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© 2019 by the authors., 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., This research was funded by Instituto de Salud Carlos III, grant PI17/01919, co-financed by the European Regional Development Fund (FEDER), and by Fundación Científica de la Asociación Española Contra el Cáncer (AECC), Grupos Clínicos Coordinados 2018. Carolina Herrero is supported by a predoctoral i-PFIS fellowship from Instituto de Salud Carlos III (IFI17/00047); Laura Muinelo is supported by Asociación Española Contra el Cáncer (AECC).

Colorectal cancer (CRC) is one of the major causes of cancer-related deaths. Early detection of tumor relapse is crucial for determining the most appropriate therapeutic management. In clinical practice, computed tomography (CT) is routinely used, but small tumor changes are difficult to visualize, and reliable blood-based prognostic and monitoring biomarkers are urgently needed. The aim of this study was to prospectively validate a gene expression panel (composed of GAPDH, VIL1, CLU, TIMP1, TLN1, LOXL3 and ZEB2) for detecting circulating tumor cells (CTCs) as prognostic and predictive tool in blood samples from 94 metastatic CRC (mCRC) patients. Patients with higher gene panel expression before treatment had a reduced progression-free survival (PFS) and overall-survival (OS) rates compared with patients with low expression (p = 0.003 and p ≤ 0.001, respectively). Patients with increased expression of CTCs markers during treatment presented PFS and OS times of 8.95 and 11.74 months, respectively, compared with 14.41 and 24.7 for patients presenting decreased expression (PFS; p = 0.020; OS; p ≤ 0.001). Patients classified as non-responders by CTCs with treatment, but classified as responders by CT scan, showed significantly shorter survival times (PFS: 8.53 vs. 11.70; OS: 10.37 vs. 24.13; months). In conclusion, our CTCs detection panel demonstrated efficacy for early treatment response assessment in mCRC patients, and with increased reliability compared to CT scan. ACIS (Axencia de Coñecemento en Saude); SERGAS. Cofinanced ERDF Funds 2007–2013

BACKGROUND: Remodeling targeted tissues for reception of tumor cells metastasizing from primary lesions is a consequence of communication between the tumor and the environment that governs metastasis. This study describes a novel approach that aims to disrupt the process of metastasis by interfering with this intense dialogue. METHODS: Proteomics and adhesion assays identified exosomes purified from the ascitic fluid of ovarian cancer patients (n = 9) as intermediaries of tumor cell attachment. A novel tumor cell capture device was fabricated by embedding exosomes onto a 3D scaffold (metastatic trap [M-Trap]). Murine models of ovarian metastasis (n = 3 to 34 mice per group) were used to demonstrate the efficacy of M-Trap to capture metastatic cells disseminating in the peritoneal cavity. Kaplan-Meier survival curves were used to estimate cumulative survival probabilities. All statistical tests were two-sided. RESULTS: The exosome-based M-Trap device promoted tumor cell adhesion with a nonpharmacological mode of action. M-Trap served as a preferential site for metastasis formation and completely remodeled the pattern of peritoneal metastasis in clinically relevant models of ovarian cancer. Most importantly, M-Trap demonstrated a statistically significant benefit in survival outcomes, with mean survival increasing from 117.5 to 198.8 days in the presence of M-Trap; removal of the device upon tumor cell capture further improved survival to a mean of 309.4 days (P < .001). CONCLUSIONS: A potent artificial premetastatic niche based on exosomes is an effective approach to impair the crosstalk between metastatic cells and their environment. In the clinical setting, the capacity to modulate the pattern of dissemination represents an opportunity to control the process of metastasis. In summary, M-Trap transforms a systemic, fatal disease into a focalized disease where proven therapeutic approaches such as surgery can extend survival.

Metastasis is the cause of most cancer deaths. Circulating tumour cells (CTCs) are cells released from the primary tumour into the bloodstream that are considered the main promoters of metastasis. Therefore, these cells are targets for understanding tumour biology and improving clinical management of the disease. Several techniques have emerged in recent years to isolate, detect, and characterise CTCs. As CTCs are a rare event, their study requires multidisciplinary considerations of both biological and physical properties. In addition, as isolation of viable cells may give further insights into metastatic development, cell recovery must be done with minimal cell damage. The ideal system for CTCs analysis must include maximum efficiency of detection in real time. In this sense, new approaches used to enrich CTCs from clinical samples have provided an important improvement in cell recovery. However, this progress should be accompanied by more efficient strategies of cell quantification. A range of biosensor platforms are being introduced into the technology for CTCs quantification with promising results. This review provides an update on recent progress in CTCs identification using different approaches based on sensor signaling.

The accuracy in the diagnosis of metastatic colorectal cancer (mCRC) represents one of the challenges in the clinical management of patients. The detection of circulating tumour cells (CTC) is becoming a promising alternative to current detection techniques, as it focuses on one of the players of the metastatic disease and it should provide with more specific and sensitive detection rates. Here, we describe an improved method of detection of CTC from mCRC patients by combining immune-enrichment, optimal purification of RNA from very low cell numbers, and the selection of accurate PCR probes. As a result, we obtained a logistic model that combines GAPDH and VIL1 normalized to CD45 rendering powerful results in the detection of CTC from mCRC patients (AUROC value 0.8599). We further demonstrated the utility of this model at the clinical setting, as a reliable prognosis tool to determine progression-free survival in mCRC patients. Overall, we developed a strategy that ameliorates the specificity and sensitivity in the detection of CTC, resulting in a robust and promising logistic model for the clinical management of metastatic colorectal cancer patients. Ministerio de Sanidad, Consumo y Bienestar Social. Beca número: CP08/00142 Programa Comisión Europea Fondo Europeo de Desarollo Regional (FEDER)

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.

Introducción El cáncer de endometrio es el más frecuente de los tumores infiltrantes del tracto genital inferior femenino. El adenocarcinoma endometrioide representa el 80% de todos los carcinomas de endometrio. Otros tipos histológicos menos frecuentes son el seroso o el de célula clara. Los tumores de endometrio se clasifican en dos grandes grupos en función de variables clínicas, histopatológicas y de su perfil de alteraciones moleculares: el cáncer de endometrio endometrioide o tumores tipo I, y cáncer de endometrio no endometrioide o tumores tipo II. El síntoma guía del cáncer de endometrio es el sangrado vaginal, que está presente en el 90% de los casos. El tratamiento primario es quirúrgico, lo que permite un correcta estadificación de la enfermedad. La radioterapia externa es el tratamiento adyuvante que ha demostrado mejores resultados, disminuyendo la tasa de recurrencias loco-regionales. La quimioterapia está indicada en casos avanzados al diagnóstico o en las recidivas. Existen diferentes modelos animales preclínicos para terapias experimentales oncológicas. Los de inoculación pueden ser ectópicos, si se desarrollan mediante la implantación de células o de tejido tumoral fuera del órgano en el que producen el tumor en el humano, u ortotópicos, si la inoculación se realiza en el mismo órgano en el que se produce el tumor en el humano. El modelo ortotópico ha demostrado que produce invasión tumoral y generación de metástasis de forma similar a la observada en el humano. Objetivos Generar y caracterizar un modelo murino ortotópico de cáncer de endometrio a partir de tejido tumoral humano de carcinoma de endometrio endometrioide y a partir de la línea celular comercial de cáncer de endometrio Hec1A. Material y métodos 1. Línea celular. Se utiliza la línea celular comercial Hec1A transfectada con el gen de la luciferasa para generar células Hec1A-Fluc. 2. Animales. Se usan ratones hembras atímicos Swiss Nude y Balb-c Nude de 5 semanas. 3. Tejido tumoral endometrial. Se seleccionan dos muestras de tejido endometrioide a partir de cirugías que se realizaron por la Unidad de Ginecología Oncológica de Hospital Vall d’Hebron. 4. Técnica de inoculación. Se estudian dos vías de inoculación diferentes: la vía transmiometrial y la transvaginal. 5. Seguimiento. Se evalúa el seguimiento mediante bioluminiscencia in vivo en el modelo derivado de células Hec1A-Fluc, y mediante ecografía transabdominal y resonancia magnética en el modelo desarrollado a partir de tejido tumoral humano. 6. Necropsia, estudio mediante bioluminiscencia ex vivo, estudio histológico e inmunohistoquímico. Resultados La técnica de inoculación transmiometrial es superior a la transvaginal. En el modelo ortotópico derivado de células Hec1A-Fluc, el 85% de los animales que presentaron tumor endometrial generaron además implantes pélvicos, diseminación linfática en el 92% de los animales e implantes abdominales en el 75%. Las metástasis hematógenas a pulmón se detectaron en el 71% de los animales. En el modelo ortotópico derivado de tejido tumoral endometrioide, el 77% de los animales presentó diseminación en la cavidad pélvica, y el 66% presentó además implantes abdominales. Conclusiones 1. Es posible generar un modelo murino ortotópico de cáncer de endometrio a partir de tejido tumoral humano endometrioide. Su perfil histológico e inmunohistoquímico es similar a los tumores de endometrio tipo I. Su perfil de diseminación metastásica representa a un tumor localmente avanzado, con afectación pélvica y ganglionar. 2. Es posible generar un modelo murino ortotópico de cáncer de endometrio a partir de células Hec1A-Fluc. El seguimiento mediante bioluminiscencia in vivo permite el estudio del crecimiento del tumor y del perfil de desarrollo de metástasis. El estudio inmunohistoquímico del tumor es similar al del cáncer de endometrio tipo II. El patrón de diseminación representa a un tumor altamente metastásico, con diseminación linfática, transcelómica y hematógena., Introduction Endometrial cancer is the most common of the tumors infiltrating the lower female genital tract. Endometrioid adenocarcinoma accounts for 80% of all endometrial carcinomas. Other less common histological types are serous or clear cell. Endometrial tumors are classified into two major groups based on clinical, histopathological and molecular alterations profile: endometrioid endometrial cancer or type I tumors, and non-endometrioid endometrial cancer or type II. The leading symptom of endometrial cancer is vaginal bleeding, which is present in 90% of cases. Primary treatment is surgery, allowing accurate staging of the disease. External radiation therapy is the adjuvant treatment that has shown better results, decreasing the rate of loco-regional recurrences. Chemotherapy is indicated in advanced cases at diagnosis or in recurrences. There are several preclinical animal models for experimental oncology. The inoculation model can be ectopic, if developed by implanting tumor tissue or cells outside the organ in which the tumor occurs in humans, or orthotopic, if the inoculation is done in the same organ or tissue in which tumor occurs in humans. Orthotopic model can generate tumor invasion and metastasis similarly to that observed in humans. Objectives Generate and characterize an orthotopic murine model of endometrial cancer derived from tumor tissue of human endometrial carcinoma and from the commercial endometrial cancer cell line HEC1A. Material and methods 1. Cell line. Using the commercial cell line HEC1A transfected with the luciferase gene to generate Fluc-HEC1A cells. 2. Animals. We use female athymic mice Swiss nude and Balb-c nude. 3. Endometrial tumor tissue. We select two endometrial tissue samples from surgeries that were performed by the Gynecologic Oncology Unit of Hospital Vall d'Hebron. 4. Inoculation technique. We study two different inoculation routes: transmiometrial and transvaginal. 5. Follow-up. Assessed by in vivo bioluminescence in the model derived from HEC1A-Fluc cells and by transabdominal ultrasound and magnetic resonance in the model developed from human tumor tissue. 6. Necropsy, ex vivo bioluminescence study, histological and immunohistochemical study. Results Transmiometrial inoculation technique is superior to the transvaginal. In the orthotopic model derived from HEC1A-Fluc cells, pelvic implants were observed in 85% of the animals that showed endometrial tumor, lymphatic dissemination in 92% of the animals and abdominal implants in 75%. Hematogenous metastases to the lung were detected in 71% of animals. In the orthotopic model derived from endometrial tumor tissue, 77% of the animals showed spread into the pelvic cavity, and 66% had abdominal implants. Conclusions 1. It is possible to generate an orthotopic mouse model of endometrial cancer from human endometrial tumor tissue. The histological and immunohistochemical profile is similar to type I endometrial tumors. It shows the metastatic spread of a locally advanced tumor with pelvic implants and lymph node involvement. 2. It is possible to generate an orthotopic murine model of endometrial cancer from HEC1A-Fluc cancer cells. The follow-up by using in vivo bioluminescence allows the study of tumor growth and metastasis development. The immunohistochemical study of the tumor is similar to the type II endometrial cancer. The pattern of spread depicts a highly metastatic tumor with lymphatic, transcoelomic and hematogenous spread.

El càncer d'endometri és la malaltia ginecològica més comuna i representa la quarta neoplàsia més freqüent en la dona en els països desenvolupats. Actualment és possible diagnosticar el 80% dels casos de EEC en estadiatge I FIGO, per tant, poder aplicar cirurgia i presentar un bon pronòstic. L'estadiatge I FIGO inclou tres subtipus: afecta l'endometri (IA), infiltra el miometri 50% (IC). El nostre grup es va centrar en la identificació de nous gens implicats en el EEC mitjançant l'estudi dels patrons d'expressió gènica diferencial entre teixit endometrial sa, hiperplàsic i tumoral, així com la cerca de nous gens associats al fenotip carcinomatós endometrial amb potencial valor diagnòstic i/o pronòstic. Es va trobar que els dos gens majorment sobreexpressats eren RUNX1/AML1 i ERM/ETV5 i alhora, es troben sobreexpressats en la fase en la qual el EEC esdevé invasiu. Partint d'aquí, vam plantejar la caracterització del mecanisme d'acció del factor de transcripció ERM/ETV5 en el EEC durant els esdeveniments inicials d'invasió i disseminació. La sobreexpressió d'ERM/ETV5 a la línia cel·lular de càncer d'endometri Hec-1A indueix dispersió cel·lular que correlaciona amb un augment de l'activitat gelatinasa de la MMP2. Tant els experiments de ChiP com amb iRNA o l'ús d'un inhibidor específic de MMP2 mostren un nexe funcional entre la sobreexpressió d'ERM/ETV5 i l'activació de MMP2. En el model animal ortotòpic de EEC es demostra que l'augment de l'activitat de MMP2 associat a la sobreexpressió d'ERM/ETV5 confereix major capacitat invasiva als tumors endometrials i aquests mostren un patró més agressiu i infiltrant. Es va confirmar que ERM/ETV5 tenia un paper en els passos inicials de la disseminació endometrial ja que es van localitzar ERM/ETV5 i MMP-2 en el front d'invasió de mostres de EEC humanes amb infiltració miometrial. Per tant podem proposar que en el EEC, ERM/ETV5 actua a través de l'activitat gelatinolítica de MMP-2 per a donar major capacitat invasiva, associat al punt d'inici de la infiltració miometrial. Tot seguit vam plantejar determinar les alteracions moleculars associades a la sobreexpressió del factor de transcripció ERM/ETV5 durant la infiltració miometrial en el EEC, identificant nous marcadors moleculars o dianes terapèutiques que podrien estar sota el control de l'activitat transcripcional d'ERM/ETV5. L'anàlisi comparatiu mitjançant 2D-DIGE i espectrometria de masses dels patrons d'expressió proteica diferencial de la línia cel·lular que sobreexpressa de forma estable GFP-ERM/ETV5 anvers la línia cel·lular sense transfectar i la que presenta el vector GFP buit, determina un llistat de proteïnes diferencialment expressades que es troben implicades en la regulació d'actina i la senyalització per TGF-beta i progesterona. Vam caracteritzar la sobreexpressió específica de la proteïna Hep27 que presenta localització mitocondrial depenent d'ERM/ETV5. Estudis funcionals van demostrar associació amb l'estrès oxidatiu. L'anàlisi dels resultats obtinguts reforcen el paper d'ERM/ETV5 com a factor regulador de la migració i invasió tumoral, i assenyala la seva implicació en la resposta a l'estrès oxidatiu associat a l'inici de la invasió en el carcinoma endometrial. Fins ara, la immunohistoquímica ha estat l'eina utilitzada per a la identificació de proteïnes implicades en la invasió del carcinoma endometrial, sense tenir en compte la percepció global del front d'invasió. En aquest últim treball hem dut a terme una aproximació proteòmica per a caracteritzar components específics del front d'invasió o l'estroma reactiu mitjançant la comparació de l'àrea invasiva d'un carcinoma endometrial anvers l'àrea tumoral superficial no invasiva i el teixit normal provinents de la mateixa pacient. Així hem pogut identificar proteïnes diferencialment expressades en el front d'invasió i que es troben implicades en la morfologia cel·lular, acoblament i moviment, així com els mecanismes moleculars relacionats amb la senyalització i interacció cèl·lula-cèl·lula i la resposta moduladora a l'estrès oxidatiu., We have recently described the Ets family transcription factor, ERM/ETV5, specifically up-regulated in EEC, and associated with myometrial infiltration. Ets family members have been correlated to tumor progression by up-regulating the expression of matrix-degrading proteases. We investigated the possibility that in EEC, ERM/ETV5 may induce the expression of genes involved in extra-cellular matrix remodeling. The overexpression of ERM/ETV5 induced scattering in the EEC cell line Hec-1A, correlating to increased MMP-2 gelatinase activity. Both ChIP and iRNA experiments and specific MMP-2 inhibitor demonstrated a functional link between ERM/ETV5 overexpression and MMP-2 activation. Orthotopically implanted overexpressing ERM/ETV5 tumors presented a more aggressive and infiltrative pattern of myometrial invasion. The specific localization of ERM/ETV5 and MMP-2 at the invasive front of myometrial infiltrating human EEC reinforced the hypothesis of a role for ERM/ETV5 in the early steps of endometrial dissemination.To understand the role of ETV5 during myometrial infiltration, we analysed by 2D-DIGE technology those proteins whose expression was altered in endometrial cell lines stably over-expressing ERM/ETV5. Pathway analysis pointed to actin regulation and TGF-beta and progesterone signalling as processes regulated by ERM/ETV5. We characterized the specific up-regulation of the nuclear dehydrogenase/reductase Hep27, its ERM/ETV5-dependent mitochondrial localization, and functional studies demonstrated a link with oxidative stress.Overall, the ETV5-related proteomic approach performed in the Hec-1A cell line reinforces a role of this transcription factor in the regulation of the migratory and invasive tumour behaviour, and points to a modulated response to oxidative stress associated with the promotion of invasion in endometrial cancer. To date, the identification of proteins involved in endometrial carcinoma invasion has been essentially conducted by immunohistochemical methods, without a global perception on the invasive front. In this work we attempted a proteomic approach to characterise specific components of the invasive front or reactive stroma by comparing the invasive area of an endometrial carcinoma with the non¬invasive superficial area and normal tissue from the same patients. This led us to identify proteins involved in cellular morphology, assembly and movement, differentially expressed at the invasive front, as well as pathways like cell-to-cell signalling and interaction and a modulated response to oxidative stress as events related to endometrial carcinoma invasion. In conclusion, we describe a novel proteomic approach that specifically deals with endometrial carcinoma invasion front, allowing the identification of new players of myometrial infiltration.