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Analyzing different tumor regions by next generation sequencing allows the assessment of intratumor genetic heterogeneity (ITGH), a phenomenon that has been studied widely in some tumor types but has been less well explored in endometrial carcinoma (EC). In this study, we sought to characterize the spatial and temporal heterogeneity of 9 different ECs using whole-exome sequencing, and by performing targeted sequencing validation of the 42 primary tumor regions and 30 metastatic samples analyzed. In addition, copy number alterations of serous carcinomas were assessed by comparative genomic hybridization arrays. From the somatic mutations, identified by whole-exome sequencing, 532 were validated by targeted sequencing. Based on these data, the phylogenetic tree reconstructed for each case allowed us to establish the tumors' evolution and correlate this to tumor progression, prognosis, and the presence of recurrent disease. Moreover, we studied the genetic landscape of an ambiguous EC and the molecular profile obtained was used to guide the selection of a potential personalized therapy for this patient, which was subsequently validated by preclinical testing in patient-derived xenograft models. Overall, our study reveals the impact of analyzing different tumor regions to decipher the ITGH in ECs, which could help make the best treatment decision

Introducción: Los tumores endometriales constituyen la neoplasia ginecológica más frecuente en mujeres occidentales, mientras que el cáncer ovárico es la de mayor tasa de mortalidad. El desarrollo de nuevos modelos preclínicos tridimensionales (organoides) que reproduzcan las características específicas del tumor permite llevar a cabo estudios de caracterización molecular y screening farmacológico, lo que podría orientar la selección de tratamientos y el pronóstico de las pacientes, de especial importancia en casos de alto riesgo, recidiva o progresión de la enfermedad a varias líneas de tratamiento. El principal objetivo de este trabajo es presentar un procedimiento del desarrollo de organoides a partir de muestras mínimamente invasivas de pacientes de cáncer de endometrio y ovario. Materiales y métodos: Se obtuvieron muestras de pacientes de cáncer endometrial (n=6) y cáncer ovárico (n=1). Se desarrolló un método de procesamiento para obtener una suspensión de célula única epitelial. Se compararon cualitativamente distintos protocolos descritos en la literatura, tanto a nivel de eficiencia de generación y rendimiento de los cultivos (BME droplet y BME bilayer organoid culture), como de mantenimiento (químico, mecánico y enzimático). Se llevó a cabo una caracterización preliminar de los organoides de tumor ovárico mediante inmunofluorescencia. Resultados: El método desarrollado permitió obtener una suspensión de células tumorales con una viabilidad >75% en todas las muestras. Se identificaron diferentes patrones morfológicos en las estructuras generadas. El método de cultivo BME droplets y el mecánico resultaron ser los más eficientes. La caracterización de los organoides de ovario demostró la expresión de PAX8, vimentina y citoqueratinas. Conclusiones: La tasa de éxito en la generación de organoides y el rendimiento sufrieron gran variabilidad, no pudiendo ser esta atribuida a las diferencias de viabilidad de la suspensión celular de partida. Se destaca la necesid, Introdución: Os tumores endometriais constitúen a neoplasia xinecolóxica máis frecuente nas mulleres occidentais, namentres que o cancro ovárico é a de maior taxa de mortalidade. O desenvolvemento de novos modelos preclínicos tridimensionais (organoides) que reproduzan as características específicas do tumor permite levar a cabo estudos de caracterización molecular e cribado farmacolóxico, o que podería orientar a selección de tratamentos e o pronóstico das pacientes, de especial importancia nos casos de alto risco, recidiva ou progresión da enfermidade a varias liñas de tratamento. O principal obxectivo deste traballo é presentar un procedemento da xeración de organoides a partir de mostras minimamente invasivas de pacientes de cancro de endometrio e ovario. Materiais e métodos: Obtivéronse mostras de pacientes de cancro endometrial (n=6) e cancro ovárico (n=1). Desenvolveuse un método de procesamento para obter unha suspensión de célula única epitelial. Comparáronse cualitativamente distintos protocolos descritos na literatura, tanto a nivel de eficiencia de xeración e rendemento dos cultivos (BME droplet e BME bilayer organoid culture), como de mantemento dos mesmos (químico, mecánico e encimático). Levouse a cabo unha caracterización preliminar dos organoides de tumor ovárico mediante inmunofluorescencia. Resultados: O método desenvolvido permitiu obter unha suspensión de células tumorais cunha viabilidade >75% en todas as mostras. Identificáronse diferentes patróns morfolóxicos nas estruturas xeradas. O método de cultivo de BME droplets e o mecánico resultaron ser os de maior eficiencia. A caracterización dos organoides de ovario demostrou a expresión de PAX8, vimentina e citoqueratinas. Conclusións: A taxa de éxito na xeración de organoides e no rendemento do cultivo sufriu unha grande variabilidade, non podendo ser atribuida ás diferenzas de viabilidade da suspensión celular de partida. Destácase a necesidade de estandarizar protocolos e desenvolver estudos q, Background: Endometrial tumors constitute the most frequent gynecological neoplasia in occidental women, whereas ovarian cancer is the one with the highest mortality rate. New 3D preclinical models (organoids) development that recapitulate specific characteristics of the original tumor allows to drive molecular characterization tests and drug screening, which could lead treatment selection and patient’s prognosis, especially important in high-risk cases, relapse or illness progression to several lines of therapy. The main objective of this project is to show a development organoid procedure from minimally invasive samples of endometrial and ovarian cancer patients. Methods: Endometrial cancer (n=6) and ovarian cancer (n=1) samples were obtained. A method for processing samples to achieve an epithelial single-cell suspension was developed. Several protocols described in literature were qualitatively compared, in terms of efficiency of the generation of structures comparing two techniques (BME droplet and BME bilayer organoid culture), and in terms of culture maintenance (chemical, mechanic and enzymatic). A preliminary characterization of ovarian organoids was conducted by immunofluorescence. Results: The developed method allowed to obtain a tumoral single-cell suspension with a viability >75% in all the samples. Different morphological patterns in the generated structures were observed. BME droplets culture and mechanic method showed to be the most efficient ones. Ovarian organoid characterization revealed PAX8, vimentin and cytokeratins expression. Conclusions: The success rate of organoids generation suffered high variability, which could not be attributed to differences in the suspension cell viability. It is highlighted the need of standardized protocols and studies to support clinical implementation

La medicina de precisión basada en la biopsia líquida es una herramienta mínimamente invasiva que permite la monitorización en tiempo real de la evolución del tumor mediante el análisis de los ácidos nucleicos libres circulantes, las células tumorales circulantes (CTC) y las vesículas extracelulares (VEs). En esta tesis, por una parte, se estudió el proceso de adhesión y anidamiento de las CTC en el contexto del carcinoma endometrial mediante modelos in vitro e in vivo y se buscaron estrategias para bloquear el proceso de metástasis. Por otra parte, se desarrolló y optimizó la tecnología ExoGAG, una nueva herramienta de aislamiento de VEs basada en la unión de un agente de precipitación a los glicosaminoglicanos presentes en la superficie de las VEs. Además, se estudiaron diferentes aplicaciones de la tecnología ExoGAG en la búsqueda de biomarcadores proteicos y genéticos presentes en las VEs. Finalmente, se establecieron los mecanismos que permitieron su comercialización y se analizaron las estrategias para el traslado de su uso a la práctica clínica.

Background: Cancer metastasis is a deathly process, and a better understanding of the different steps is needed. The shedding of circulating tumor cells (CTCs) and CTC-cluster from the primary tumor, its survival in circulation, and homing are key events of the metastasis cascade. In vitro models of CTCs and in vivo models of metastasis represent an excellent opportunity to delve into the behavior of metastatic cells, to gain understanding on how secondary tumors appear. Methods: Using the zebrafish embryo, in combination with the mouse and in vitro assays, as an in vivo model of the spatiotemporal development of metastases, we study the metastatic competency of breast cancer CTCs and CTC-clusters and the molecular mechanisms. Results: CTC-clusters disseminated at a lower frequency than single CTCs in the zebrafish and showed a reduced capacity to invade. A temporal follow-up of the behavior of disseminated CTCs showed a higher survival and proliferation capacity of CTC-clusters, supported by their increased resistance to fluid shear stress. These data were corroborated in mouse studies. In addition, a differential gene signature was observed, with CTC-clusters upregulating cell cycle and stemness related genes. Conclusions: The zebrafish embryo is a valuable model system to understand the biology of breast cancer CTCs and CTC-clusters.

UDHEBRON, The incidence and mortality of endometrial cancer (EC) have risen in recent years, hence more precise management is needed. Therefore, we combined different types of liquid biopsies to better characterize the genetic landscape of EC in a non-invasive and dynamic manner. Uterine aspirates (UAs) from 60 patients with EC were obtained during surgery and analyzed by next-generation sequencing (NGS). Blood samples, collected at surgery, were used for cell-free DNA (cfDNA) and circulating tumor cell (CTC) analyses. Finally, personalized therapies were tested in patient-derived xenografts (PDXs) generated from the UAs. NGS analyses revealed the presence of genetic alterations in 93% of the tumors. Circulating tumor DNA (ctDNA) was present in 41.2% of cases, mainly in patients with high-risk tumors, thus indicating a clear association with a more aggressive disease. Accordingly, the results obtained during the post-surgery follow-up indicated the presence of ctDNA in three patients with progressive disease. Moreover, 38.9% of patients were positive for CTCs at surgery. Finally, the efficacy of targeted therapies based on the UA-specific mutational landscape was demonstrated in PDX models. Our study indicates the potential clinical applicability of a personalized strategy based on a combination of different liquid biopsies to characterize and monitor tumor evolution, and to identify targeted therapies

Background: Current therapeutic options in the course of metastatic castration-resistant prostate cancers (mCRPC) reinforce the need for reliable tools to characterize the tumor in a dynamic way. Circulating tumor cells (CTCs) have emerged as a viable solution to the problem, whereby patients with a variety of solid tumors, including PC, often do not have recent tumor tissue available for analysis. The biomarker characterization in CTCs could provide insights into the current state of the disease and an overall picture of the intra-tumor heterogeneity. Methods: in the present study, we applied a global gene expression characterization of the CTC population from mCRPC (n = 9), with the goal to better understand the biology of these cells and identify the relevant molecules favoring this tumor progression. Results: This analysis allowed the identification of 50 genes specifically expressed in CTCs from patients. Six of these markers (HOXB13, QKI, MAOA, MOSPD1, SDK1, and FGD4), were validated in a cohort of 28 mCRPC, showing clinical interest for the management of these patients. Of note, the activity of this CTC signature was related to the regulation of MYC, a gene strongly implicated in the biology of mCRPC. Conclusions: Overall, our results represent new evidence on the great value of CTCs as a non-invasive biopsy to characterize PC

Background: Late state colorectal cancer treatments have important side effects that should be avoided in patients where drug effectiveness is not adequate. PrediCTC is a new biomarkers blood test developed to determinate the chemotherapy response in unresectable metastatic colorectal cancer patients that could allow to obviate unnecessary treatments. Aim: To assess from the Spanish Societal Perspective the cost-utility of the test PrediCTC compared to the computed tomography in aim to evaluate chemotherapy treatment response in late stage colorectal cancer patients. Methods: Based on the results of Barbazán et al., a Markov model has been developed, in which the different lines and cycles that the colorectal patient can receive and how they can move between them according to the computed tomography or PrediCTC have been represented. The effectiveness has been expressed in quality adjusted life years (QALYs), avoiding adverse events. Results: Base case analysis shows savings in different types of costs for PrediCTC (per patient): €14.30 in those arise from adverse events, €22,345.73 in chemotherapy costs, €4849.61 in other direct costs, and €306.21 in indirect costs. Although computed tomography 12-week assessed patients gain 0.17 QALYs compared with PrediCTC. Conclusions: From the Spanish Societal Perspective, PrediCTC is not a cost-utility option but allows to identify earlier patients who are not benefiting from first-line chemotherapy avoiding unnecessary side effects and costs.

Tumor-derived extracellular vesicles (EVs) are secreted in large amounts into biological fluids of cancer patients. The analysis of EVs cargoes has been associated with patient´s outcome and response to therapy. However, current technologies for EVs isolation are tedious and low cost-efficient for routine clinical implementation. To explore the clinical value of circulating EVs analysis we attempted a proof-of-concept in endometrial cancer (EC) with ExoGAG, an easy to use and highly efficient new technology to enrich EVs. Technical performance was first evaluated using EVs secreted by Hec1A cells. Then, the clinical value of this strategy was questioned by analyzing the levels of two well-known tissue biomarkers in EC, L1 cell adhesion molecule (L1CAM) and Annexin A2 (ANXA2), in EVs purified from plasma in a cohort of 41 EC patients and 20 healthy controls. The results demonstrated the specific content of ANXA2 in the purified EVs fraction, with an accurate sensitivity and specificity for EC diagnosis. Importantly, high ANXA2 levels in circulating EVs were associated with high risk of recurrence and non-endometrioid histology suggesting a potential value as a prognostic biomarker in EC. These results also confirmed ExoGAG technology as a robust technique for the clinical implementation of circulating EVs analyses

Understanding hemodynamics in blood circulation is crucial in order to unveil the mechanisms underlying the formation of stenosis and atherosclerosis. In fact, there are experimental evidences pointing out to the existence of some given vessel configurations that are more likely to develop the above mentioned pathologies. Along this manuscript, we performed an exhaustive investigation in a simplified model aiming to characterize by means of physical quantities those regions and configurations in vessel bifurcations that are more likely to develop such pathologies. The two-fold analysis is based, on the one hand, on numerical simulations (via CFD) and, on the other hand, on experiments realized in an ad-hoc designed polydimethylsiloxane (PDMS) channel with the appropriate parameters and appropriate fluid flows. The results obtained demonstrate that low velocity regions and low shear stress zones are located in the outer walls of bifurcations. In fact, we found that there is a critical range of bifurcation angles that is more likely to vascular disease than the others in correspondence with some experimental evidence. The effect of the inflow velocity on this critical range is also analyzed

Background Zebrafish (Danio rerio) is a model organism that has emerged as a tool for cancer research, cancer being the second most common cause of death after cardiovascular disease for humans in the developed world. Zebrafish is a useful model for xenotransplantation of human cancer cells and toxicity studies of different chemotherapeutic compounds in vivo. Compared to the murine model, the zebrafish model is faster, can be screened using high-throughput methods and has a lower maintenance cost, making it possible and affordable to create personalized therapies. While several methods for cell proliferation determination based on image acquisition and quantification have been developed, some drawbacks still remain. In the xenotransplantation technique, quantification of cellular proliferation in vivo is critical to standardize the process for future preclinical applications of the model. Methods This study improved the conditions of the xenotransplantation technique – quantification of cellular proliferation in vivo was performed through image processing with our ZFtool software and optimization of temperature in order to standardize the process for a future preclinical applications. ZFtool was developed to establish a base threshold that eliminates embryo auto-fluorescence and measures the area of marked cells (GFP) and the intensity of those cells to define a ‘proliferation index’. Results The analysis of tumor cell proliferation at different temperatures (34 °C and 36 °C) in comparison to in vitro cell proliferation provides of a better proliferation rate, achieved as expected at 36°, a maintenance temperature not demonstrated up to now. The mortality of the embryos remained between 5% and 15%. 5- Fluorouracil was tested for 2 days, dissolved in the incubation medium, in order to quantify the reduction of the tumor mass injected. In almost all of the embryos incubated at 36 °C and incubated with 5-Fluorouracil, there was a significant tumor cell reduction compar

Circulating tumour cells (CTCs) play a key role in the metastasis process, as they are responsible for micrometastasis and are a valuable tool for monitoring patients in real-time. Moreover, efforts to develop new strategies for CTCs isolation and characterisation, and the translation of CTCs into clinical practice needs to overcome the limitation associated with the sole use of Epithelial Cell Adhesion Molecule (EpCAM) expression to purify this tumour cell subpopulation. CTCs are rare events in the blood of patients and are believed to represent the epithelial population from a primary tumour of epithelial origin, thus EpCAM immunoisolation is considered an appropriate strategy. The controversy stems from the impact that the more aggressive mesenchymal tumour phenotypes might have on the whole CTC population. In this work, we first characterised a panel of cell lines representative of tumour heterogeneity, confirming the existence of tumour cell subpopulations with restricted epithelial features and supporting the limitations of EpCAM-based technologies. We next developed customised polystyrene magnetic beads coated with antibodies to efficiently isolate the phenotypically different subpopulations of CTCs from the peripheral blood mononuclear cells (PBMCs) of patients with metastatic cancer. Besides EpCAM, we propose Epidermal Growth Factor Receptor (EGFR) as an additional isolation marker for efficient CTCs detection.

Background Metastases are the most common reason of cancer death in patients with solid tumors. Lymph nodes, once invaded by tumor cells, act as reservoirs before cancer cells spread to distant organs. To address the limited access of intravenously infused chemotherapeutics to the lymph nodes, we have developed PEGylated polyglutamic acid nanocapsules (PGA-PEG NCs), which have shown ability to reach and to accumulate in the lymphatic nodes and could therefore act as nanotransporters. Once in the lymphatics, the idea is that these nanocapsules would selectively interact with cancer cells, while avoiding non-specific interactions with immune cells and the appearance of subsequent immunotoxicity. Results The potential of the PGA-PEG NCs, with a mean size of 100 nm and a negative zeta potential, to selectively reach metastatic cancer cells, has been explored in a novel 3D model that mimics an infiltrated lymph node. Our 3D model, a co-culture of cancer cells and lymphocytes, allows performing experiments under dynamic conditions that simulate the lymphatic flow. After perfusion of the nanocarriers, we observe a selective interaction with the tumor cells. Efficacy studies manifest the need to develop specific therapies addressed to treat metastatic cells that can be in a dormant state. Conclusions We provide evidence of the ability of PGA-PEG NCs to selectively interact with the tumor cells in presence of lymphocytes, highlighting their potential in cancer therapeutics. We also state the importance of designing precise in vitro models that allow performing mechanistic assays, to efficiently develop and evaluate specific therapies to confront the formation of metastasis

Knowledge on the molecular mechanisms underlying metastasis colonization in Non-Small Cell Lung Cancer (NSCLC) remains incomplete. A complete overview integrating driver mutations, primary tumour heterogeneity and overt metastasis lacks the dynamic contribution of disseminating metastatic cells due to the inaccessibility to the molecular profiling of Circulating Tumour Cells (CTCs). By combining immunoisolation and whole genome amplification, we performed a global gene expression analysis of EpCAM positive CTCs from advanced NSCLC patients. We identified an EpCAM+ CTC-specific expression profile in NSCLC patients mostly associated with cellular movement, cell adhesion and cell-to-cell signalling mediated by PI3K/AKT, ERK1/2 and NF-kB pathways. NOTCH1 emerged as a driver connecting active signalling pathways, with a reduced number of related candidate genes (NOTCH1, PTP4A3, LGALS3 and ITGB3) being further validated by RT-qPCR on an independent cohort of NSCLC patients. In addition, these markers demonstrated high prognostic value for Progression-Free Survival (PFS). In conclusion, molecular characterization of EpCAM+ CTCs from advanced NSCLC patients provided with highly specific biomarkers with potential applicability as a “liquid biopsy” for monitoring of NSCLC patients and confirmed NOTCH1 as a potential therapeutic target to block lung cancer dissemination.

La metástasis está implicada en la mayoría de muertes vinculadas a procesos tumorales. La metástasis constituye una secuencia de eventos complejos en el que la célula tumoral alcanza la circulación y coloniza tejidos distantes. Este proceso no es meramente un proceso invasivo, sino que va acompañado de una gran plasticidad influenciada por el ambiente tumoral. El trabajo desarrollado en esta tesis doctoral tiene como objetivo profundizar en el conocimiento del modo en el que se desarrolla la diseminación metastásica a partir de dos de sus participantes: la Célula Tumoral Circulante (CTC) y el exosoma tumoral como vesícula de comunicación entre la célula y su entorno. Para ello hemos elaborado un perfil de expresión específico de CTCs en cáncer de pulmón NSCLC a la vez que hemos llevado a cabo la caracterización proteómica del exosoma de la línea de cáncer de endometrio HEC-1A englobada dentro de un marco de EMT (Transición Epitelio-Mesénquima). Los resultados arrojan un perfil de expresión de EMT asociado a la supervivencia y movilidad celular en las CTCs de cáncer de pulmón NSCLC; a la vez que la caracterización proteómica destaca la influencia del proceso de EMT en el acondicionamiento del nicho premetastásico y el anidamiento de la célula tumoral.