No description available.

No description available.

No description available.

Inadequate metabolic conditions in solid tumors lead to the formation of quiescent cancer cells that are suspended in a transient cell cycle arrest. When conditions change, quiescent cancer cells can re-enter the cell cycle and cause recurrence. Drug screening efforts have revealed mitochondrial oxidative phosphorylation as a unique metabolic dependency in quiescent cancer cells. The anthelmintic drug nitazoxanide is an inhibitor of oxidative phosphorylation and preferentially active against quiescent cancer cells in multicellular tumor spheroids.  In this thesis, we employed current and developed new models of quiescent cancer cells and applied live cell imaging for improved preclinical evaluation of cancer drugs in hepatocellular and colorectal carcinoma cell lines. As part of this work, a new assay to measure mitochondrial membrane potential in three-dimensional cell models was developed, an application of the JC-1 assay, and we demonstrated that the preferential activity against quiescent cancer cells of nitazoxanide is shared by two kinase inhibitors: sorafenib and regorafenib. The sensitivity of quiescent cancer cells to nitazoxanide, sorafenib, and regorafenib correlated with the disruption of the mitochondrial membrane potential. Nitazoxanide and sorafenib, in combination, caused an additive decrease in viability, mitochondrial membrane potential, and colony regrowth capacity.  Furthermore, we developed a quiescent hollow fiber assay and implemented an improved analysis using live cell imaging and adenosine triphosphate analysis. Hypoxia and cancer cell quiescence were enriched in hollow fiber macrocapsules over time, and the culture conditions affected nitazoxanide sensitivity. Additionally, we used basement membrane extract gel to support cell growth in hollow fiber macrocapsules and implanted macrocapsules in mice. We observed that the in vivo environment was favorable to cell growth. Through this characterization of the quiescent hollow fiber assay, we were able to outline important paths for future research.

RNA-bindande proteiner (RBP:er) har framträtt som kritiska regulatorer i post-transkriptionell genreglering, och deras dysreglering bidrar till cancerpatogenes. CAPRIN1, ett cytoplasmiskt protein, har identifierats som ett onkofetalt RBP med potentiell betydelse i hepatocellulärt karcinom (HCC), en mycket malign form av levercancer med dålig prognos. Detta examensarbete syftade till att använda gensredigeringsteknologier som CRISPR-interferens (CRISPRi) och RNA-interferens (RNAi) för att störa CAPRIN1-uttryck i HCC-cellinjer och analysera dess effekter på cellproliferation och mängden av CAPRIN1:s främsta RNA-mål. CRISPRi-konstruktioner som riktade sig mot transkriptionsstartstället (TSS) av CAPRIN1 designades och validerades, men trots lyckad kloning och uttryck av guideRNA:er (gRNA:er) resulterade CRISPRi-medierad gensläckning (KD) inte i någon signifikant minskning av CAPRIN1-uttryck. Ineffektiviteten hos CRISPRi tillskrevs låg transfektionseffektivitet, vilket bekräftades av fluorescensmikroskopi med EGFP-expressande plasmider i HepG2- och Huh7-celler. Detta kräver ytterligare optimering av elektroporationsparametrar eller utforskning av alternativa leveransmetoder. I kontrast uppnådde RNAi-medierad KD en betydande minskning av CAPRIN1-uttryck, vilket visade hög KD-effektivitet (90%) enligt RT-qPCR. Denna nivå av gensläckning gav en robust modell för vidare analyser. Efterföljande RT-qPCR av sex topp-RNA-mål identifierade av RAPseq visade varierande svar, med oväntad uppreglering av MYC och Cyclin D2, vilket går emot litteratur som antyder att deras stabilitet beror på CAPRIN1. Denna diskrepans kan förklaras av betydande standardavvikelse i uttrycksnivåerna, vilket understryker behovet av att upprepa experimenten samt genomföra en transkriptomanalys med RNA-seq för att identifiera meningsfulla mål såsom långa icke-kodande RNA (lncRNA:er). Ytterligare, MTT-proliferationstester, visade ökad cellproliferation efter CAPRIN1-KD, även om experimentella inkonsekvenser såsom ojämna cellantal begränsade slutsatsernas pålitlighet. För att erhålla tillförlitliga resultat behöver dock MTT-testet upprepas för att säkerställa konsistens med den positiva kontrollen och samla in statistiskt signifikanta data.Sammanfattningsvis, trots att CRISPRi-försöket stötte på betydande utmaningar, kan gRNA-designen och klonade plasmider användas i framtida studier när elektroporationsprotokollet har optimerats. Dessutom visade den framgångsrika RNAi-medierade KD av CAPRIN1 en alternativ metod som kan antas för vidare studier med andra RBP-kandidater. Framtida studier bör syfta till att optimera elektroporationssystemet, validera KD på proteinnivå och använda RNA-seq för en omfattande analys av CAPRIN1:s inverkan på transkriptomet i HCC-cellinjer. Dessa ansträngningar kommer att förbättra förståelsen av CAPRIN1:s regulatoriska mekanismer och dess potential som terapeutiskt mål vid HCC. / RNA-binding proteins (RBPs) play a crucial role in post-transcriptional gene regulation, and their dysregulation is implicated in cancer pathogenesis. CAPRIN1, a cytoplasmic activation/proliferation-associated protein, identified as an oncofetal RBP with potential significance in hepatocellular carcinoma (HCC), is a worthy candidate to be studied. Objective: This project aimed to establish a protocol for silencing RBPs gene expression via CRISPR interference (CRISPRi) technology using CAPRIN1 as a case study, as the roles of CAPRIN1 in tumorigenesis remains unknown. Results: Despite successful cloning of the designed gRNAs, CRISPRi-mediated knockdown did not reduce CAPRIN1 expression, attributed to low transfection efficiency as indicated by fluorescence microscopy. RNAi-mediated knockdown, however, achieved substantial reduction in CAPRIN1 expression (90%). Subsequent analysis of CAPRIN1 top RNA interactors showed substantial standard deviations, highlighting the need for repeating the experiments. The MTT assay showed uneven initial seeding densities, necessitating further repetitions of the assay. Conclusions: The study highlights the challenges associated with CRISPi-mediated knockdown, especially in transfecting HepG2 cells with large plasmids using electroporation. However, the designed gRNAs and cloned plasmids have the potential to be used in future studies, provided that the transfection process is further optimized. RNAi proved to be an effective alternative for gene silencing, achieving high knockdown efficiency and enabling subsequent analysis of CAPRIN1 target RNAs. Prioritizing further optimization of CRISPRi transfection protocols in upcoming projects will facilitate the adoption of this workflow for investigating other RBP candidates. Additionally, performing a comprehensive RNA-seq analysis could provide a deeper understanding of CAPRIN1’s role in HCC.

No / Human aging involves a gradual decrease in cellular integrity that contributes to multiple complex disorders such as neurodegenerative disorders, cancer, diabetes, and cardiovascular diseases. Genome-wide association studies (GWAS) play a key role in discovering genetic variations that may contribute towards disease vulnerability. However, mostly disease-associated SNPs lie within non-coding part of the genome; majority of the variants are also present in linkage disequilibrium (LD) with the genome-wide significant SNPs (GWAS lead SNPs). Overall 600 SNPs were analyzed, out of which 291 returned RegulomeDB scores of 1-6. It was observed that just 4 out of those 291 SNPs show strong evidence of regulatory effects (RegulomeDB score < 3), while none of them includes any GWAS lead SNP. Nevertheless, this study demonstrates that by combining ENCODE project data along with GWAS reported information will provide important insights on the impact of a genetic variant-moving from GWAS towards understanding disease pathways. It is noteworthy that both genome-wide significant SNPs as well as the SNPs in LD must be considered for future studies; this may prove to be crucial in deciphering the potential regulatory elements involved in complex disorders and aging in particular.

No description available.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Le carcinome hépatocellulaire (CHC) est un cancer au pronostic sombre, car il est souvent diagnostiqué trop tardivement pour entreprendre un traitement curatif. Il se développe dans 80-90% des cas sur fond de cirrhose. On connait mal comment la fibrose, étape préliminaire à la cirrhose, et son principal constituant, le collagène de type 1 (COL1), peuvent jouer un rôle dans le processus du CHC. Nous avons tout d’abord étudié le développement de la fibrose dans un modèle utilisant la souris nue. Nous avons déterminé qu’après 16 semaines d’administration de thioacétamide dans l’eau de boisson, il est possible d’obtenir une fibrose suffisante pour induire une hépatoprotection en présence de différents hépatotoxiques (AST dans le sérum de souris fibrotiques vs non-fibrotiques : Anti-Fas JO2 (4665 ± 2596 vs. 13953 ± 2260 U/L; P<0.05), acétaminophène (292 ± 66 vs. 4087 ± 2205 U/L; P<0.01) et CCL4 (888 ± 268 vs. 15673 ± 2782 U/L; P<0.001)). Ces résultats confirment que la présence de COL1 et de fibrose favorise la survie des hépatocytes normaux tel qu’observé précédemment au laboratoire. Par la suite, nous avons sélectionné in vivo, par injection intrasplénique de la lignée de CHC Hepa1-6, une lignée à forte tumorigénicité nommée dt-Hepa1-6 (28±12 lésions vs. 0±0 lésions à 21 jours). Cette lignée était composée d’une sous-population cellulaire arborant la protéine de surface EpCAM (34.0±0.1%). Par tri cellulaire, nous avons démontré que ces cellules étaient partiellement responsables de la tumorigénicité accrue (EpCAM + (86.7±2.3%) :1093±74 lésions vs. EpCAM- (15.3±1.0%) :473±100 lésions; P<0.01). Nous avons alors démontré que la présence de fibrose favorise le développement de la lignée dt-Hepa1-6 in vivo (604±242 vs 22±9 lésions; P<0.05). De plus, la présence de fibrose réduit l’efficacité du traitement au cisplatin in vivo (44.5±4.9 vs. 78.7±6.9%; P<0.01) confirmant les résultats obtenus in vitro (Apoptose : COL1 13.75±0.44% vs. plastique 31.45±1.37%; P<0.001). En conclusion, la présence de fibrose et de son principal constituant, le COL1, favorise la survie et la progression du CHC. / Hepatocellular carcinoma (HCC) is a dreadful pathology, often diagnosed too late to be cured. In 80-90% of cases, it arises in the context of liver cirrhosis. Little is known on the implication of liver fibrosis, one of the key elements of cirrhosis, and its major constituent, type I collagen (COL1), on the development of HCC. We first studied the development of fibrosis in a nude mouse model. We determined that, after 16 weeks of thioacetamide administration in drinking water, we obtained a sufficient degree of fibrosis to reach a hepatoprotective state when animals were exposed to different hepatotoxic agents (Serum AST of fibrotic vs non-fibrotic mice : Anti-Fas JO2 (4665 ± 2596 vs. 13953 ± 2260 U/L; P<0.05), acetaminophen (292 ± 66 vs. 4087 ± 2205 U/L; P<0.01) et CCL4 (888 ± 268 vs. 15673 ± 2782 U/L; P<0.001)). This confirmed that COL1 and the presence of fibrosis protects normal hepatocytes as observed previously in our laboratory. Next, we selected in vivo, by intrasplenic injection of the murine HCC cell line Hepa1-6, a highly tumorigenic cell line that we named dt-Hepa1-6 (28±12 lesions vs. 0±0 lesions at 21 days). This cell line was constituted of cell subsets expressing EpCAM protein at their surface (34.0±0.1%). Through cell sorting, we demonstrated that these cells were partially responsible for the enhanced tumorigenicity observed (EpCAM + (86.7±2.3%) :1093±74 lesions vs. EpCAM- (15.3±1.0%) :473±100 lesions; P<0.01). We then showed that the presence of liver fibrosis increases the development of dt-Hepa1-6 cell line in vivo (604±242 vs 22±9 lesions; P<0.05). Moreover, fibrosis reduced the anti-neoplastic efficacy of cisplatinum in vivo (44.5±4.9 vs. 78.7±6.9%; P<0.01) confirming in vitro results (Apototic index : COL1 13.75±0.44% vs. plastic 31.45±1.37%; P<0.001). In conclusion, fibrosis and its major constituent, COL1, favor the survival and progression of HCC.

Le Carcinome Hépatocellulaire a une incidence mondiale élevée et est associé à un mauvais pronostic. Les traitements curatifs existants ne sont applicables qu’à une minorité de patients. La radiothérapie sélective interne (SIRT) est un traitement palliatif de plus en plus utilisé. Elle consiste à l’injection sélective intra-tumorale de microsphères d’yttrium-90 par infusion intra-artérielle, et repose sur deux étapes : une étape pré-thérapeutique de simulation du traitement avec l’injection de macroagrégats d’albumines marqués au 99mTc et le traitement en lui-même. Cependant les caractéristiques de ces deux vecteurs diffèrent et peuvent conduire à des variations de biodistribution et à une dosimétrie approximative. Ce travail a pour but de développer un vecteur radiothéranostique unique pour la SIRT : les microparticules à base d’amidon (SBMP), afin de pallier aux différents problèmes rencontrés en clinique. L’optimisation du radiomarquage par le 68Ga et le 188Re sous forme de kits lyophilisés prêts-à-l’emploi, a permis d’obtenir une pureté radiochimique > 98 % et > 95 % respectivement. Une étude préliminaire par imagerie TEP/TDM in vivo chez le rat, suite à l’injection intraartérielle des 68Ga-SBMP a montré une biodistribution spécifique des microparticules avec plus de 95 % de l’activité retrouvée dans le foie et plus particulièrement dans les tumeurs. Les SBMP offrent plusieurs avantages répondant à différents problèmes actuels et constituent un agent théranostique prometteur pour la SIRT. Une présentation de la SIRT, des différentes microparticules en développement pour la SIRT et des modèles animaux de tumeur hépatique existants seront également développées dans ce travail. / The Hepatocellular Carcinoma has a high incidence worldwide and is associated with a bad prognostic. The existing curative treatments can only be apply in a minority of cases. The selective internal radiation therapy (SIRT) is a palliative treatment that is increasingly used. This technique is define by the selective intratumoral injection of yttrium-90microspheres via intra-arterial infusion. It involves two steps : a pre-therapeutic one for treatment simulation purpose with the injection of serum albumin macroaggregates radiolabeled with 99mTc and the treatment itself. However the characteristics of these two vectors are different and can lead to variations in biodistribution and approximate dosimetry. This works aims to develop a unique radiotheranostic vector for the SIRT: the starch-basedmicroparticles (SBMP), in order to overcome the different currents clinical problems. The optimization of the radiolabeling by the 68Ga and the 188Re in the form of ready-to-use radiolabeling kits allowed to obtain a radiochemical purity > 98 % and > 95 % respectively. A preliminary in vivo study by PET/CT imaging in rat, following the intra-arterial injection of 68Ga-SBMP displayed a specific biodistribution of the microparticles with more than 95 % of the activity found in the liver and mostly in the tumors. The SBMP offer several advantages that answer different current issues and area promising theranostic agent for the SIRT. A presentation of the SIRT, the different microparticles in development and the existing animal models of hepatic tumor will also be developed in this work.