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During the last decade, ultrasonic manipulation has matured into an important tool with a wide range of applications, from fundamental cell biological research to clinical and industrial implementations. The contactless nature of ultrasound makes it possible to manipulate living cells in a gentle way, e.g., for positioning, sorting, and aggregation. However, when manipulating cells using ultrasound, especially using high acoustic amplitudes, a great deal of heat can be generated. This constitutes a challenge, since the viability of cells is dependent on a stable physiological temperature around 37°C.      In this Thesis we present applications of ultrasonic manipulation of fluids, particles, and cells in temperature-controlled micrometer-sized devices fabricated using well established etching techniques, directly compatible with high-resolution fluorescence microscopy. Furthermore, we present ultrasonic manipulation in larger up to centimeter-sized devices optimized for fluid mixing and cell lysis. In the present work, two new ultrasonic manipulation platforms have been developed implementing temperature control. These platforms are much improved with increased performance and usability compared to previous platforms. Also, two new ultrasonic platforms utilizing low-frequency ultrasound for solubilization and cell lysis of microliter-volumed and milliliter-volumed samples have been designed and implemented.      We have applied ultrasound to synchronize the interaction between large numbers of immune, natural killer cells, and cancer cells to study the cytotoxic response, on a single cell level. A heterogeneity was found among the natural killer cell population, i.e., some cells displayed high cytotoxic response while others were dormant. Furthermore, we have used temperature-controlled ultrasound to form up to 100, in parallel, solid cancer HepG2 tumors in a glass-silicon multi-well microplate. Next, we investigated the immune cells cytotoxic response against the solid tumors. We found a correlation between the number of immune cells compared to the size of the tumor and the cytotoxic outcome, i.e., if the tumor could be defeated.             Finally, the effect of high acoustic pressure amplitudes in the MPa-range on cell viability has been studied in a newly developed platform optimized for long-term stable temperature control, independent on the applied ultrasound power. Lastly, we present two applications of ultrasonic fluid mixing and lysis of cells. One platform is optimized for small microliter-sized volumes in plastic disposable chips and another is optimized for large milliliter-sized volumes in plastic test tubes. The latter platform has been implemented for clinical sputum sample solubilization and cell lysis for genomic DNA extraction for subsequent pathogen detection / Ultraljudsmanipulering har under de senaste tio åren mognat och utvecklats till ett verktyg med ett brett användningsområde. Idag kan man finna applikationer inom allt från cellbiologisk grundforskning till industri samt sjukvård. Ultraljudsmanipuleringens kontaktlösa natur gör det till en varsam metod för att manipulera celler, till exempel inom positionering, sortering och aggregering. När ultraljud med hög amplitud används kan värmeutvecklingen, som är oundviklig, bli ett problem. För att kunna säkerställa hög cellviabilitet krävs temperaturkontroll som kan hålla en fysiologisk, stabil temperatur på 37°C.      I denna avhandling presenterar vi tillämpningar av temperaturkontrollerad ultraljudsmanipulering i mikrometerstora anordningar fabricerade med väletablerade etsningstekniker.  Dessa anordningar är optimerade för att vara fullt kompatibla med högupplöst fluorescensmikroskopi.  Vi demonstrerar även ultraljudsmanipulering i centimeterstora anordningar optimerade för omrörning och blandning av vätskor samt lysering av celler. Två nya plattformar för ultraljudsmanipulering med inbyggd temperaturkontroll har utvecklats. Dessa två plattformar erbjuder ökad prestanda, flexibilitet samt även användarvänlighet. Utöver dessa plattformar har ytterligare två anordningar för lågfrekvent ultraljudssolubilisering och cellysering av mikroliter- och milliliterstora prover konstruerats.      I denna avhandling har vi tillämpat ultraljud för att synkronisera interaktionen mellan populationer utav immunceller (natural killer-celler) och cancerceller för att på cellnivå studera det cytotoxiska gensvaret. Vi fann en heterogenitet hos immuncellspopulationen. Det manifesterade sig i en fördelning av immuncellerna, från celler med stort cytotoxiskt gensvar till inaktiva immunceller. Vi har dessutom använt temperaturkontrollerad ultrasljudsmanipulering för att skapa solida cancertumörer utav HepG2-cancerceller, upp till 100 stycken parallellt, i en multihåls-mikrotiterplatta bestående av glas och kisel. Med hjälp av dessa tumörer har vi studerat det cytotoxiska gensvaret från immuncellerna. Vi fann att förhållandet mellan antalet immunceller och storleken på tumören bestämde utfallet, det vill säga om tumören kunde bekämpas.      Vi presenterar dessutom effekten utav högamplitudsultraljudsexponering av cancerceller i en plattform speciellt designad för höga tryckamplituder med implementerad ultraljudseffektsoberoende temperaturkontroll. Slutligen presenterar vi två tillämpningar av ultraljud för vätskeblandning och cellysering. Den första tillämpningen är anpassad för små volymer i plastchip för engångsbruk och den andra är optimerad för större volymer i plastprovrör. Den senare tillämpningen är speciellt framtagen för ultraljudssolubilisering och cellysering utav kliniska sputumprover för att möjliggöra DNA-extrahering för detektion av smittämnen. / <p>QC 20150522</p>

O carcinoma epidermóide representa mais de 90% das neoplasias malignas de cabeça e pescoço, apresentando taxas elevadas de morbi-mortalidade, porém pouco se sabe sobre as vias de sinalização que estão envolvidas na progressão tumoral. Têm sido relatado na literatura, que alguns estímulos podem ativar a holoenzima PI3K que, por sua vez, desencadeia um processo que induz a fosforilação da proteína Akt (pAkt) que leva a ativação e a translocação do NF-B do citoplasma para o núcleo, onde ocorre a transcrição de genes envolvidos na proliferação e invasão celular. Assim, esse estudo analisou, através dos métodos de Imunofluorescência e Western Blot, a expressão das proteínas pAkt, NF-B e Ciclina D1 em três linhagens de células de carcinoma epidermóide de cabeça e pescoço (HN6, HN30 e HN31) submetidas a cultivo tridimensional e ensaio de invasão (gerando clones invasivos HN6.1, HN30.1 e HN31.1), ambos realizados com Matrigel®. O pAkt apresentou marcação citoplasmática e nuclear nas linhagens celulares HN6, HN30, HN6.1 e na HN30 submetida ao cultivo tridimensional, todavia, as linhagens celulares HN31, HN30.1, HN31.1 e HN6/HN31 cultivadas tridimensionalmente, apresentaram positividade predominantemente nuclear. No caso do NF-B, a localização foi marcadamente citoplasmática em todas as linhagens celulares cultivadas bi ou tridimensionalmente, porém, com exceção da HN31.1, o padrão de marcação foi citoplasmático e nuclear nos clones invasivos. Por fim, a Ciclina D1 exibiu imunopositividade apenas nuclear. A técnica de Western Blot mostrou diminuição nos níveis de expressão das proteínas analisadas quando as células foram cultivadas em Matrigel®, sendo, na maioria das vezes, essa redução estatisticamente significante, exceto no caso da linhagem celular HN6, que apresentou um aumento significativo de Ciclina D1. Os clones invasivos HN6.1 e HN30.1 exibiram elevação significativa dos níveis de expressão de pAkt e NF-B porém, a HN31.1 apresentou aumento de pAkt e discreta diminuição de NF-B, mas ambos os valores não estatisticamente significantes. Em relação à Ciclina D1, houve um aumento dos seus níveis em todas as linhagens invasivas, sendo que apenas na HN6.1 foi estatisticamente significante. Esses resultados mostraram que, nas linhagens celulares avaliadas, quando cultivadas em ambiente tridimensional, a significativa redução dos níveis de expressão de proteínas da via de sinalização PI3K ocorreu devido a uma possível fase adaptativa dessas células ao Matrigel® que seria seguida provavelmente por uma etapa de aumento do potencial proliferativo e posterior transdiferenciação de algumas células para ganho de fenótipo mais agressivo. Além disso, este estudo mostrou a participação da via de sinalização Akt/NF-B/Ciclina D1 no processo de invasão das células de carcinoma epidermóide de cabeça e pescoço (HN6.1 e HN30.1). / Squamous cell carcinoma represents more than 90% of the head and neck malignant tumors, with high mortality rates. Nevertheless, the knowledge about signaling pathways involved in tumor progression remains unclear. The relationship between pAkt and NF-B is well established in carcinogenesis. It is known that the activation of PI3K can be induced by some factors what leads to Akt phosphorilation (pAkt). This further event starts an activation cascade that induces NF-B translocation from the cytoplasm into the nucleus where the transcription of genes enrolled in cellular proliferation and invasion will be done. Therefore, this study analyzed the status of pAkt, NF-B and Cyclin D1 proteins by Immunofluorescence and Western Blot methods in three head and neck squamous cell carcinoma cell lines (HN6, HN30 and HN31) submitted to three-dimensional culture model and an in vitro invasion assay (invasive clones HN6.1, HN30.1 e HN31.1), both with Matrigel®. pAkt expression was detected in cytoplasm and nucleus in HN6, HN30, HN6.1 and HN30 cultured with Matrigel®, however, HN31, HN30.1, HN31.1 cell lines and HN6/HN31 submitted to three-dimensional culture model showed nuclear expression. NF-B localization was strongly cytoplasmatic in all cell lines cultured with or without Matrigel®, but, regarding HN31.1, NF-B protein expression was nuclear and cytoplasmatic in the invasive clones. Cyclin D1 was nuclear in all cell lines analyzed. Western blot assays showed a decrease in pAkt, NF-B and Cyclin D1 expression levels in all cells lines in three-dimensional culture model, most of them statistically significant, but It was detected a considerable increase in Cyclin D1 expression in HN6 cultured in threedimensional model. Moreover, HN6.1 and HN30.1 showed a significant enhance in pAkt and NF-B levels but, HN31.1 demonstrated a raise in pAkt and a slight decline in NF-B, both were not statistically significant. Finally, an increase in Cyclin D1 expression levels was illustrated in all cell lines but, only HN6.1 showed a statistic signal. These results suggest that, in the cell lines studied, when cultured in threedimensional model, a noteworthy reduction in expression levels of PI3K signaling pathway proteins happened for the reason that a possibly adaptation phase of these cells to Matrigel® was occurring, in the first moment, but it would be probably followed by an increase proliferative potential stage and subsequent transdifferentiation of some cells that could show, consequently, a more aggressive phenotype. At last, this study revealed the participation of Akt/NF-B/Cyclin D1 signaling pathway in invasion process of head and neck squamous cell carcinoma (HN6.1 and HN30.1).

O carcinoma epidermóide representa mais de 90% das neoplasias malignas de cabeça e pescoço, apresentando taxas elevadas de morbi-mortalidade, porém pouco se sabe sobre as vias de sinalização que estão envolvidas na progressão tumoral. Têm sido relatado na literatura, que alguns estímulos podem ativar a holoenzima PI3K que, por sua vez, desencadeia um processo que induz a fosforilação da proteína Akt (pAkt) que leva a ativação e a translocação do NF-B do citoplasma para o núcleo, onde ocorre a transcrição de genes envolvidos na proliferação e invasão celular. Assim, esse estudo analisou, através dos métodos de Imunofluorescência e Western Blot, a expressão das proteínas pAkt, NF-B e Ciclina D1 em três linhagens de células de carcinoma epidermóide de cabeça e pescoço (HN6, HN30 e HN31) submetidas a cultivo tridimensional e ensaio de invasão (gerando clones invasivos HN6.1, HN30.1 e HN31.1), ambos realizados com Matrigel®. O pAkt apresentou marcação citoplasmática e nuclear nas linhagens celulares HN6, HN30, HN6.1 e na HN30 submetida ao cultivo tridimensional, todavia, as linhagens celulares HN31, HN30.1, HN31.1 e HN6/HN31 cultivadas tridimensionalmente, apresentaram positividade predominantemente nuclear. No caso do NF-B, a localização foi marcadamente citoplasmática em todas as linhagens celulares cultivadas bi ou tridimensionalmente, porém, com exceção da HN31.1, o padrão de marcação foi citoplasmático e nuclear nos clones invasivos. Por fim, a Ciclina D1 exibiu imunopositividade apenas nuclear. A técnica de Western Blot mostrou diminuição nos níveis de expressão das proteínas analisadas quando as células foram cultivadas em Matrigel®, sendo, na maioria das vezes, essa redução estatisticamente significante, exceto no caso da linhagem celular HN6, que apresentou um aumento significativo de Ciclina D1. Os clones invasivos HN6.1 e HN30.1 exibiram elevação significativa dos níveis de expressão de pAkt e NF-B porém, a HN31.1 apresentou aumento de pAkt e discreta diminuição de NF-B, mas ambos os valores não estatisticamente significantes. Em relação à Ciclina D1, houve um aumento dos seus níveis em todas as linhagens invasivas, sendo que apenas na HN6.1 foi estatisticamente significante. Esses resultados mostraram que, nas linhagens celulares avaliadas, quando cultivadas em ambiente tridimensional, a significativa redução dos níveis de expressão de proteínas da via de sinalização PI3K ocorreu devido a uma possível fase adaptativa dessas células ao Matrigel® que seria seguida provavelmente por uma etapa de aumento do potencial proliferativo e posterior transdiferenciação de algumas células para ganho de fenótipo mais agressivo. Além disso, este estudo mostrou a participação da via de sinalização Akt/NF-B/Ciclina D1 no processo de invasão das células de carcinoma epidermóide de cabeça e pescoço (HN6.1 e HN30.1). / Squamous cell carcinoma represents more than 90% of the head and neck malignant tumors, with high mortality rates. Nevertheless, the knowledge about signaling pathways involved in tumor progression remains unclear. The relationship between pAkt and NF-B is well established in carcinogenesis. It is known that the activation of PI3K can be induced by some factors what leads to Akt phosphorilation (pAkt). This further event starts an activation cascade that induces NF-B translocation from the cytoplasm into the nucleus where the transcription of genes enrolled in cellular proliferation and invasion will be done. Therefore, this study analyzed the status of pAkt, NF-B and Cyclin D1 proteins by Immunofluorescence and Western Blot methods in three head and neck squamous cell carcinoma cell lines (HN6, HN30 and HN31) submitted to three-dimensional culture model and an in vitro invasion assay (invasive clones HN6.1, HN30.1 e HN31.1), both with Matrigel®. pAkt expression was detected in cytoplasm and nucleus in HN6, HN30, HN6.1 and HN30 cultured with Matrigel®, however, HN31, HN30.1, HN31.1 cell lines and HN6/HN31 submitted to three-dimensional culture model showed nuclear expression. NF-B localization was strongly cytoplasmatic in all cell lines cultured with or without Matrigel®, but, regarding HN31.1, NF-B protein expression was nuclear and cytoplasmatic in the invasive clones. Cyclin D1 was nuclear in all cell lines analyzed. Western blot assays showed a decrease in pAkt, NF-B and Cyclin D1 expression levels in all cells lines in three-dimensional culture model, most of them statistically significant, but It was detected a considerable increase in Cyclin D1 expression in HN6 cultured in threedimensional model. Moreover, HN6.1 and HN30.1 showed a significant enhance in pAkt and NF-B levels but, HN31.1 demonstrated a raise in pAkt and a slight decline in NF-B, both were not statistically significant. Finally, an increase in Cyclin D1 expression levels was illustrated in all cell lines but, only HN6.1 showed a statistic signal. These results suggest that, in the cell lines studied, when cultured in threedimensional model, a noteworthy reduction in expression levels of PI3K signaling pathway proteins happened for the reason that a possibly adaptation phase of these cells to Matrigel® was occurring, in the first moment, but it would be probably followed by an increase proliferative potential stage and subsequent transdifferentiation of some cells that could show, consequently, a more aggressive phenotype. At last, this study revealed the participation of Akt/NF-B/Cyclin D1 signaling pathway in invasion process of head and neck squamous cell carcinoma (HN6.1 and HN30.1).

The majority of cancer research projects mainly focus on the epithelial cancer cell, while the role of the tumour stroma has been largely neglected. Conventional 2D techniques, such as well plates and other kinds of tissue culture plastic, and animal models are mainly used to broaden our understanding of how tumours arise, develop, and induce metastasis. However, there is accumulating evidence suggesting a tremendous impact of the non‐cancerous tumour stroma on carcinogenesis, while other publications illustrate the great importance of advanced 3D in vitro models for cancer research. The overall goal of this work was to investigate how cancer associated fibroblasts (CAFs; the most abundant component in the tumour stroma) and normal prostate fibroblasts (NPFs), isolated from patients diagnosed with aggressive forms of prostate cancer, contribute to angiogenesis, an important hallmark of cancer progression. For this purpose, a 3D in vitro angiogenesis co‐culture model was established. At first, two (semi‐) synthetic hydrogel platforms, gelatine methacrylate (GelMA) and star‐shaped (star)PEG‐heparin hydrogels were characterised and their physicochemical properties were compared with each other. Interestingly, GelMA gels shrank while starPEG‐heparin gels swelled in cell culture medium over the course of 24 hours. The cell concentration, in addition to the stiffness, was critical for the formation of endothelial networks, and the knowledge of swelling behaviour enabled the adjustment of initial cell density to ensure the density between both gel types was comparable. Moreover, preliminary tests with mesenchymal stem cells demonstrated that the hydrogel can be actively remodelled, as evaluated by stiffness parameters at day one and seven of incubation. Growth factors (GFs) affect cellular fate and behaviour, and storage, presentation and administration of such chemokines can be critical for certain cellular applications. Due to the high anionic charge density of heparin, starPEG‐heparin hydrogels are known to reversibly immobilise several GFs and thereby might mimic the GF reservoir of the extra cellular matrix. Thus, transport processes of GFs with low and high heparin affinity inside these hydrogels were analysed by fluorescence correlation spectroscopy and a bulk diffusion approach. Results indicated that diffusion constants were synergistically decreased with increasing size and heparin affinity of the diffusant. Next, the capability of endothelial cells (ECs) to self‐assemble and organise into 3D capillary networks was tested in GelMA, starPEG‐heparin and Matrigel hydrogels. Only starPEG‐heparin hydrogels allowed the formation of interconnected capillaries in macroscopic hydrogel samples. However, as it is widely used to test for pro‐ and anti‐angiogenic agents, the 2D Matrigel angiogenesis assay was included for subsequent co‐culture experiments of ECs and fibroblasts in order to investigate how the stromal cells influence the formation of endothelial networks. For a detailed characterisation of 3D structures, a conventionally applied 2D method (Maximum Intensity Projection for 3D reconstructed images, MIP) was compared to an optimised 3D analysing tool. As a result, it was discovered that MIP analysis did not allow for an accurate determination of 3D endothelial network parameters, and can result in misleading interpretations of the data set. Indirect co‐cultures of hydrogel‐embedded ECs with a 2D layer of fibroblasts showed that fibroblast‐derived soluble factors, including stromal cell‐derived factor 1 and interleukin 8, affected endothelial network properties. However, only co‐encapsulation of ECs and fibroblasts in starPEG‐heparin hydrogel discs revealed remarkable changes in endothelial network parameters between CAF and NPF samples. In detail, the total length and branching of the capillaries was increased. For two donor pairs, the diameter of capillaries was decreased in CAF samples compared to NPF samples, underlining the high physiological relevance of this model. In contrast, significant differences in 2D Matrigel assays were not detected between, CAF, NPF and control (ECs only) samples. In summary, a 3D angiogenesis co‐culture system was successfully developed and used to characterise stromal‐endothelial interactions in detail. The combination of advanced biomaterials (starPEG‐heparin) and 3D analysing techniques goes beyond conventional 2D in vitro cancer research, and opens new avenues for the development of more complex models to further improve the acquisition of more biologically relevant data.

Le neuroblastome pédiatrique à haut risque est incurable malgré l’intensification des traitements. Chez le patient, les cellules de neuroblastome échappent à l’activité anticancéreuse des cellules immunitaires Natural Killer (NK). Or, lorsque cultivées in vitro en monocouche (2D), les cellules de neuroblastomes redeviennent sensibles à l’activité cytotoxique des cellules NK ce qui ne reflètent pas leur résistance dans les tumeurs in situ. Nous faisons l'hypothèse que lorsque cultivées en 3D sous forme de sphéroïdes, les cellules de neuroblastome pourraient retrouver certaines caractéristiques qui les rendraient plus représentatives des tumeurs in situ au niveau immunologique. Ainsi, un tel modèle préclinique pourrait mieux refléter la résistance aux cellules NK et servir de modèle de criblage pour la découverte de médicaments potentialisant la cytotoxicité des cellules NK. Pour répondre à cette question, nous avons développé un système de culture cellulaire 3D utilisant plusieurs lignées cellulaires de neuroblastome. À ce système, une co-culture en 3D avec une lignée de cellules Natural Killer (NK92) a été mise en place. Nous avons mis en évidence que les sphéroïdes de neuroblastome présentent des changements d’expression de certains gènes qui sont retrouvées chez les patients ainsi qu’une plus grande résistance à l’activité cytotoxique des cellules NK92 en comparaison avec les lignées en monocouche. Les co cultures de sphéroïdes ont été exposées à des inhibiteurs de protéines impliquées à différents niveaux de l’épigénome afin de découvrir des médicaments qui sensibiliseraient les cellules de neuroblastome à l’activité cytotoxique des NK92. Une différence dans la sensibilité aux médicaments entre les sphéroïdes et les cellules en 2D ainsi qu’en monoculture ou en co-culture a été observée et certains composés ont été identifiés en vue de potentialiser l’activité des cellules NK92. Ainsi, nos études ont permis de mieux comprendre les mécanismes impliqués dans la résistance des cellules du neuroblastome à l’activité cytotoxique des cellules NK dans un modèle plus représentatif de la tumeur in situ. / High-risk pediatric neuroblastoma remains incurable despite intensified treatments. In patients, neuroblastoma cells evade the anti-cancer activity of Natural Killer (NK) immune cells. However, when cultured in vitro in a monolayer (2D), neuroblastoma cells become sensitive to the cytotoxic activity of NK cells, which does not reflect their resistance in tumors in situ. We hypothesize that when cultured in 3D in the form of spheroids, neuroblastoma cells could regain certain characteristics that would make them representative of tumors in situ at the immunological level. Thus, such a preclinical model could better reflect NK cell resistance and serve as a screening model for drug discovery to discover a treatment that can potentiate NK cell cytotoxicity. To answer this question, we developed a 3D cell culture system using several neuroblastoma cell lines. To this system, a 3D coculture model with a Natural Killer (NK92) cell line was set up. We have shown that neuroblastoma spheroids develop changes in the expression of certain genes that are found in patients as well as greater resistance to NK92 cells compared to monolayer cell lines. Spheroid cocultures were exposed to inhibitors of proteins involved at different levels of the epigenome to discover drugs that would sensitize neuroblastoma cells to the cytotoxic activity of NK92. A difference in drug sensitivity between spheroids and cells in 2D as well as in monoculture or coculture was observed and some compounds were identified to potentiate the activity of NK92 cells. Thus, our studies have provided a better understanding of the mechanisms involved in the resistance of neuroblastoma cells to the cytotoxic activity of NK cells in a more representative model of the tumor in situ.

Le cancer du sein est une maladie complexe et difficile à caractériser. Parmi les différents sous-types moléculaires, les tumeurs du sein Triple-Négatives (TN) sont particulièrement agressives et de mauvais pronostic. Elles sont caractérisées par une absence d’expression des récepteurs aux œstrogènes (ER), à la progestérone (PR), l’absence de surexpression du récepteur Human Epidermal growth factor 2 (HER2) et de fréquentes mutations sur les gènes BRCA1/2 (profil « BRCAness »). En absence de thérapies ciblées efficaces, de nombreux traitements ciblés notamment les inhibiteurs de poly-ADP-ribose polymérases (anti-PARPs) sont actuellement en cours de développement, en recherche préclinique et clinique. Basés sur le principe de létalité synthétique, les anti-PARPs ciblent les propriétés BRCAness des tumeurs TN. Dans ce contexte, ces travaux de recherche ont été orientés sur le développement d’outils diagnostics afin d’optimiser l’efficacité des anti-PARPs sur des tumeurs TN. Pour ce faire, dans un premier temps, des cultures cellulaires en 3D via la technique Liquid Overlay ainsi que des tests de cytotoxicités associés ont été développés, à partir des lignées cellulaires MDA-MB-231 et SUM1315 de phénotype TN. Ces deux modèles de sphéroïdes ont ensuite été optimisés/normalisés dans un milieu de culture synthétique intitulé OPTIPASS (BIOPASS). Dans un deuxième temps, l’efficacité d’un co-traitement combinant l’anti-PARP1 Olaparib à faibles et à fortes doses et la radiothérapie fractionnée (5x2 Gy) a été modélisée sur les deux lignées MDA-MB-231 et SUM1315, en conditions 2D et 3D. Ces expériences ont clairement mis en évidence un effet potentialisateur de l’Olaparib sur la radiothérapie (i) en présence de faibles doses de cet anti-PARP (5 µM ou inférieur) (ii) à long terme et (iii) en présence d’un fractionnement maximum (5x2 Gy). De plus, les lignées tumorales TN étudiées présentaient des différences de sensibilité vis-à-vis du co-traitement. Ainsi, une analyse transcriptomique in silico a mis en évidence des profils très différents de ces lignées hautement métastatiques et très agressives. Notamment, la lignée SUM1315 semblait présenter un engagement neuronal, suggérant son origine métastatique cérébrale. Ces résultats encourageants pourraient ouvrir de nouvelles perspectives pour le traitement des métastases cérébrales de tumeurs mammaires TN, très fréquentes chez ce sous-type. Dans un troisième temps, afin de mieux caractériser le mode d’action de l’Olaparib sur ces modèles de sphéroïdes, un dérivé fluorescent de l’Olaparib, l’Ola-FL, a été synthétisé et caractérisé. L’analyse de la pénétration et de la distribution de l’Ola-FL au sein des sphéroïdes MDA-MB-231 et SUM1315 a mis en évidence une distribution rapide et homogène du composé ainsi que sa persistance après 3h d’incubation, dans toute la profondeur des sphéroïdes et notamment dans les zones hypoxiques centrales. Enfin, l’analyse de la co-expression de deux pompes Multidrug Resistance (MDR) majeures, la MRP7 et la P-gp après le traitement des deux lignées TN avec l’Olaparib, a mis en évidence sur les cultures 2D, une expression de type relai de la MRP7 et la P-gp. Sur les sphéroïdes traités avec une faible dose d’Olaparib à long terme, une expression basale de la MRP7 et une surexpression de la P-gp ont été détectées, au sein des cellules résiduelles périphériques des sphéroïdes. Ces résultats mettent clairement en évidence l’implication des pompes d’efflux dans les mécanismes de résistances à l’Olaparib, dans ces tumeurs agressives. L’ensemble des résultats issus de la modélisation de l’action de l’Olaparib sur des sphéroïdes MDA-MB-231 et SUM1315 laissent supposer sa plus grande efficacité à faible dose et à long-terme, notamment dans les zones hypoxiques des sphéroïdes, probablement aussi à l’origine de son effet potentialisateur avec la radiothérapie. / Breast cancer is a very complex and heterogeneous disease. Among the different molecular subtypes, Triple-Negative (TN) breast cancers are particularly aggressive and of poor prognosis. TN tumours are characterized by a lack of estrogen receptors expression (ER), progesterone receptors expression (PR), the absence of Human Epidermal growth factor receptor 2 overexpression (HER2) of the frequent mutations on BRCA1 / 2 genes ("BRCAness" phenotype). In the absence of effective targeted therapies, many targeted therapies including poly-ADP-ribose polymerase inhibitors (anti-PARPs) are currently under development in preclinical and clinical studies. Based on the synthetic lethality concept, the anti-PARPs specifically target the BRCAness properties of TN tumors. In this context, these works were focused on the development of diagnostic tools for the optimization of TN tumours treatment with anti-PARPs. For this, firstly, 3D cell cultures formed with the Liquid Overlay technique as well as associated cytotoxicity tests were developed, from the TN breast cancer cell lines MDA-MB-231 and SUM1315. These two spheroid models were then optimized and standardized in a synthetic culture medium called OPTIPASS (BIOPASS). Secondly, the efficacy of a co-treatment combining anti-PARP1 Olaparib at low and high doses and fractioned radiotherapy (5x2 Gy) was analyzed on the two cell lines MDA-MB-231 and SUM1315 cultured in 2D and 3D conditions. These experiments clearly demonstrated a potentiating effect of Olaparib on radiotherapy (i) in presence of low doses of this anti-PARP (5 μM or inferior) (ii) at long term and (iii) in presence of the maximum fractionation (5x2 Gy). In addition, these two TN cell lines showed a heterogeneous sensitivity to the co-treatment. Thus, an in silico transcriptomic analysis revealed very different profiles of these highly metastatic and highly aggressive cell lines. Notably, the SUM1315 cell line presented a neuronal commitment, suggesting its cerebral metastatic origin. These promising results could open up new perspectives for the treatment of TN tumours brain metastases, which are very common in this subtype. Thirdly, in order to better characterize the mode of action of Olaparib on these spheroid models, a fluorescent derivative of Olaparib, Ola-FL, was synthesized and characterized. The analysis of Ola-FL penetration and distribution in MDA-MB-231 and SUM1315 spheroids showed a rapid and homogeneous distribution of the compound as well as its persistence after 3h of incubation, in all the depth of the spheroids and especially in the central hypoxic zones. Finally, the analysis of the co-expression of two major Multidrug Resistance (MDR) pumps, MRP7 and P-gp after the treatment of the two TN lines with Olaparib, revealed on 2D cultures, a relay type expression of the MRP7 and the P-gp. On spheroids treated with a low dose of Olaparib art long term (10 days), a basal expression of MRP7 and an overexpression of P-gp were detected in the peripheral residual cells of the spheroids. These results clearly highlighted the involvement of these efflux pumps in Olaparib resistance mechanisms, in these aggressive tumors. All the results resulting from the modeling of the action of Olaparib on MDA-MB-231 and SUM1315 spheroids suggest its greater efficacy at low dose and at long-term, especially in the hypoxic zones of the spheroids. This parameter might be probably at the origin of its potentiating effect with radiotherapy.

Despite the possible common histopathological features at diagnosis, cancer cells present within breast carcinomas are highly heterogeneous in their molecular signatures. This heterogeneity is responsible for disparate clinical behaviors, treatment responses and long-term outcomes in breast cancer patients. Although the few histopathological markers can partially describe the diversity of cells found in tumor tissue sections, the full molecular characterization of individual cancer cells is currently impossible in routine clinical practice. In this respect, Fourier transform infrared (FTIR) microspectroscopic imaging of histological sections allows obtaining, for each pixel of tissue images, hundreds of independent potential markers, which makes this technique a particularly powerful tool to distinguish cell types and subtypes. As a complement to the conventional clinicopathological evaluation, this spectroscopic approach has the potential to directly reveal molecular descriptors that should allow identifying different clonal lineages found within a single tumor and therefore provide knowledge relevant to diagnosis, prognosis and treatment personalization. Yet, interpretation of infrared (IR) spectra acquired on tissue sections requires a well-established calibration, which is currently missing. Conventionally, mammary epithelial cells are studied in vitro as adherent two-dimensional (2D) monolayers, which lead to the alteration of cell-microenvironmental interplay and consequently to the loss of tissue structure and function. A number of key in vivo-like interactions may be re-established with the use of three-dimensional (3D) laminin-rich extracellular matrix (lrECM)-based culture systems. The aim of this thesis is to investigate by FTIR imaging the influence of the in vitro growth environment (2D culture versus 3D lrECM culture and 3D monoculture versus 3D co-culture with fibroblasts) on a series of thirteen well-characterized human breast cancer cell lines and to determine culture conditions generating spectral phenotypes that are closer to the ones observed in malignant breast tissues. The reference cell lines cultured in a physiologically relevant basement membrane model and having undergone formalin fixation, paraffin embedding (FFPE), a routine treatment used to preserve clinical tissue specimens, could contribute to the construction of a spectral database. The latter could be ultimately employed as a valuable tool to interpret IR spectra of cells present in tumor tissue sections, particularly through the recognition of unique spectral markers.To achieve the goal, we developed and optimized, in a first step, the preparation of samples derived from traditional 2D and 3D lrECM cell cultures in order to preserve their morphological and molecular relevance for FTIR microspectroscopic analysis. We then highlighted the importance of the influence of the growth environment on the cellular phenotype by comparing spectra of 2D- and 3D-cultured breast cancer cell lines between them. A particular focus was placed to establish a correlation between FTIR spectral data and publicly available microarray-based gene expression patterns of the whole series of breast cancer cell lines grown in 2D and 3D lrECM cultures. Our results revealed that, although based on completely different principles, gene expression profiling and FTIR spectroscopy are similarly sensitive to both the cell line identity and the phenotypes induced by cell culture conditions. We also identified by FTIR imaging changes in the chemical content occurring in the microenvironment surrounding cell spheroids grown in 3D lrECM culture model. Finally, we illustrated the impact of the in vivo-like microenvironment on the IR spectra of breast cancer cell lines grown in 3D lrECM co-culture with fibroblasts and compared them with spectra of cell lines grown in 3D lrECM monoculture. Unsupervised statistical data analyses reported that cells grown in 3D co-cultures produce spectral phenotypes similar to the ones observed in FFPE tumor tissue sections from breast carcinoma patients. Altogether, our results suggest that FFPE samples prepared from 3D lrECM cultures of breast cancer cell lines and studied by FTIR microspectroscopic imaging provide reliable information that could be integrated in the setting up of a recognition model aiming to identify and interpret specific spectral signatures of cells present in breast tumor tissue sections. / Le cancer du sein est une maladie très hétérogène, tant au niveau clinique que biologique. Cette hétérogénéité rend impossible la caractérisation moléculaire complète des cellules cancéreuses individuelles dans la pratique clinique courante. Dans ce contexte, l’imagerie infrarouge à transformée de Fourier (FTIR) des coupes tissulaires permet d'obtenir pour chaque pixel d'une image de tissu des centaines de marqueurs potentiels indépendants, ce qui pourrait faire de cette technique un outil particulièrement puissant pour identifier des différents types et sous-types cellulaires. L'interprétation des spectres infrarouges (IR) enregistrés à partir des coupes histologiques nécessite cependant une calibration qui fait actuellement défaut. Cette calibration pourrait être obtenue à partir de lignées cellulaires tumorales bien caractérisées. Traditionnellement, les cellules épithéliales mammaires sont étudiées in vitro sous forme de monocouches adhérentes bidimensionnelles (2D), ce qui conduit à l'altération de la communication entre les cellules et leur environnement et, par conséquent, à la perte de l’architecture et de la fonction du tissu épithélial. Un certain nombre d'interactions physiologiques clés peuvent être rétablies en utilisant des systèmes de culture tridimensionnelle (3D) dans une matrice extracellulaire riche en laminine (lrECM). L'objectif de cette thèse consiste à étudier par imagerie FTIR l'influence du microenvironnement (via une comparaison entre les cultures 2D et 3D lrECM ou les cultures 3D lrECM en présence ou en l’absence de fibroblastes) sur une série de treize lignées de cellules tumorales mammaires humaines bien caractérisées et à déterminer les conditions de culture générant des phénotypes spectraux qui se rapprochent le plus de ceux observés dans les tissus tumoraux. Au cours de ce travail, nous avons mis au point la culture des lignées cellulaires dans un modèle 3D lrECM ainsi qu’une méthodologie de préparation des échantillons offrant la possibilité de les comparer de manière pertinente avec les cellules cancéreuses présentes dans les coupes histologiques. De même, nous avons étudié par imagerie FTIR les effets du microenvironnement sur les lignées de cellules tumorales et inversement. Pour les lignées investiguées, le passage d’une culture 2D à une culture 3D lrECM s’accompagne, en effet, de modifications du spectre IR étroitement corrélées aux modifications du transcriptome. Les marqueurs spectraux indiquent également que l’environnement 3D génère un phénotype cellulaire proche de celui trouvé dans les coupes histologiques. De manière intéressante, cette proximité est d’autant plus renforcée en présence de fibroblastes dans le milieu de culture. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Le cancer du sein de phénotype « Basal-like » triple négatif (BLTN) est particulièrement agressif et de mauvais pronostic. Il est insensible aux traitements hormonaux laissant pour seule stratégie de traitement la chimiothérapie conventionnelle. De ce fait, de nouvelles thérapeutiques ciblées sont en développement, tels que les inhibiteurs de la Poly-ADP-Ribose-Polymerase (PARP). Dans ce contexte, nos travaux de recherche ont été orientés sur l’optimisation du traitement des cancers du sein BLTN en modélisant l’action d’un anti-PARP modèle, l’Olaparib sur la lignée SUM1315 de phénotype BLTN. Dans un premier temps, l’étude de la coexpression de la BCRP et de la P-gp, deux protéines « Multidrug Resistance » (MDR) majeures en présence de 50 µM d’Olaparib® a montré une induction de leurs expressions chez les cellules SUM1315, avec une réponse de type relais. La BCRP établirait une première ligne de défense cellulaire et son action serait ensuite relayée par la P-gp durant 24h de traitement. Ce mécanisme est en corrélation avec la concentration intracellulaire d’Olaparib mesurée par HPLC. L’ensemble de nos résultats suggère qu’il serait possible de contourner le mécanisme de résistance induit par les protéines MDR si une concentration stable en Olaparib est maintenue dans les cellules à long terme. Nous avons ensuite étudié la potentialisation de l’action de l’Olaparib en le combinant avec un traitement par radiothérapie à basse et haute énergie, sur la viabilité des cellules de la lignée SUM1315. La comparaison des résultats avec un traitement Olaparib seul ou irradiation seule et ceux des traitements combinés Olaparib/radiothérapie a alors mis en évidence un effet synergique des deux traitements sur la viabilité cellulaire. L’effet synergique de cette combinaison fonctionne même avec de faibles doses d’Olaparib. De cette manière, il serait possible de réduire les doses d’anti-PARP utilisées tout en gardant les bénéfices du traitement. Enfin, nous avons développé deux techniques de cultures cellulaires en trois dimensions (i) « hanging drop » et (ii) « liquid overlay », permettant de mimer plus fidèlement les conditions des tumeurs in vivo. L’observation en microscopie électronique à transmission et à balayage des sphéroïdes obtenus par ces deux techniques a permis de démontrer l’intégrité des cellules au sein des sphéroïdes ainsi que la formation de jonctions cellulaires. Cependant, les sphéroïdes obtenus en « liquid overlay » ont montré une meilleure intégrité ultra-structurale. / « Triple Negative Basal-Like » (BLTN) breast cancer is particularly aggressive and of poor prognosis. It is insensitive to hormone-targeted therapies leaving conventional chemotherapy as the only treatment strategy. Therefore, new promising targeted therapies are being developed, such as Poly-ADP-Ribose-Polymerase inhibitors (anti-PARPs). In this context, our research has been directed towards optimizing the treatment of BLTN breast cancer by modelling the action of an anti-PARP model, Olaparib®, on BLTN cell line SUM1315. Firstly, the study of the co-expression of BCRP and P-gp, two major “Multidrug Resistance” proteins (MDR) in the presence of 50 µM Olaparib® showed an induction of their expression in SUM1315 cells, with a relay-type response. BCRP would establish a first line of cellular defense and its action would then be taken over by P-gp, for 24h of treatment. This mechanism is correlated with the intracellular concentration of Olaparib® measured by HPLC. All of our results suggest that it would be possible to circumvent the induced MDR resistance mechanism if a stable concentration of Olaparib® is maintained in cells in the long term. Secondly, we studied the potentiation of the action of Olaparib® combining it with low and high-energy radiations on the viability of SUM1315 cells. Comparison of the results with single Olaparib®, single irradiation, or the combination of Olaparib®/radiotherapy then demonstrated a synergistic effect of the two treatments when delivered concomitantly, on cell viability. The synergistic effect of this combination works even with low doses of Olaparib®. In this way it would be possible to reduce the anti-PARP doses while maintaining the benefits of this treatment. Finally, we have developed two techniques of cell culture in three dimensions: (i) "hanging drop" and (ii) "liquid overlay", in order to mimic more accurately the conditions of tumours in vivo. Observations of spheroids obtained by these two techniques by transmission and scanning electron microscopy demonstrated the integrity of cells within as well as the formation of cell junctions. However, the spheroids obtained by "liquid overlay" showed better ultra-structural integrity.

High-throughput drug screening (HTS) in live cells is often a vital part of the preclinical anticancer drug discovery process. So far, two-dimensional (2D) monolayer cell cultures have been the most prevalent model in HTS endeavors. However, 2D cell cultures often fail to recapitulate the complex microenvironments of in vivo tumors. Monolayer cultures are highly proliferative and generally do not contain quiescent cells, thought to be one of the main reasons for the anticancer therapy failure in clinic. Thus, there is a need for in vitro cellular models that would increase predictive value of preclinical research results. The utilization of more complex three-dimensional (3D) cell cultures, such as multicellular tumor spheroids (MCTS), which contain both proliferating and quiescent cells, has therefore been proposed. However, difficult handling and high costs still pose significant hurdles for application of MCTS for HTS. In this work, we aimed to develop novel assays to apply MCTS for HTS and drug evaluation. We also set out to identify cellular processes that could be targeted to selectively eradicate quiescent cancer cells. In Paper I, we developed a novel MCTS-based HTS assay and found that nutrient-deprived and hypoxic cancer cells are selectively vulnerable to treatment with inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). We also identified nitazoxanide, an FDA-approved anthelmintic agent, to act as an OXPHOS inhibitor and to potentiate the effects of standard chemotherapy in vivo. Subsequently, in Paper II we applied the high-throughput gene-expression profiling method for MCTS-based drug screening. This led to discovery that quiescent cells up-regulate the mevalonate pathway upon OXPHOS inhibition and that the combination of OXPHOS inhibitors and mevalonate pathway inhibitors (statins) results in synergistic toxicity in this cell population. In Paper III, we developed a novel spheroid-based drug combination-screening platform and identified a set of molecules that synergize with nitazoxanide to eradicate quiescent cancer cells. Finally, in Paper IV, we applied our MCTS-based methods to evaluate the effects of phosphodiesterase (PDE) inhibitors in PDE3A-expressing cell lines. In summary, this work illustrates how MCTS-based HTS yields potential to reveal and exploit previously unrecognized tumor-specific vulnerabilities. It also underscores the importance of cell culture conditions in preclinical drug discovery endeavors.