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Shedding lights on cancer cells and their microenvironment : development of 3D in vitro tumor models to shorten the translation of nanomedicines from the bench to the bedside / Cellules tumorales et leur micro-environnement : développement de modèles 3D in vitro pour l’évaluation préclinique de nouveaux nanomédicamentsLazzari, Gianpiero 06 November 2018 (has links)
Au cours des dernières décennies, des systèmes de taille nanométrique chargés en principes actifs (nanomédicaments) et des nouvelles stratégies thérapeutiques ont été développés afin de surmonter les limitations liées à la chimiothérapie conventionnelle telles qu’une distribution non spécifique, une mauvaise accumulation dans les tissus cibles ainsi qu’une métabolisation rapide. Cependant, le succès des nouveaux médicaments en clinique reste encore limité et seulement un faible nombre de nanomédicaments est actuellement commercialisé.Une divergence entre les résultats précliniques in vitro et les performances obtenues in vivo est souvent observée dans la première étape du développement d'un médicament. Cet écart pourrait être attribué au manque de modèles pertinents, représentatifs de la pathologie observée chez l’Homme et qui soient de bons prédicteurs de la réponse thérapeutique chez les patients. En effet, les modèles utilisés aujourd’hui (généralement culture cellulaire en deux dimensions, 2D) ne reproduisent pas la structure complexe de la tumeur in vivo. Ainsi, ils ne permettent pas une évaluation fiable du potentiel thérapeutique réel des médicaments. Dans cette optique, les méthodologies de culture de cellules en trois dimensions (3D) sont extrêmement avantageuses. Ces méthodologies permettent, en effet, la construction de systèmes cellulaires pertinents qui reproduisent in vitro la relation entre les cellules cancéreuses et leur microenvironnement. Parmi ces modèles, l'assemblage de cellules sous forme de sphéroïdes multicellulaires a été largement exploré. Néanmoins, les sphéroïdes décrits jusqu'à présent correspondent à des nodules formés exclusivement de cellules cancéreuses, ce qui constitue une vraie limitation. En effet, ces sphéroïdes ne reproduisent pas l’organisation de la tumeur et l'hétérogénéité du microenvironnement, et par conséquent ils ne parviennent pas à mimer les multiples barrières biologiques que les médicaments doivent traverser pour atteindre les cellules cibles.Dans cet esprit, l'objectif de cette thèse de doctorat était de surmonter ces limitations et de construire des modèles pertinents qui reproduisent in vitro la relation entre les cellules cancéreuses et leur microenvironnement afin de i) mieux comprendre les mécanismes de passage des nanomédicaments et ii) mieux prédire l’efficacité des nouveaux traitements.Au cours de cette thèse nous nous sommes intéressés au cancer du pancréas qui est caractérisé par la présence d'un abondant stroma formant un bloc fibreux (réaction desmoplastique) qui limite la pénétration des médicaments et réduit ainsi leur efficacité. Cette tumeur représente donc un bon exemple de barrière biologique tumorale.La partie principale de ce travail de recherche repose sur la construction et la caractérisation complète d’un nouveau type de sphéroïde multicellulaire, capable de reproduire in vitro la relation entre les cellules cancéreuses et leur microenvironnement, grâce à la co-culture de cellules cancéreuses pancréatiques, de fibroblastes et de cellules endothéliales. Les études de cytotoxicité in vitro nous ont permis d’investiguer la capacité de ce modèle à reproduire la résistance des cellules cancéreuses aux traitements observés in vivo. Grâce à la Microscopie de Fluorescence à Feuillet de Lumière nous avons pu étudier la pénétration de la doxorubicine, soit en forme libre, soit encapsulée dans des nanoparticules, au sein des sphéroïdes. Ensuite, afin de mieux comprendre comment les médicaments et nanomédicaments interagissent avec la tumeur, nous avons cherché à combiner la culture 3D avec des conditions dynamiques contrôlées dans un dispositif microfluidique. Pour atteindre cet objectif, nous avons conçu et fabriqué une puce sur mesure, adaptée pour loger à la fois le sphéroïde et des canaux dans lesquels les cellules endothéliales pourront s’organiser sous forme de vaisseaux. / In the last decades, various engineered systems for drug delivery (i.e., nanomedicines) have been developed with the aim to overcome the limits associated to conventional chemotherapy, such as non-specific drug distribution, poor delivery to the target tissue and rapid metabolism. However, the success of new therapeutic strategies in the clinic is still suboptimal and only a limited number is currently marketed.A discrepancy between promising preclinical in vitro results and the in vivo performances is often observed in the early stage of drug development and might be ascribed to the lack of capacity of the models commonly used for in vitro studies to faithfully reproduce the pathophysiology of solid tumors. These models mainly consist of cancer cells cultured as flat (two dimensional, 2D) monolayers or assembled to form three dimensional (3D) multicellular tumor spheroids (MCTS).However, being composed exclusively of one cell type, these models are too simplistic. They do not allow to reproduce the heterogeneous cellular composition, as well as, the complex architecture of the tumor and its surrounding microenvironment. Thus, they fail to replicate the multiple biological barriers that drugs and nanomecidines have to cross in order to reach the target cells.The aim of this PhD thesis was to overcome these limitations and construct a reliable tool for an appropriate in vitro evaluation of the therapeutic potential of nanomedicines and other chemotherapies. Attention has been focused on the pancreatic ductal adenocarcinoma (PDAC) whose strong fibrotic reaction represents a well-known example of a tumor biological barrier responsible of the limited efficacy of the treatments. The main part of this research work relies on the construction and complete characterization of novel hetero-type MCTS based on a triple co-culture of pancreatic cancer cells, fibroblasts and endothelial cells, and thus capable to integrate the cancerous component and the microenvironment of the tumor. The constructed 3D model has demonstrated the capacity to reproduce in vitro the influence of the microenvironment on the sensitivity of cancer cells to chemotherapy. In addition, by combining the 3D model and the innovative Light Sheet Fluorescence Microscopy (LSFM), we have been able to investigate the penetration of the anticancer drug doxorubicin (in a free form and loaded into nanoparticles (NPs)) in a high informative manner. Then, in order to acquire a better understanding on how nanomedicines and other anticancer chemotherapies interact with the tumor, we sought to combine the hetero-type 3D culture with controlled flow conditions in a microfluidic device. To reach this goal we have designed and fabricated a tailor-made chip suitable to host both a MCTS and a perfusable microvascular network (i.e., MCTS-on-a-chip).
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Engineered microsystems and their application in the culture and characterization of three-dimensional (3D) breast tumor modelsMenon, Nidhi 26 May 2021 (has links)
Microsystems are a broad category of engineered technologies in the micro and nano scale
that have a diverse range of applications. They are emerging as a powerful tool in the field
of biomedical research, drug discovery, as well as clinical diagnostics and prognostics, especially
with regards to cancer. One of the major challenges in precision and personalized
medicine in cancer lies in the technical difficulties of ex-vivo cell culture and propagation
of the limited number of primary cells derived from patients. Therefore, our aims are to
1. Develop a biologically relevant platform for culturing cancer cells and characterize how
it influences the cell growth and phenotype compared to conventional 2-dimensional(2D)
cell culturing techniques, 2. Isolate secondary metabolites from endophytic fungi and screen
them on the platform for potential anticancer properties in a preliminary drug discovery
pipeline, 3. Design and develop biosensors for quantifying cell responses in real-time within
these systems.
Several biomaterial scaffolds with microscale architectures have been utilized for engineering
the tumor extracellular matrix, but very few studies have thoroughly characterized the
phenotypic changes in their cell models, which is critical for translational applications of biomaterial
systems. The overall objective of these studies is to engineer a biomimetic platform
for the culture of breast cancer cells in vitro and to quantify and profile their phenotypic
changes. In order to do this, we first evaluated a blank-slate matrix consisting of thiolated
collagen, hyaluronic acid and heparin, cross-linked chemically via Michael addition reaction
using diacrylate functionalized poly (ethylene glycol). The hydrogel network was used with
triple-negative breast cancer cells and showed significant changes in characteristics, with
cells self-assembling to form a 3D spheroid morphology, with higher viability, and exhibiting
significantly lower cell death upon chemotherapy treatment, as well as had a decrease in proliferation.
Furthemore, the transcriptomic changes quantified using RNA-Seq and Next-Gen
Sequencing showed the dramatic changes in some of the commonly targeted pathways in cancer
therapy. Furthermore, we were able to show the importance of our biomimetic platform
in the process of drug discovery using fungal endophytes and their secondary metabolites as
the source for potential anticancer molecules. Additionally, we developed gold nanoparticle
and antibody-based (ICAM1 and CD11b) sensors to quantify cell responses spatiotemporally
on our platform. We were able to show quenching of the green fluorescent fluorophores due
to the Förster Resonance Energy Transfer mechanism between the fluorophore and the gold
nanometal surface. We also observed antigen-dependent recovery of fluorescence and inhibition
of energy transfer upon the antibody binding to the cell-surface receptors. Future efforts
are directed towards incorporating the hydrogel system with antigen-dependent sensors in a
conceptually-designed microfluidic platform to spatiotemporally quantify the expression of
surface proteins in various cells of the tumor stroma. This includes the migration,infiltration,
and polarization of specific immune cells. This approach will provide further insight into the
heterogeneity of cells at the single-cell resolution in defined spaces within the 3D microfluidic
platform. / Doctor of Philosophy / Microsystems are a broad category of engineered technologies in the micro and nano scale
that have a diverse range of applications. They are emerging as a powerful tool in the field
of biomedical research, drug discovery, as well as clinical diagnostics and prognostics, especially
with regards to cancer. However, a major challenge in being able to offer personalized
medicine to cancer patients comes from the difficulty of growing cells from the patient's
tumor biopsy in a laboratory for further screening and analysis. There are also limited resources
available for real-time expression of proteins on cell-surfaces, that could be potential
biomarkers and targets for treatment.
Various natural and synthetic polymers are biocompatible and have been used widely in
engineering the tumor extracellular matrix. However, the effect of hydrogels derived from
these polymers on the specific tumor cells are not always well characterized. Our studies
explore the influence of a biohybrid hydrogel on breast cancer cells and our results show that
the microscale architecture of the hydrogel platform works as a suitable scaffold for recapitulating
the 3-dimensional(3D) breast tumor microenvironment, and can also be employed in
the drug discovery process. Additionally, we developed a nano-scale biosensor to enable the
quantification of specific cell-surface proteins in real-time. Ongoing and future efforts are focused
on designing and fabricating a microfluidic device with precise control over the design
of space and special chambers for cell culture. These will be used for studying interactions of
various cells in the tumor microenvironment that influence cancer progression. Integrating
these micro-scale systems, including sensors will allow researchers to quantify cell behavior
in response to the variable factors they are exposed to, as well as provide insight to answer
fundamental questions about cancer biology that are limited by the conventional 2D cell
culture systems.
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