Caracterización de células madre tumorales en cáncer de pulmón no microcítico

[EN] Background: Despite the advances in the molecular characterization of lung cancer, chemoresistance, tumor progression and metastasis make of lung cancer the first cause of death cancer-related worldwide. Cancer stem cells (CSCs) are small subpopulations of stem-like cells with self-renewal, differentiation and tumorigenic properties that constitute a promising target, but remain largely unknown. The aim of this study was to isolate and analyze gene expression of CSCs from lung cancer cell-lines and tumor-tissue from resectable NSCLC patients. Methods: This study was performed on cells from NSCLC tumor samples and cell lines (H1650, H1993, A549 and PC9) grown in monolayer and as spheroids. The expression of: CSC-markers (CD133, EPCAM1, ALDH1A1, CD166, ABCG2, CD44, MUC1, BMI1); pluripotency (KLF4, OCT4, NANOG, SOX2, MYC, CCND1); cell cycle (CDKN1A, CDKN2A, MDM2, WEE1); invasiveness (CDH1, VIM, SNAI1, MMP2, MMP9, CEACAM5); Notch pathway (NOTCH1, NOTCH2, NOTCH3, DLL1, DLL4, HEY1, HES1); Wnt pathway (WNT1, WNT2, WNT3, WNT5A, CTNBB1, DKK1, FZD7) and Hedgehog pathway (SMO, PTCH1, SHH, GLI1) were analyzed by quantitative real-time PCR (qPCR). Housekeeping genes ACTB, CDKN1B and GUSB were used as endogenous controls for relative expression calculation. Results: Lung tumorspheres had increased expression of EPCAM1, CD44, ALDH1A1 and CDKN1A (p= 0.028, p= 0.021, p= 0.043 and p= 0.021, respectively) when compared to their paired-adherent cells. In addition, epithelial to mesenquimal transition (EMT) inducer SNAI1 was overexpressed (p= 0.011) in tumorspheres. Regarding Notch pathway, DLL4, NOTCH1 and NOTCH2 showed higher expression in spheroids (p= 0.028, p= 0.038 and p= 0.036, respectively). In Wnt pathway, we found higher expression levels of WNT3, CTNBB1 and GSK3B (p= 0.021, p= 0.008 and p= 0.021, respectively) in lungspheres, whereas the activator of the non-canonical Wnt pathway, WNT5A, tended to be less expressed in spheroids compared to adherent-cultured cells. No significant differences were found in other analyzed genes. Conclusions: Lung spheroids from cancer cell lines and primary tumors showed increased levels of CSC-markers. Genes related to Notch and Wnt were found to be more expressed in tumorspheres, suggesting these pathways as interesting lung-CSC targets.[ES] Introducción: A pesar de los avances en la caracterización molecular del cáncer de pulmón, la resistencia a la quimioterapia, la progresión tumoral y la metástasis hacen del mismo la primera causa de muerte debida a cáncer a nivel mundial. Las células madre tumorales (CSC) son pequeñas subpoblaciones de células con capacidad de autorenovación, diferenciación y tumorigenicidad que constituyen una diana terapéutica prometedora, pero cuya caracterización es aún un campo poco explorado. El objetivo de este trabajo es aislar y analizar la expresión génica de CSCs procedentes de líneas celulares de cáncer de pulmón y de tejido tumoral de pacientes con cáncer de pulmón no microcítico (CPNM) en estadios resecables. Material y Métodos: El estudio se realizó en líneas celulares (H1650, H1993, A549 and PC9) y muestras tumorales de pacientes resecados con CPNM crecidas en monocapa y en placas de baja adherencia con medio sin suero (tumoresferas). La expresión de marcadores de CSC (CD133, EPCAM1, ALDH1A1, CD166, ABCG2, CD44, MUC1, BMI1), genes de pluripotencia (KLF4, OCT4, NANOG, SOX2, MYC, CCND1), genes reguladores del ciclo celular (CDKN1A, CDKN2A, MDM2, WEE1), genes asociados a metástasis (CDH1, VIM, SNAI1, MMP2, MMP9, CEACAM5); y genes de las vías de señalización Notch (NOTCH1, NOTCH2, NOTCH3, DLL1, DLL4, HEY1, HES1); Wnt (WNT1, WNT2, WNT3, WNT5A, CTNBB1, DKK1, FZD7) y Hedgehog (SMO, PTCH1, SHH, GLI1) fue analizada mediante PCR cuantitativa a tiempo real (qPCR), normalizándose frente a la expresión de tres genes controles seleccionados: ACTB, CDKN1B y GUSB , utilizados para el cálculo de la expresión relativa. Resultados: Las tumoresferas de pulmón presentan una expresión incrementada de EPCAM1, CD44, ALDH1A1 y CDKN1A (p= 0.028, p= 0.021, p= 0.043 and p= 0.021, respectivamente) comparadas con sus correspondientes células crecidas en adherencia. Además, el inductor de la transición epitelio-mesenquimal (EMT), SNAI1, está sobreexpresado (p= 0.011) en tumoresferas. Los genes de la vía Notch: DLL4, NOTCH1 y NOTCH2 también muestran mayor expresión en esferoides (p= 0.028, p= 0.038 and p= 0.036, respectivamente) que en células crecidas en monocapas. En cuanto a los genes de la ruta de Wnt, se observan mayores niveles de expresión de WNT3, CTNBB1 and GSK3B (p= 0.021, p= 0.008 and p= 0.021, respectivamente) en esferoides, mientras que el activador de la vía no canónica de Wnt, WNT5A, tiende a estar menos expresado en las células cultivadas en suspensión frente a las células cultivadas en adherencia. No se encontraron diferencias significativas en el resto de genes analizados. Conclusiones: Las tumoresferas de pulmón obtenidas a partir de líneas celulares y tumores primarios de pacientes muestran mayores niveles de marcadores de CSC. Además, genes pertenecientes a las rutas de señalización de Notch y Wnt están mayoritariamente expresados en tumoresferas, sugiriendo estas vías de señalización como dianas potenciales contra las CSC en cáncer de pulmón.Herreros Pomares, A. (2016). Characterization of cancer stem cells from non-small cell lung cancer. http://hdl.handle.net/10251/146816TFG

Tumorspheres as an in vitro model for cancer stem-like cell characterization in non-small cell lung cancer. Prognostic implications

[ES] El cáncer de pulmón es el tipo de cáncer más frecuentemente diagnosticado y la principal causa de muerte debida a cáncer en el mundo, con sólo un 15% de pacientes con una supervivencia mayor a 5 años tras el diagnóstico. La resección quirúrgica es el tratamiento estándar para los pacientes en estadios tempranos con un buen ECOG, pero el 75% de los pacientes son diagnosticados en estadios avanzados, cuando la intervención quirúrgica no es posible y entre un 35% y un 50% de los pacientes operados recaen tras una cirugía aparentemente exitosa. En los últimos años, se han logrado importantes avances en el desarrollo de la inmunoterapia y de tratamientos contra mutaciones conductoras, pero muchos pacientes todavía desarrollan resistencia, progresan y mueren. Esta resistencia terapéutica ha sido asociada a las células madre tumorales (CMTs), una población tumoral con propiedades de célula madre capaz de sobrevivir a las terapias convencionales y regenerar el tumor incluso cuando son indetectables. En esta tesis doctoral, se establecieron cultivos primarios de pacientes de cáncer de pulmón no microcítico (CPNM) resecados, usando ensayos de formación de tumoresferas para el enriquecimiento en CMTs y condiciones de adherencia para los controles. Las tumoresferas derivadas de pacientes mostraron capacidad de autorenovación y crecimiento exponencial ilimitado, alta resistencia a agentes quimioterápicos, capacidad de invasión y diferenciación in vitro y un elevado potencial tumorigénico in vivo. Usando PCR cuantitativa, se analizaron los perfiles de expresión de los cultivos y se determinó que NANOG, NOTCH3, CD44, CDKN1A, SNAI1 e ITGA6 eran los genes más diferencialmente expresados entre tumoresferas y células adherentes. Los análisis de inmunoblot e inmunofluorescencia confirmaron que las proteínas codificadas por estos genes se encuentran aumentadas en tumoresferas de los pacientes con adenocarcinoma y mostraron patrones de expresión y localización diferencial entre éstas y los controles en adherencia. El valor pronóstico de los genes significativamente sobreexpresados en tumoresferas fue evaluado in silico en una cohorte de 661 pacientes con CPNM procedente del TCGA. De todos ellos, CDKN1A, SNAI1 y ITGA6 mostraron estar relacionados con el pronóstico de los pacientes de acuerdo a un análisis de regresión de Cox y fueron seleccionados para construir una firma de expresión génica, denominada firma de CMTs. Los análisis de supervivencia por Kaplan-Meier mostraron que los pacientes con valores elevados de la firma tienen una supervivencia global (SG) menor para la cohorte completa de CPNM [37,7 vs. 60,40 meses, p = 0,001] y para la subcohorte de adenocarcinoma (ADC) [36,6 vs. 53,5 meses, p = 0,003], pero no para la de los epidermoides. Además, el análisis multivariante mostró que la firma de CMTs es un marcador pronóstico independiente para la SG de los pacientes en la cohorte completa [hazard ratio (HR): 1,498; intervalo de confianza (IC) 95%, 1,167-1,922; p = 0,001] y la subcohorte de ADC [HR: 1,869; IC 95%, 1,275-2,738; p = 0,001]. Esta firma fue también analizada en un grupo independiente de 245 pacientes procedentes del Consorci Hospital General Universitari de València, confirmando su valor pronóstico en los pacientes con ADC [42,90 vs. no alcanzado (NA) meses, p = 0,020]. En resumen, nuestros hallazgos aportan información pronóstica relevante para los pacientes con ADC de pulmón y establecen las bases para el desarrollo de nuevos tratamientos.[CA] El càncer de pulmó és el tipus de càncer més diagnosticat i la principal causa de mort deguda a càncer en el món, amb només un 15% de pacients amb una supervivència major a 5 anys després del diagnòstic. La resecció quirúrgica és el tractament estàndard per als pacients en estadis primaris amb un bon ECOG, però el 75% dels pacients són diagnosticats en estadis avançats, quan la intervenció quirúrgica no és possible i entre un 35% i un 50% dels pacients operats recauen després d'una cirurgia aparentment satisfactòria. En els últims anys, s'han aconseguit importants avanços en el desenvolupament de la immunoteràpia i de tractaments contra mutacions conductores, però molts pacients encara desenvolupen resistència, progressen i moren. Aquesta resistència a les teràpies ha estat relacionada amb les cèl·lules mare tumorals (CMTs), una població tumoral amb propietats de cèl·lula mare capaç de sobreviure a les teràpies convencionals i regenerar el tumor fins i tot quan són indetectables. En aquesta tesi doctoral, es van establir cultius primaris de pacients de càncer de pulmó no microcític (CPNM) ressecats, usant assajos de formació de tumoresferes per a l'enriquiment en CMTs i condicions d'adherència per als controls. Les tumoresferes derivades de pacients van mostrar capacitat d'autorenovació, creixement exponencial il·limitat, alta resistència a agents quimioteràpics, capacitat d'invasió i diferenciació in vitro i un elevat potencial tumorigènic in vivo. Usant PCR quantitativa, es van analitzar els perfils d'expressió dels cultius i es va determinar que NANOG, NOTCH3, CD44, CDKN1A, SNAI1 i ITGA6 eren els gens més diferencialment expressats entre tumoresferes i cèl·lules adherents. Les anàlisis de immunoblot i immunofluorescència van confirmar que les proteïnes codificades per aquests gens es troben augmentades en tumoresferes dels pacients amb adenocarcinoma i van mostrar patrons d'expressió i localització diferencial entre aquestes i els controls en adherència. El valor pronòstic dels gens significativament sobreexpressats en tumoresferes va ser avaluat in silico en una cohort de 661 pacients amb CPNM procedent del TCGA. De tots ells, CDKN1A, SNAI1 i ITGA6 van mostrar estar relacionats amb el pronòstic dels pacients d'acord a una anàlisi de regressió de Cox i van ser seleccionats per a construir una signatura d'expressió gènica, denominada signatura de CMTs. Les anàlisis de supervivència per Kaplan-Meier van mostrar que els pacients amb valors elevats de la signatura tenen una supervivència global (SG) menor per a la cohort completa de CPNM [37,7 vs. 60,40 mesos, p = 0,001] i per a la subcohort d'adenocarcinoma (ADC) [36,6 vs. 53,5 mesos, p = 0,003], però no per a la dels escamosos. A més, l'anàlisi multivariant va mostrar que la signatura de CMTs és un marcador pronòstic independent per a la SG dels pacients en la cohort completa [hazard ratio, (HR): 1,498; interval de confiança (IC) 95%, 1,167-1,922; p = 0,001] i la subcohort d'ADC [HR: 1,869; IC 95%, 1,275-2,738; p = 0,001]. Aquesta signatura va ser també analitzada en un grup independent de 245 pacients procedents del Consorci Hospital General Universitari de València, confirmant el seu valor pronòstic en els pacients amb ADC [42,90 vs. no arribat (NA) mesos, p = 0,020]. En resum, els nostres resultats aporten informació pronòstica rellevant per als pacients amb ADC de pulmó i estableixen les bases per al desenvolupament de nous tractaments.[EN] Lung cancer is the most commonly diagnosed type of cancer and the leading cause of cancer-related death worldwide, with approximately 15% of patients surviving 5 years after diagnosis. Curative surgery is the standard of care for early-stage patients with a good performance status, but 75% are diagnosed at advances stages, when surgery is not possible, and 35-50% of the resected patients relapse after an apparently successful surgical treatment. Significant advances in the development of therapies against driver mutations and immune-based treatments for these patients have been achieved in recent years, but many patients still develop treatment resistance, progress, and die. The high resistance against these therapies has been associated to cancer stem-like cells (CSCs), a population with stem properties which is able to survive after conventional treatments and regenerate tumor even when are undetectable. In this thesis, primary cultures from early-stage non-small cell lung cancer (NSCLC) patients were established, using sphere-forming assays for CSCs enrichment and adherent conditions for the control counterparts. Patient-derived tumorspheres showed self-renewal and unlimited exponential growth potentials, resistance against chemotherapeutic agents, invasion and differentiation capacities in vitro, and superior tumorigenic potential in vivo. Using RTqPCR, gene expression profiles were analyzed, and NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 were selected as the best contributors to distinguish tumorspheres from adherent cells. Immunoblot and immunofluorescence analyses confirmed that proteins encoded by these genes were consistently increased in tumorspheres from adenocarcinoma patients and showed differential localization and expression patterns. The prognostic role of genes significantly overexpressed in tumorspheres was evaluated in silico in a cohort of 661 NSCLC patients from TCGA. Based on a Cox regression analysis, CDKN1A, SNAI1 and ITGA6 were found to be associated with prognosis and used to calculate a gene expression score, named CSCs score. Kaplan-Meier survival analysis showed that patients with high CSCs score have shorter overall survival (OS) in the entire cohort [37.7 vs. 60.4 months, p = 0.001] and in the adenocarcinoma (ADC) subcohort [36.6 vs. 53.5 months, p = 0.003], but not in the squamous cell carcinoma one. Multivariate analysis indicated that this gene expression score is an independent biomarker of prognosis for OS in both, the entire cohort [hazard ratio (HR): 1.498; 95% confidence interval (CI), 1.167-1.922; p = 0.001], and the ADC subcohort [HR: 1.869; 95% CI, 1.275-2.738; p = 0.001]. This score was also analyzed in an independent group of 245 patients from Consorci Hospital General Universitari de València, confirming its prognostic value in the ADC subtype [42.90 vs. not reached (NR) months, p = 0.020]. In conclusion, our findings provide relevant prognostic information for lung ADC patients and the basis for developing novel therapies.Herreros Pomares, A. (2020). Tumorspheres as an in vitro model for cancer stem-like cell characterization in non-small cell lung cancer. Prognostic implications [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137036TESI

Efecto de las gonadotropinas recombinantes humanas rhFSH y rhLH sobre el desarrollo embrionario en conejo

[ES] La obtención de un gran número de óvulos y embriones es interesante tanto en la práctica clínica como en la producción animal, pues reduce costes y facilita la mejora y el desarrollo de herramientas reproductivas y genéticas. Sin embargo, los procedimientos de superovulación presentan una serie de limitaciones que han impulsado los estudios sobre foliculogénesis y ovogénesis y el desarrollo de nuevos productos y modos de administración. En este sentido, los resultados obtenidos sobre el uso de pequeñas dosis de la hormona luteinizante (LH) en los tratamientos de superovulación con la hormona folículo-estimulante (FSH) son contradictorios, por lo que su efecto sigue sin esclarecerse de forma definitiva. El objetivo de este trabajo es evaluar el efecto de las gonadotropinas recombinantes humanas rhFSH y rhLH, sobre la estimulación ovárica, usando el conejo como modelo animal, y determinar el impacto sobre la calidad de los embriones, empleando como indicadores el desarrollo in vitro hasta blastocisto y la expresión de los genes octamer-binging transcription factor (OCT4), NANOG homeobox (NANOG) y sex determining región Y-box 2 (SOX2). Tanto los tratamientos que emplean rhFSH, como aquellos que utilizan rhFSH combinada con un 10% de rhLH, inducían una respuesta superovulatoria, siendo 37’50 IU de rhFSH suplementada con 3’75 IU de rhLH la combinación que produjo una mayor respuesta ovulatoria. La utilización de bajas concentraciones de rhFSH con 10% de rhLH (18’75 IU de rhFSH y 1’87 IU de rhLH) provocaba una disminución significativa de la tasa de fecundación que no se observó para concentraciones elevadas (37’50 IU de rhFSH + 10% de rhLH). Asimismo, la calidad de los embriones no fue afectada en términos de capacidad de desarrollo in vitro desde el estadio de 8-16 células hasta blastocisto ni de expresión embrionaria de los genes OCT4, NANOG y SOX2.[EN] Obtaining a large amount of oocytes and embryos is relevant for clinical practice and animal production, in order to reduce costs and easily improve and develop reproductive and genetic tools. However, superovulation protocols have several limitations, leading researchers to further explore the areas of folliculogenesis and oogenesis, and the development of new products and methods of administration. In this respect, the current results about the use of small doses of the luteinizing hormone (LH) in superovulation treatments with follicle-stimulating hormone (FSH) are contradictory and its effect is still unknown. This study aimed to evaluate the effect of recombinant human gonadotropins, rhFSH and rhLH, upon ovarian stimulation, using rabbit does as an animal model, and to determinate their impact on embryo quality, marked by the ability to develop to the blastocyst stage in vitro and the gene expression of the following genes: octamer-binging transcription factor (OCT4), NANOG homeobox (NANOG) and sex determining región Y-box 2 (SOX2). The results indicated that superovulation treatment with rhFSH alone or in combination with 10% rhLH induced a superovulatory response in doe rabbits; the combination of 37.50 IU of rhFSH supplemented with 3.75 IU of rhLH produced the highest ovarian response. Low concentration of rhFSH with 10% rhLH (18.75 IU of rhFSH and 1.87 IU rhLH) produced a significant decrease of the fecundation rate that is not observed for high concentrations (37.50 IU of rhFSH + 10% rhLH). Moreover, embryo quality is not affected in terms of their ability to develop in vitro from 8-16 cells to blastocyst stage and neither affects embryonic gene expression of the genes OCT4, NANOG and SOX2.[CA] L’obtenció d’un gran nombre d’òvuls i embrions és interesant tant en la pràctica clínica com en la producció animal, doncs redueix costos i facilita la millora i el desenvolupament de ferramentes reproductives i genètiques. No obstant, els procediments de superovulació presenten una sèrie de limitacions que han impulsat els estudis sobre foliculogènesi i ovogènesi i el desenvolupament de nous productes i mètodes d’administració. En aquest sentit, els resultats obtinguts sobre l’ús de xicotetes dosi de l’hormona luteïnitzant (LH) en els tractaments de superovulació amb l’hormona fol·licle-estimulant (FSH) són contradictoris, per la qual cosa el seu efecte segueix sense esclarir-se de forma definitiva. L’objectiu d’aquest treball és avaluar l’efecte de les gonadotropines recombinats humanes rhFSH i rhLH, sobre l’estimulació ovàrica, utilitzant el conill com a model animal, i determinar l’impacte sobre la qualitat dels embrions, emprant com a indicadors el desenvolupament in vitro fins blastòcit i l’expressió dels gens octamer-binging transcription factor (OCT4), NANOG homeobox (NANOG) i sex determining region Y-box 2 (SOX2). Tant els tractaments que empraren rhFSH, com aquells que utilitzen rhFSH combinada amb un 10% de rhLH, induïen una resposta superovulatoria, essent 37’50 IU de rhFSH suplementada amb 3’75 IU de rhLH la combinació que produeix una major resposta ovulatoria. La utilització de baixes concentracions de rhFSH amb 10% de rhLH (18’75 IU de rhFSH i 1’87 IU de rhLH) provocava una disminució significativa de la taxa de fecundació que no es va observar per a concentracions elevades (37’50 IU de rhFSH + 10% de rhLH). Així mateix, la qualitat dels embrions no va ser afectada en termes de capacitat de desenvolupament in vitro des de l’estadi de 8-16 cèl·lules fins blastòcit ni d’expressió embrionària dels gens OCT4, NANOG i SOX2.Herreros Pomares, A. (2014). Efecto de las gonadotropinas recombinantes humanas rhFSH y rhLH sobre el desarrollo embrionario en conejo. http://hdl.handle.net/10251/39791.Archivo delegad

Lung tumorspheres as a drug screening platform against cancer stem cells

Treatment resistance and metastasis are linked to cancer stem cells (CSCs). This population represents a promising target, but remains unexplored in lung cancer. The main objective of this study was to characterize lung CSCs and discover new therapeutic strategies

Effect of luteinizing hormone on rabbit ovarian superstimulation and embryo developmental potential

[EN] Assisted reproduction technologies require ovarian stimulation to increase the number of oocytes and embryos. Currently, superstimulation is achieved by gonadotropin treatment, but the embryo yield and quality are highly variable. Commonly, commercial preparations derived from pituitary and urinary origin are used to superovulate. Hence, ovarian superstimulation protocols have usually included both FSH and LH. The appearance of recombinant gonadotropins manufactured by genetic engineering techniques has ensured high quality and batch-to-batch consistency. Moreover, this enables us to assess the importance of LH in the ovarian stimulation. The main aim of this study was to evaluate the effect of recombinant human LH supplementation (10%) on embryonic development produced by rabbit does superovulated with low or high concentration (18.75 or 37.50 IU) of recombinant human FSH (rhFSH). Females treated with rhFSH increased the ovulation rate, and it was significantly higher when the high FSH dose was supplemented with LH. The superstimulation treatment used did not significantly affect in vitro development rate until the expanded blastocyst stage. The results of this study seem to suggest that, in terms of superovulatory response, when rabbit does are treated with 37.5-IU rhFSH, the use of LH supplementation allows an increase in the number of follicles recruited and the quality of embryos, in terms of ability to develop in vitro until blastocyst, and the expression profile of OCT4, NANOG, and SOX2 genes is not affected.This research was supported in part by the Valencian regional government research project PROMETEOII/2014/036. The authors would like to thank Neil Macowan Language Services for revising the English version of the article.Viudes De Castro, MP.; Herreros Pomares, A.; Saenz De Juano Ribes, MDLD.; Marco Jiménez, F.; Vicente Antón, JS. (2015). Effect of luteinizing hormone on rabbit ovarian superstimulation and embryo developmental potential. Theriogenology. 84(3):446-451. https://doi.org/10.1016/j.theriogenology.2015.04.001S44645184

Soluble galectin-3 as a microenvironment-relevant immunoregulator with prognostic and predictive value in lung adenocarcinoma

Despite the success of therapies in lung cancer, more studies of new biomarkers for patient selection are urgently needed. The present study aims to analyze the role of galectin-3 (GAL-3) in the lung tumor microenvironment (TME) using tumorspheres as a model and explore its potential role as a predictive and prognostic biomarker in non-small cell lung cancer (NSCLC) patients. For in vitro studies, lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) primary cultures from early-stage patients and commercial cell lines were cultured, using tumorsphere-forming assays and adherent conditions for the control counterparts. We analyzed the pattern of secretion and expression of GAL-3 using reverse transcription-quantitative real-time PCR (RTqPCR), immunoblot, immunofluorescence, flow cytometry and immunoassay analysis. Our results using three-dimensional (3D) models of lung tumor cells revealed that soluble GAL-3 (sGAL-3) is highly expressed and secreted. To more accurately mimic the TME, a co-culture of tumorspheres and fibroblasts was used, revealing that GAL-3 could be important as an immunomodulatory molecule expressed and secreted in the TME, modulating immunosuppression through regulatory T cells (TREGS). In the translational phase, we confirmed that patients with high expression levels of GAL-3 had more TREGS, which suggests that tumors may be recruiting this population through GAL-3. Next, we evaluated levels of sGAL-3 before surgery in LUAD and LUSC patients, hypothesizing that sGAL-3 could be used as an independent prognostic biomarker for overall survival and relapse-free survival in early-stage LUAD patients. Additionally, levels of sGAL-3 at pretreatment and first response assessment from plasma to predict clinical outcomes in advanced LUAD and LUSC patients treated with first-line pembrolizumab were evaluated, further supporting that sGAL-3 has a high efficiency in predicting durable clinical response to pembrolizumab with an area under curve (AUC) of 0.801 (p=0.011). Moreover, high levels might predict decreased progression-free survival and overall survival to anti-PD-1 therapy, with sGAL-3 being a prognosis-independent biomarker for advanced LUAD

Analysis of Exosomal Cargo Provides Accurate Clinical, Histologic and Mutational Information in Non-Small Cell Lung Cancer

Lung cancer is a malignant disease with high mortality and poor prognosis, frequently diagnosed at advanced stages. Nowadays, immense progress in treatment has been achieved. However, the present scenario continues to be critical, and a full comprehension of tumor progression mechanisms is required, with exosomes being potentially relevant players. Exosomes are membranous vesicles that contain biological information, which can be transported cell-to-cell and modulate relevant processes in the hallmarks of cancer. The present research aims to characterize the exosomes' cargo and study their role in NSCLC to identify biomarkers. We analyzed exosomes secreted by primary cultures and cell lines, grown in monolayer and tumorsphere formations. Exosomal DNA content showed molecular alterations, whereas RNA high-throughput analysis resulted in a pattern of differentially expressed genes depending on histology. The most significant differences were found in XAGE1B, CABYR, NKX2-1, SEPP1, CAPRIN1, and RIOK3 genes when samples from two independent cohorts of resected NSCLC patients were analyzed. We identified and validated biomarkers for adenocarcinoma and squamous cell carcinoma. Our results could represent a relevant contribution concerning exosomes in clinical practice, allowing for the identification of biomarkers that provide information regarding tumor features, prognosis and clinical behavior of the disease. Keywords: non-small cell lung cancer; liquid biopsy; exosomes; extracellular vesicles; cell cultures; adenocarcinoma; squamous cell carcinoma; biomarker; tumorsphere

Increased levels of NETosis biomarkers in high-grade serous ovarian cancer patients’ biofluids: Potential role in disease diagnosis and management

Introduction: High-grade serous ovarian cancer (HGSOC) is the second most frequent gynecological malignancy but the most lethal, partially due to the spread of the disease through the peritoneal cavity. Recent evidence has shown that, apart from their role in immune defense through phagocytosis and degranulation, neutrophils are able to participate in cancer progression through the release of neutrophil extracellular traps (NETs) in a process called NETosis. NETs are composed of DNA, histones, calprotectin, myeloperoxidase (MPO) and elastase and the NETosis process has been proposed as a pre-requisite for the establishment of omental metastases in early stages of HGSOC. Nevertheless, its role in advanced stages remains to be elucidated. Therefore, our principal aim is to characterize a NETosis biomarker profile in biofluids from patients with advanced HGSOC and control women. Methods: Specifically, five biomarkers of NETosis (cell-free DNA (cfDNA), nucleosomes, citrullinated histone 3 (citH3), calprotectin and MPO) were quantified in plasma and peritoneal fluid (PF) samples from patients (n=45) and control women (n=40). Results: Our results showed that HGSOC patients presented a higher concentration of cfDNA, citH3 and calprotectin in plasma and of all five NETosis biomarkers in PF than control women. Moreover, these biomarkers showed a strong ability to differentiate the two clinical groups. Interestingly, neoadjuvant treatment (NT) seemed to reduce NETosis biomarkers mainly systemically (plasma) compared to the tumor environment (PF). Discussion: In conclusion, NETosis biomarkers are present in the tumor environment of patients with advanced HGSOC, which might contribute to the progression of the disease. Besides, plasma cfDNA and calprotectin could represent minimally invasive surrogate biomarkers for HGSOC. Finally, NT modifies NETosis biomarkers levels mainly at the systemic level

Identification, Culture and Targeting of Cancer Stem Cells

Chemoresistance, tumor progression, and metastasis are features that are frequently seen in cancer that have been associated with cancer stem cells (CSCs). These cells are a promising target in the future of cancer therapy but remain largely unknown. Deregulation of pathways that govern stemness in non-tumorigenic stem cells (SCs), such as Notch, Wnt, and Hedgehog pathways, has been described in CSC pathogenesis, but it is necessary to conduct further studies to discover potential new therapeutic targets. In addition, some markers for the identification and characterization of CSCs have been suggested, but the search for specific CSC markers in many cancer types is still under development. In addition, methods for CSC cultivation are also under development, with great heterogeneity existing in the protocols used. This review focuses on the most recent aspects of the identification, characterization, cultivation, and targeting of human CSCs, highlighting the advances achieved in the clinical implementation of therapies targeting CSCs and remarking those potential areas where more research is still required