mTHPC, différentes stratégies de vectorisation ont été développées. La formulation liposomale de mTHPC non PEGylée (Foslip®) améliore la biodistribution ainsi que les propriétés pharmacocinétiques de la mTHPC. Cependant, une rapide destruction des liposomes en circulation ainsi qu’une rapide libération de la mTHPC sont des inconvénients majeurs. Une alternative possible est l’utilisation de vésicules extracellulaires (VE). Dérivées des cellules, les VE possèdent une stabilité naturelle dans la circulation sanguine et une capacité à transporter et délivrer leur contenu de manière spécifique aux cellules cancéreuses. Cette vectorisation est intéressante en PDT en raison d’une importante capacité d’encapsulation des porphyrines. Le but de cette étude était d’évaluer l’intérêt des VE en tant que nanovecteur de la mTHPC dans divers modèles précliniques comparé au Foslip®. Contrairement au Foslip®, l’intégrité membranaire des VE est conservée en présence de 20% de plasma. In vitro, les mTHPC-VE ont montré une internalisation cellulaire par un mécanisme actif d’endocytose. Dans un modèle cellulaire en 3D de sphéroïdes multicellulaires, les mTHPC-VE ont permis d’accroitre l’accumulation cellulaire, la diffusion au sein de ce modèle ainsi que l’efficacité PDT. In vivo, les mTHPC-VE apparaissent plus efficace au niveau PDT avec un retard de croissance tumorale significativement augmenté. En conclusion, l’intégration de la mTHPC au sein des VE améliore l’efficacité PDT dans les différents modèles d’étude. Le suivi des mTHPC-VE à l’aide d’un traceur radioactif chez la souris ainsi que l’étude du ciblage de la vascularisation tumorale seront étudiés dans la suite du travail / Photodynamic therapy (PDT) is an alternative treatment to surgery in oncology using photosensitizer (PS), light and oxygen. Meta-tetra(hydroxylphenyl)chlorin (mTHPC) is one of the most used PS in clinics due to its high absorption in the deep red and high 1O2 quantum yield. In order to improve the mTHPC tumor selectivity different attempts of nanovectorisation were conducted. Non-PEGylated liposomal mTHPC (Foslip®) increase biodistribution and pharmacokinetic properties. However, the rapid liposome destruction during circulation and rapid mTHPC release are obvious shortcomings. Alternatively, mTHPC vectorization could be realized by extracellular vesicles (EVs). Derived from the cell, EVs possess a natural stability in bloodstream and ability to transport and deliver cargo molecules into cancer cells. This formulation is interesting for PDT due to the ability to encapsulate porphyrins. The aim of the present study was to determine the interest of EVs as mTHPC nanocarriers in various preclinical models compared to Foslip®. In contrast to Foslip®, membrane integrity of mTHPC-EVs was conserved in 20% of plasma. In vitro, mTHPC-EVs showed cellular internalization by an active endocytosis mechanism. In a 3D model of spheroids, mTHPC-EVs have improved cellular uptake, better diffusion inside spheroid and increased PDT efficacy. In vivo, mTHPC-EVs appeared to be more potent in terms of PDT efficacy, with a tumor growth delay significantly higher. In conclusion, integration of mTHPC in EVs improves PDT efficacy in various preclinical models. The tracking of mTHPC-EVs using a radioactive tracer in xenografted rodents as well as the study of vascularization targeting will be studied in the next step of this work

Cell contractility is mainly imagined as a force dipole-like interaction based on actin stress fibers that pull on cellular adhesion sites. Here, we present a different type of contractility based on isotropic contractions within the actomyosin cortex. Measuring mechanosensitive cortical contractility of suspended cells among various cell lines allowed us to exclude effects caused by stress fibers. We found that epithelial cells display a higher cortical tension than mesenchymal cells, directly contrasting to stress fiber-mediated contractility. These two types of contractility can even be used to distinguish epithelial from mesenchymal cells. These findings from a single cell level correlate to the rearrangement effects of actomyosin cortices within cells assembled in multicellular aggregates. Epithelial cells form a collective contractile actin cortex surrounding multicellular aggregates and further generate a high surface tension reminiscent of tissue boundaries. Hence, we suggest this intercellular structure as to be crucial for epithelial tissue integrity. In contrast, mesenchymal cells do not form collective actomyosin cortices reducing multicellular cohesion and enabling cell escape from the aggregates.