O câncer de pulmão é um dos mais incidentes e letais, e, assim, a busca de novos fármacos é necessária. Atualmente o desenvolvimento de fármacos conta com abordagens computacionais que otimizam este processo. Dado que a fosfatidiletanolamina desempenha importantes papeis fisiológicos e uma das enzimas envolvidas na sua síntese, a CTP:fosfoetanolamina-citidilil-transferase (Pcyt2) é frequentemente superexpressa em células de câncer de pulmão, no presente trabalho, foram avaliados o potencial terapêutico de CHY-1, um análogo da miltefosina desenvolvido como inibidor da enzima Pcyt2, e os mecanismos inerentes à sua atividade antitumoral. O CHY-1 apresentou citotoxicidade superior ao seu protótipo e a outro inibidor da enzima Pcyt2, a meclizina. Além disso, as células malignas foram mais sensíveis ao CHY-1 do que as células não-tumorigênicas. Em conclusão, o presente trabalho evidencia o potencial do CHY-1 como um inibidor da enzima Pcyt2 e candidato a fármaco com atividade preferencial para câncer de pulmão. / Lung cancer is one of the most incident and lethal cancers, thus, the pursuit for new drugs is necessary. Nowadays, new drugs development has computational tools that improves this process. Once that phosphatidylethanolamine plays several important physiological roles and one of the enzymes of its production pathway, CTP:phosphoethanolamine cytidylyltransferase (Pcyt2), is usually overexpressed in lung cancer cells, therefore, this study aimed was to evaluate the antitumor effects of CHY-1, a miltefosine analogue developed as an inhibitor of Pcyt2 enzyme, and to investigate the mechanisms related to its antitumor action. CHY-1 was more cytotoxicity than its prototype, miltefosine, and was more cytotoxic than another inhibitor Pcyt2 enzyme, meclizine. Morevover, malignant cells were more sensitive to CHY-1 effects than non-tumorigenic cells. In conclusion, this work presents CHY-1 as an inhibitor of Pcyt2 enzyme and new candidate a drug with preferential activity on NSCLC cells.

Non-small-cell lung cancer (NSCLC) accounts for 80–85% of all lung cancers and is associated with significant mortality. As epidermal-growth-factor receptor (EGFR) is over-expressed in 80-90% of NSCLC, its inhibition via EGFR-Tyrosine Kinase inhibitors (EGFR-TKIs) is a main therapeutic strategy. However, patients with mutations in KRAS are resistant to EGFR-TKIs. A study in mutant KRAS-driven lung cancer in transgenic mice showed that tumor growth was dependent on the activity of focal adhesion kinase (FAK). Therefore, we hypothesized that KRAS-mutant NSCLC will be sensitive to FAK-TKIs and, given known FAK-EGFR cross-talk, FAK inhibition will sensitize KRAS-mutant NSCLC to EGFR-TKIs. We performed cell viability assays of WT versus mutant KRAS NSCLC cell lines following treatment with FAK-TKI alone or in combination with a clinically relevant EGFR-TKI. We found that KRAS-mutant cells were more sensitive to FAK-TKI than KRAS-WT NSCLC. In addition, we found that the combination treatment including FAK and EGFR TKIs resulted in reduced tumor cell viability as compared to treatment with either drug alone. This enhanced anti-tumor response could be due to FAK-TKI’s ability to down-regulate EGFR downstream targets. Our preliminary data suggests that in KRAS-mutant cells the drug combination appears to more effectively inhibit Akt activity than single drug treatment alone. This suggests an enhanced ability to impair cell survival following treatment with the drug combination. We also found that treatment with FAK TKI in KRAS mutant NSCLC cells resulted in increased activation of EGFR which was due in part to modulation of EGFR recycling and production of endogenous EGFR ligands. Thus, the combination of FAK- and EGFR-TKIs may be more effective in KRAS mutant NSCLC as treatment with EGFR-TKI overcomes the unexpected ‘side effect’ of treatment with FAK-TKI, namely activation of the EGFR pathway by this drug. The findings of our study are novel and have uncovered previously unrecognized outcomes of FAK inhibition on EGFR activity. Moreover, our data support the notion that the combination of FAK- and EGFR-TKIs could be an effective treatment for KRAS mutant NSCLC patients.

Au travers d’études cliniques et biologiques, de méta-analyses et de revues systématiques de la littérature, nous avons étudié les CBNPC de stade III sur le plan thérapeutique et cherché des facteurs pronostiques pour la survie dans le but d’améliorer la classification internationale et, à terme, de permettre une meilleure prise en charge des patients inclus dans ce groupe hétérogène de tumeurs.<p>Dans le cadre d’essais randomisés, nous avons montré qu’un abord multimodal et multidisciplinaire permettait d’améliorer le pronostic des patients atteints d’un CBNPC de stade III. Le traitement des tumeurs non résécables implique une combinaison de chimiothérapie et de radiothérapie, dont l’administration concomitante doit être proposée aux patients aptes à la tolérer. La chimiothérapie doit être incluse dans le schéma thérapeutique des tumeurs potentiellement résécables. Elle permet une résection chirurgicale complète chez des patients sélectionnés dont la tumeur était initialement non résécable.<p>Nous avons déterminé que des caractéristiques cliniques (l’indice de performance et l’âge), biologiques (les taux sanguins de polynucléaires neutrophiles, d’hémoglobine et de plaquettes, la bilirubinémie) et propres à la tumeur (l’extension locale [T3-4] et ganglionnaire [N3]) avaient une valeur pronostique indépendante pour la survie. Ceci nous a permis d’aboutir à une proposition de modification de la classification internationale concernant les CBNPC de stade III.<p>Bien que pris individuellement, les facteurs biologiques que nous avons étudiés (p53, EGF-R, TTF-1, Mdm2) n’aient pas de valeur pronostique pour la survie, nous avons montré que la combinaison EGF-R+/TTF1- était un facteur pronostique indépendant en analyse multivariée pour la survie spécifique au cancer bronchique.<p>Nous avons finalement évalué le rôle pronostique de la tomodensitométrie par émission de positrons et de la mesure semi-quantitative de captation du 18F-FDG (SUV) sur la survie des patients atteints de CBNPC et montré qu’un SUV élevé était un facteur de mauvais pronostic pour la survie. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Cancer cells have been known for some time to have very different metabolismas compared to that of normal non proliferating cells. As metabolism is involvedin almost every aspect of cell function, there has been a recent resurgence ofinterest in inhibiting cancer metabolism as a therapeutic strategy. Inhibitors thatspecifically target altered metabolic components in cancer cells are being developedas antiproliferative agents. However, many such inhibitors have not progressedinto the clinic due to limited efficacy either in vitro or in vivo. In this study weexplore the hypothesis that this is often due to the robustness of the metabolicnetwork and the differences between individual cancer cell lines in their metaboliccharacteristics. We take a systems biology approach. We investigate the cellular bioenergetic profiles of a panel of five non-small celllung cancer cell lines before and after treatment with a novel inhibitor of theglutaminase-1 (GLS1) enzyme. Additionally, we explore the effects of this inhibitoron intracellular metabolism of these cell lines as well as on the uptake and secretionof glucose, lactate and amino acids. To be able to do the latter robustly, wehad to modify the experimental assay considerably from procedures that seemto be standard in the literature; using these earlier procedures the metabolicenvironment of the cells was highly variable, leading to misleading results onthe metabolic effects of the inhibitor. We reduced cell density, altered mediumvolume and changed the time-window of the assay. This led to the cells growingexponentially, appearing indifferent to the few remaining changes. In this newassay, the metabolic effects of the glutaminase inhibitor became robust. One of the most significant results of this study is the metabolic heterogeneitydisplayed across the cell line panel under basal conditions. Differences in themetabolic functioning of the cell lines were observed in terms of both theirbioenergetic and metabolic profile. The amount of respiration attributed tooxidative phosphorylation differed between cell lines and respiratory capacity wasattenuated in most cells. However, the rate of glycolysis was similar betweencell lines in this assay. These results suggest that the Warburg effect arisesthrough a greater diversity of mechanisms than traditionally assumed, involvingvarious combinations of changes in the expression of glycolytic and mitochondrialmetabolic enzymes. The effects of GLS1 inhibition on cellular bioenergetics and metabolism alsodiffered between cell lines, even between resistant cell lines, indicating that theremay also be a diversity of resistance mechanisms. The metabolomic response ofcell lines to treatment suggests potential resistance mechanisms through metabolicadaptation or through the prior differences in the metabolic function of resistantcell lines. Part of the metabolome response to GLS1 inhibition was quite specificfor sensitive cells, with high concentrations of IMP as the strongest marker. Our results suggest that the metabolome is a significant player in what determinesthe response of cells to metabolic inhibitors, that its responses differ between cancercells, that responses are not beyond systems understanding, and that thereforethe metabolome should be taken into account in the design of and therapy withanti-cancer drugs.

Les revues systématiques sont des outils indispensables à la synthèse des connaissances en évaluation thérapeutique. Il est désormais fréquent que plusieurs traitements soient disponibles pour une même indication. L’objectif des patients et des cliniciens est alors de savoir quels sont, parmi l’ensemble des traitements disponibles, le(s) meilleur(s). Compte tenu de la nécessité de synthétiser les données disponibles pour tous les traitements et de maintenir cette synthèse à jour, notre objectif était d’évaluer les limites du système actuel de synthèse et de développer une méthodologie alternative. Nous avons d’abord évalué la capacité des revues systématiques à prendre en compte l’ensemble des preuves disponibles sur l’effet des multiples traitements. Nous avons utilisé l’exemple des traitements de deuxième ligne du cancer bronchique non à petites cellules métastatique non muté pour EGFR ou de statut inconnu. Nous avons montré que les 29 revues systématiques publiées jusque 2015 sur cette question, considérées collectivement, fournissaient une synthèse fragmentée et non à jour de la preuve disponible. Au moins 40% des 77 essais, des 45 traitements, des 54 comparaisons de traitements et des 28 636 patients n’étaient constamment pas pris en compte dans les revues systématiques. Nous avons discuté les raisons pour lesquelles le système de synthèse des données actuel ne permettait pas de couvrir l’ensemble des données disponibles. Nous avons ensuite développé une nouvelle forme de synthèse de la preuve disponible au cours du temps, la méta-analyse en réseau cumulative et dynamique. Elle consiste à passer d’une série de méta-analyses à une méta-analyse en réseau unique, incluant l’ensemble des traitements disponibles pour une indication donnée, avec une mise à jour du réseau d’essais et de la synthèse des données dès que les résultats d’un nouvel essai deviennent disponibles. Elle débute par une méta-analyse en réseau initiale suivie d’une succession de mises à jour répétées à intervalles réguliers. Nous avons décrit les étapes méthodologiques, et développé le protocole d’une étude de preuve de concept, appliquée aux traitements de deuxième ligne du cancer bronchique non à petites cellules. Enfin, nous avons réalisé la méta-analyse en réseau initiale sur ce même exemple. Nous avons inclus 98 essais randomisés évaluant 60 traitements chez 34 179 patients. Nous avons montré que les traitements par immunothérapie (nivolumab et pembrolizumab) avaient un effet sur la survie globale supérieur aux chimiothérapies et thérapeutiques ciblées actuellement recommandées (nivolumab versus docetaxel HR=0,68 (IC95% 0,55-0,83) ; versus pemetrexed HR=0,65 (0,5-0,83) ; versus erlotinib HR=0,66 (0,51-0,84) and versus gefitinib HR=0,65 (0,51-0,82)). Les résultats étaient similaires pour le pembrolizumab. Pour la survie sans progression, le nivolumab avait aussi un effet supérieur aux quatre traitements recommandés. La méta-analyse en réseau cumulative et dynamique pourrait devenir l’outil permettant de changer de paradigme dans la synthèse des connaissances afin d’améliorer la prise de décision médicale. / Systematic reviews are essential tools to synthesize available evidence for therapeutic evaluation. Multiple treatments are now frequently available for a given condition. Patients and physicians want to know which one is the best among all treatments. Thus we need to retrieve and synthesize all available evidence across all treatments and furthermore to maintain it updated when new evidence and new treatments become available. Our objective was to evaluate the limits of the current ecosystem of evidence synthesis and to develop an alternative methodology. We have first assessed the capacity of systematic reviews to cover all available evidence of multiple treatments. We took the example of second-line treatments of advanced non-small cell lung cancer with EGFR wild-type or unknown status. We have shown that the 29 systematic reviews published in this condition up to 2015, considered collectively, failed to provide a complete and updated synthesis of all available evidence. Almost 40% of the 77 trials, of the 45 treatments, of the 54 treatment comparisons and of the 28,636 patients were always missing from systematic reviews. We have discussed the reasons why the ecosystem of evidence synthesis fails to encompass all available evidence. We then developed a new paradigm to synthesize evidence over time called live cumulative network meta-analysis. This new concept consists in switching from a series of standard meta-analyses to a single network meta-analysis covering all treatments and systematically updated as soon as the results of a new trial become available. Live cumulative network meta-analysis is initiated with a network meta-analysis which is iteratively updated. We have described the methodological steps, developed the protocol of a proof-of-concept study applied to second-line treatments of advanced non-small cell lung cancer. Finally, we have performed the initial network meta-analysis in this condition. We have included 98 trials including 34,179 patients and assessing 60 treatments. We have shown that nivolumab was more effective in term of overall survival compared to docetaxel HR=0.68 (IC95% 0.55-0.83), to pemetrexed HR=0.65 (0.5-0.83), to erlotinib HR=0.66 (0.51-0.84) and to gefitinib HR=0.65 (0.51-0.82). Similar results were found with pembrolizumab. In progression free survival, nivolumab had a more important treatment effect compared to the four recommended treatments. Live cumulative network meta-analysis should become a paradigmatic shift for systematic reviews and meta-analysis in order to improve medical decision making.

Lung cancer claims the highest mortality and the second-most estimated new cases among all oncological diseases [1]. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all newly diagnosed lung cancers [2]. Approximately 40% of newly diagnosed lung cancer patients are stage IV. For stage IIIB/IV NSCLC, cytotoxic combination chemotherapy is standard first-line chemotherapy. A regimen of platinum (cisplatin or carboplatin) plus paclitaxel, gemcitabine, docetaxel, vinorelbine, irinotecan, or pemetrexed is the recommended clinical treatment [3]. Cisplatin is the first-generation platinum-based anti-cancer drug. Although cisplatin is much more effective than other platinum drugs at the same dosage [4], accumulating reports have shown the failure of conventional platinum-based chemotherapy due to various side effects and drug resistance [5]. Miriplatin, a member of platinum drug family, has been approved in Japan in 2009 for transcatheter arterial chemoembolization treatment of hepatocellular carcinoma (HCC) [6]. Miriplatin is a lipophilic platinum drug that contains myristates (14-carbon chains) as leaving groups and diamino cyclohexane as the non-leaving carrier ligand. The application of miriplatin in clinic is limited because it has very poor solubilities both in water and in common organic solvents [7]. The structure of solid tumors and tumor microenvironment (TME) in lung cancer constitute a barrier to the deep penetration of chemotherapy agents, which limits the effectiveness of chemotherapy [8]. Nanoparticles with appropriate properties provide a promising delivery system to overcome the biological and physiochemical barriers that hinder anti-cancer activity [9]. Lipid-based nanoparticles such as liposomes, micelles, and solid lipid nanoparticles (SLNs) can delivery anti-cancer drugs to improve their anti-cancer activities. In this study, we formulated miriplatin into various micelles, liposomes, and SLNs by film-hydration and evaluated their physicochemical properties and anti-cancer activity against NSCLC cells in culture. Miriplatin-loaded formulations with different compositions were successfully prepared by the film-hydration method. Most miriplatin-loaded micelles were more homogeneous and much smaller than miriplatin-loaded liposomes and SLNs. The majority of miriplatin-loaded micelles were about 15 nm in diameter, while SLNs were around 120 nm, and liposomes were about 180 nm. Formulations with a higher molar ratio of PE-PEG2000 had smaller sizes. SLNs loaded with a higher molar ratio of miriplatin in the compositions showed smaller sizes. Inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES) techniques were attempted to quantify the platinum element in the formulations. Formulations with a higher molar ratio of PE-PEG2000 had higher recovery of platinum element. Most miriplatin-loaded formulations had higher than 80% platinum recovery. The recovery of intact miriplatin was characterized by HPLC. Miriplatin-loaded micelles had much higher intact miriplatin recovery (about 100%) than SLNs (about 30%). By TEM imaging, the micelles showed the morphology of spherical dots of about 10 nm in diameter while SLNs showed both spherical and rodlike structures of about 120 nm in diameter. The TEM results were consistent with the size and PDI results by the Zetasizer. Three-dimensional multicellular spheroids (3D MCS) of A549 and A549-iRFP cell lines were successfully established as cell culture models to evaluate activity against non-small cell lung cancer. The viability of 3D MCS after 7-days treatment with miriplatin-loaded micelles was about 0%, which was similar to cisplatin. Miriplatin-loaded formulations with a higher molar ratio of PE-PEG2000 in the compositions had higher anti-cancer activity against 3D MCS. The anticancer activity of miriplatin-loaded formulations against 3D MCS was positively associated with the recovery of intact miriplatin from the formulations. The IC50 value of miriplatin-loaded micelles against A549-iRFP 3D MCS was around 25 µM, while that of cisplatin was 84.78 µM. In summary, the reported lipid-based nano-formulations represent a promising delivery system of miriplatin against NSCLC.

[ES] El cáncer de pulmón es el tumor más frecuente y también el que presenta mayor mortalidad en términos absolutos, suponiendo hasta más del 18% de los fallecimientos por cáncer en el mundo al año. El cáncer de pulmón no microcítico (CPNM) representa casi el 85% de todos los tumores de pulmón. Tanto las alteraciones genéticas como la heterogeneidad y el microambiente tumoral influyen en la agresividad de los tumores de CPNM. Un factor que contribuye a la heterogeneidad tumoral es la presencia de células madre tumorales (CSC, Cancer Stem Cells). La metástasis y recurrencia tumoral después del tratamiento han sido atribuidas al crecimiento y supervivencia de esta subpoblación celular. La falta de marcadores específicos de CSC de pulmón representa una dificultad para identificarlas, y los marcadores de superficie conocidos hasta el momento no son válidos para separar poblaciones de CSC, por lo que se hace necesaria la generación de nuevos ensayos experimentales para aislar CSC de manera más robusta. La identificación de CSC podría ser la base para el diseño de nuevas estrategias terapéuticas personalizadas, basadas en la selección de una combinación más racional de fármacos, que tendría como objetivo eliminar la población de CSC en los tumores de CPNM. Los objetivos de esta tesis doctoral han sido el aislamiento y caracterización de CSC derivadas de tumores de pacientes con CPNM, y de líneas de cultivo establecidas; la caracterización de proteínas involucradas en la transición epitelial mesenquimal (EMT) en CPNM que pueden ser relevantes para la adquisición y el mantenimiento de características de las células madre y para la progresión de la enfermedad; y el desarrollo de modelos experimentales de ratón a partir de xenoinjertos derivados de tumores de pacientes con CPNM (PDX) para la identificación de nuevos biomarcadores y para estudiar el fenotipo y evolución de los tumores de pacientes. En este trabajo se han optimizado los protocolos para la obtención de tumoresferas (ESF) con propiedades de células madre a partir de tejido tumoral de pacientes con CPNM y de líneas células de CPNM establecidas. Se ha confirmado el fenotipo característico de célula madre en las ESF mediante el análisis de la expresión de marcadores de superficie de CSC como CD326, CD166, CD44 y CD133, su capacidad de autorrenovación y diferenciación, así como la capacidad de iniciar tumores en ratones inmunodeprimidos. Se ha observado además que la señalización mediada por TGF-ß1 podría estar contribuyendo a un aumento de la población CSC, y que habría una relación entre el proceso de promoción de la EMT inducida por TGF-ß1 y las propiedades de CSC. El análisis de genes involucrados en la EMT y CSC en las ESF mostró que la expresión de CDKN1A, NOTCH3, CD44, NANOG, SNAI1 e ITGA6 es característica de las ESF, y por tanto la expresión combinada de estos genes podría identificar la subpoblación de CSC. Al correlacionar la expresión de estos genes con la supervivencia de los pacientes, se obtuvo una firma con valor pronóstico en ADC basada en la expresión de los genes CDKN1A, SNAI1 e ITGA6. Se ha estudiado además el papel de proteínas involucradas en la EMT cuya expresión podría promover la migración e invasión celular en CPNM. De esta manera se ha identificado que el factor de transcripción JunB y el factor de traducción eIF5A2 están involucrados en la iniciación de la EMT mediada por TGF-ß1. Finalmente, se han establecido 9 modelos de xenoinjertos derivados de tumores de pacientes con CPNM (PDX). La correlación del éxito de implantación del tumor en ratón con variables clínico-patológicas de los pacientes mostró que los tumores primarios que generaron PDX derivaban de pacientes con peor pronóstico. También se observó que la elevada expresión de Vimentina, Ezrina y Ki67 en tumores sugiere una mayor agresividad y, por tanto, estudiar su expresión podría utilizarse en / [CA] El càncer de pulmó és el tumor més frequent i també el que presenta major mortalitat en termes absoluts, suposant fins més del 18% de les defuncions per càncer en el món a l'any. El càncer de pulmó no microcític (CPNM) representa quasi el 85% de tots els tumors de pulmó. Tant les alteracions genètiques com l'heterogeneïtat i el microambient tumoral influeixen en l'agressivitat dels tumors de CPNM. Un factor que contribueix a l'heterogeneïtat tumoral és la presència de cèl·lules mare tumorals (CSC, Cancer Stem Cells). La metàstasi i recurrència tumoral després del tractament han sigut atribuïdes al creixement i supervivència d'aquesta subpoblació cel·lular. La falta de marcadors específics de CSC de pulmó representa una dificultat per a identificar-les, i els marcadors de superfície coneguts fins al moment no són vàlids per a separar poblacions de CSC, per la qual cosa es fa necessària la generació de nous assajos experimentals per a aïllar CSC de manera més robusta. La identificació de CSC podria ser la base per al disseny de noves estratègies terapèutiques personalitzades, basades en la selecció d'una combinació més racional de fàrmacs, que tindria com a objectiu eliminar la població de CSC en els tumors de CPNM. Els objectius d'aquesta tesi doctoral han sigut l'aïllament i caracterització de CSC derivades de tumors de pacients amb CPNM, i de línies de cultiu establides de CPNM; la caracterització de proteïnes involucrades en la transició epitelial mesenquimal (EMT) en CPNM que poden ser rellevants per a l'adquisició i el manteniment de característiques de les cèl·lules mare i per a la progressió de la malaltia; i el desenvolupament de models experimentals de ratolí a partir de xenotrasplantaments derivats de tumors de pacients amb CPNM (PDX) per a la identificació de nous biomarcadors i per a estudiar el fenotip i evolució dels tumors de pacients. En aquest treball s'han optimitzat els protocols per a l'obtenció de tumoresferes (ESF) amb propietats de cèl·lules mare a partir de teixit tumoral de pacients amb CPNM i de línies cel·lulars de CPNM establides. S'ha confirmat el fenotip característic de cèl·lula mare en les ESF mitjançant l'anàlisi de l'expressió de marcadors de superfície de CSC com CD326, CD166, CD44 i CD133, la seua capacitat d'autorenovació i diferenciació, així com la capacitat d'iniciar tumors en ratolins immunodeprimits. S'ha observat a més que la senyalització mediada per TGF-ß1 podria estar contribuint a un augment de la població CSC, i que hi hauria una relació entre el procés de promoció de l'EMT induïda per TGF-ß1 i les propietats de CSC. L'anàlisi de gens involucrats en l'EMT i CSC en les ESF va mostrar que l'expressió de CDKN1A, NOTCH3, CD44, NANOG, SNAI1 i ITGA6 és característica de les ESF, i per tant l'expressió combinada d'aquests gens podria identificar la subpoblació de CSC. En correlacionar l'expressió d'aquests gens amb la supervivència dels pacients, es va obtindre una signatura amb valor pronòstic en ADC basada en l'expressió dels gens CDKN1A, SNAI1 i ITGA6. S'ha estudiat a més el paper de proteïnes involucrades en l'EMT que la seua expressió podria promoure la migració i invasió cel·lular en CPNM. D'aquesta manera s'ha identificat que el factor de transcripció JunB i el factor de traducció eIF5A2 estan involucrats en la iniciació de l'EMT mediada per TGF-ß1. Finalment, s'han establit 9 models de xenotrasplantaments derivats de tumors de pacients amb CPNM (PDX). La correlació de l'èxit d'implantació del tumor en ratolí amb variables clinicopatològiques dels pacients va mostrar que els tumors primaris que van generar PDX derivaven de pacients amb pitjor pronòstic. També es va observar que l'elevada expressió de Vimentina, Ezrina i Ki67 en tumors suggereix una major agressivitat i, per tant, l'avaluació de la seua expressió podria utilitzar-se en combinació com a marcador de pr / [EN] Lung cancer is the most frequent and the most mortal tumor in absolute terms, accounting for up to more than 18% of cancer deaths worldwide per year. Non-small cell lung cancer (NSCLC) accounts for almost 85% of all lung tumors. Apart from genetic alterations, both tumor heterogeneity and tumor microenvironment influence the aggressiveness of NSCLC tumors. A factor that contributes to tumor heterogeneity is the presence of cancer stem cells (CSC). Tumor metastasis and recurrence after treatment have been attributed to the growth and survival of this cell subpopulation. The lack of specific markers for lung CSC represents a drawback for their identification, and the surface markers known to date are not valid for separating CSC subpopulations, so it is necessary to generate new experimental assays to isolate CSC in a more robust way. The identification of CSC could be the basis for the design of new personalized therapeutic strategies, based on the selection of a more rational combination of drugs, which would aim to eliminate the CSC population in NSCLC tumors. The objectives of this doctoral thesis have been the isolation and characterization of CSC derived from tumors of patients with NSCLC, and from established NSCLC cell lines; the characterization of proteins involved in the epithelial-mesenchymal transition (EMT) in NSCLC that may be relevant for the acquisition and maintenance of stem cell characteristics and for disease progression; and the development of experimental mouse models from patient-derived xenografts (PDX) of NSCLC for the identification of new biomarkers and to study the phenotype and evolution of patient tumors. In this work, the protocols for obtaining tumorspheres (SPH) with stem cell properties from tumor tissue of NSCLC patients and from established NSCLC cell lines have been optimized. The characteristic stem cell phenotype in SPH has been confirmed by analyzing the expression of CSC surface markers such as CD326, CD166, CD44 and CD133, their capacity for self-renewal and differentiation, as well as the ability to initiate tumors in immunosuppressed mice. It has also been observed that TGF-ß1-mediated signaling could be contributing to an increase in CSC population, and that there would be a relationship between the process of promoting TGF-ß1-induced EMT and CSC features. The analysis of genes involved in EMT and CSC in the SPH showed that the expression of CDKN1A, NOTCH3, CD44, NANOG, SNAI1 and ITGA6 is characteristic of the SPH, and therefore the combined expression of these genes could identify the CSC subpopulation in NSCLC tumors. By correlating the expression of these genes with the survival of the patients, a signature with prognostic value in ADC was obtained based on the expression of CDKN1A, SNAI1 and ITGA6. The role of proteins involved in EMT whose expression could promote cell migration and invasion in NSCLC has also been studied. Thus, it has been identified that the JunB transcription factor and the eIF5A2 translation factor are involved in the initiation of EMT mediated by TGF-ß1. Finally, 9 PDX models from tumors of NSCLC patients have been established. The correlation of implantation success of the tumor in mice with the clinical-pathological variables of the patients showed that primary tumors that generated PDX were derived from patients with bad prognosis. It was also observed that higher expression of Vimentin, Ezrin and Ki67 in tumors suggests higher aggressiveness and, therefore, the study of their expression could be used in combination as a prognostic marker to evaluate the progression of the disease. / Pardo Sánchez, JM. (2021). Aproximaciones moleculares para la identificación de nuevos biomarcadores y dianas terapéuticas del cáncer de pulmón no microcítico: células madre tumorales, transición epitelial-mesenquimal y modelos de xenoinjertos derivados de pacientes [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172736 / TESIS

Non-small cell lung cancer belongs to most frequent malignant tumours at all worldwide. Despite significant progress in knowledge about etiopathogenesis and targeted anticancer therapy, basic scientific research in this particular field and development of more effective treatment remains challenging. In case of its inadequate activation, the Hedgehog signaling pathway is involved in non-small cell cancer development. P53 is well known tumour suppressor gene, that serves as anticancer barrier. Its activity is mostly determined by the transcriptional activation of many pro-apoptotic genes, one of which is SIVA-1. Recently, it has been surprisingly shown, that SIVA-1 has also pro-oncogenic properties in a mouse model of non-small cell lung cancer. The aim of this study was to clarify the importance of Hedgehog signaling pathway and protein SIVA-1 and their potential relationship in development and progression of human non-small cell lung cancer. In selected cell lines of human non-small cell lung cancer, expression of each single component of Hedgehog signalign pathway was detected. In the tissue samples of tumour obtained from 39 patients that underwent surgery for non-small cell lung cancer and selected cell lines of the same tumour, expression of SIVA-1 protein was revealed. These findings indicate...

La prise en charge des cancers bronchiques localisés et localement avancés reste un challenge thérapeutique en cas de traitement par radiothérapie ou radio-chimiothérapie avec des taux d’échec importants. Il a été démontré que la Tomographie par Emission de Positons (TEP) au FDG était indispensable dans la stadification et la planification de la radiothérapie des cancers bronchiques non à petites cellules. De nombreux travaux ont proposé d’adapter le traitement de radiothérapie aux données de la TEP du métabolisme (FDG) mais également à partir de la TEP de l’hypoxie. Nous avons au cours des dernières années essayé de définir les différentes stratégies d’adaptation de la radiothérapie à partie des données de l’imagerie fonctionnelle. Dans le premier travail (travail 1), nous avons analysé les différentes méthodes de segmentation de traceurs à faible contraste pour obtenir des méthodes reproductibles et utilisables au cours d’essais thérapeutiques multicentriques. Ce travail a permis de définir une méthode de segmentation pour le FMISO (traceur de l’hypoxie) mais également pour la FLT qui permet de définir la prolifération. Le second travail (travail 2a et 2b) est le résultat de l’étude multicentrique d’augmentation de dose de radiothérapie à partir des données de la TEP FMISO. Dans ce travail, nous avons proposé de réaliser un boost de radiothérapie chez les patients présentant des tumeurs hypoxiques. Il a été démontré qu’une augmentation modérée de la dose de radiothérapie permettait d’obtenir le même contrôle local à 3 mois pour des tumeurs pourtant plus volumineuses et une tendance à un contrôle supérieur à 3 ans chez les patients ayant pu bénéficié d’un boost par rapport à ceux traités à 66Gy (26.5 mois versus 15.3 mois). Les travaux suivants (travaux 3 et 4) s’intéressent à l’hétérogénéité de fixation de la TEP-FDG et aux méthodes de segmentation de ce traceur en per-radiothérapie. Ces données permettent d’envisager des doses de radiothérapie hétérogènes avec des augmentations ciblées sur les volumes les plus hypermétaboliques en pré-traitement ou sur les volumes pour lesquels il persiste une fixation pathologique en cours de traitement. Les travaux 5 et 6 s’intéressent aux corrélations entre les zones les plus hypermétaboliques (FDG) et les zones hypoxiques (FMISO). Dans un premier temps, nous mettons en évidence le manque de corrélation entre ces 2 traceurs puis dans un second temps l’impact dosimétrique des différentes stratégies de radiothérapie adaptative basée sur la TEP du métabolisme ou de l’hypoxie. Dans le dernier travail (travail 7), nous avons comparé les résultats de deux traceurs de l’hypoxie chez des patients traités par chirurgie pour un cancer bronchique ; ces données ont été comparées aux données d’immunohistochimie pour permettre une meilleure connaissance des traceurs de l’hypoxie. L’ensemble de ces travaux doit permettre une meilleure identification des stratégies de radiothérapie adaptative basée sur l’imagerie fonctionnelle par TEP du métabolisme ou de l’hypoxie. / Résumé en anglais non disponible

<p>A multitude of neurologic diseases are increasing in patients that both diminish quality and quantity of life. My dissertation research focused on unraveling the blood-brain barrier’s alterations (BBB), primarily in lung cancer brain metastases, the most common brain metastasis in patients. We optimized a reliable and reproducible mouse model for creating brain metastases using patient derived brain seeking cells of non-small lung cancer (NSCLC) using ultrasound-guided intracardiac injection. I then evaluated brain tissue with qualitative and quantitative immunofluorescence for individual components of the BBB. Using this experimental method, I was able to identify the specific shift of each BBB component over time in NSCLC brain metastases. I then used human brain metastases specimens to demonstrate the clinical relevance of my findings. These results show distinct alterations in the BBB, which have the potential for targeting therapeutic delivery to extend patient survival. I was also able to characterize a novel epithelial-mesenchymal (EMT) phenotype in vertebral metastases of NSCLC in our model, with features similar to those seen in human patients. Most recently, I analyzed patterns of paracellular permeability associated with each BBB component of NSCLC brain metastases which may provide direct passageways for therapeutic delivery. Altogether, this research offered foundational evidence for the future development of targeted novel therapeutics, including nanoparticles. Outside of the brain metastases field, we used an experimental framework to successfully characterize the BBB alterations in a traumatic brain injury model (bTBI). These findings provided the first description of this unique pathology and the framework for developing therapeutics in other neurologic diseases. Although my research work has focused on animal models of disease, future directions based on my research work include the developing a novel 3D BBB-on-chip device to enable high throughput novel therapeutic delivery through the BBB. Long-term, identifying targetable alterations in the restrictive BBB using <i>in vitro</i> and <i>in vivo</i> models provides a potential conduit for effective prevention and treatment of a myriad of neurologic diseases to prolong patient survival and quality of life.</p>