Liquid Biopsy in non-small cell lung cancer: exosomes as a tool for the study of biomarkers.

[ES] A pesar de los nuevos avances en el tratamiento del cáncer de pulmón, su tasa de incidencia y mortalidad siguen en cabeza en todo mundo. Concretamente, el cáncer de pulmón no microcítico (CPNM) representa casi el 85% de todos los cánceres de pulmón, siendo su supervivencia a 5 años muy reducida. En base a dicho escenario, el objetivo principal de este trabajo es el de caracterizar de manera exhaustiva los exosomas secretados por las células del CPNM. Se sabe que estas microvesículas están involucradas en números procesos celulares, por lo que pueden contener gran cantidad de información acerca de las características moleculares del tumor. Para ello se han empleado cultivos primarios y líneas comerciales crecidas en diferentes condiciones, así como muestras de sangre periférica obtenida de los pacientes con CPNM. Un primer screening llevado a cabo en los exosomas secretados in vitro, ha permitido obtener un gran número de mRNAs y miRNAs relacionados con diferentes procesos biológicos y vías de señalización. Además, algunos genes como FDFT1 y SNAI1 han destacado por su sobreexpresión en exosomas procedentes de las células crecidas en formación de tumoresferas (modelos 3D), las cuales están enriquecidas en población de células madre tumorales. A su vez, otros marcadores presentes en el interior de estas microvesículas, se han mostrado relacionados con dos de los subtipos histológicos más frecuentes: adenocarcinoma (LUAD) y carcinoma escamoso (LUSC). Posteriormente, para validar los hallazgos obtenidos en exosomas, los marcadores más significativos fueron analizados in silico en una cohorte de muestras de tejido, compuesta por 661 pacientes con CPNM (TCGA database). Estos resultados han revelado una asociación entre la expresión del gen SNAI1 y la supervivencia de estos pacientes (OS y RFS p<0.05). Además, los genes XAGE1B, SEPP1 y TTF-1 (previamente determinados en exosomas), mantienen una relación significativa con el grupo de pacientes LUAD; mientras que CABYR, RIOK3 y CAPRIN1 se mantienen sobrexpresados en LUSC (Mann-Whitney test p<0.05). Estos marcadores también se han analizado en una cohorte de 186 pacientes con CPNM procedentes del Hospital General Universitario de Valencia, donde se corroboró la asociación de SNAI1 con la supervivencia de los pacientes en estadios tempranos (RFS en pacientes LUAD, p<0.05), así como la sobreexpresión de CABYR y RIOK3 en pacientes LUSC, y de XAGE1B y TTF-1 en LUAD. Por otra parte, el aislamiento de los exosomas presentes en la sangre periférica de pacientes en estadios avanzados, ha permitido identificar otros marcadores asociados a caracterísiticas clínico-patológicas relevantes. A su vez, el contenido de estas microvesículas ha sido empleado para la detección de mutaciones génicas ligadas al manejo clínico del CPNM. En resumen, los resultados obtenidos en este trabajo ponen de manifiesto el potencial de los exosomas como fuente de biomarcadores para el estudio de las diferentes etapas de desarrollo del CPNM. Estas microvesículas ofrecen una visión completa y en tiempo real, de las características de la enfermedad, pudiendo ser aisladas de forma repetida y mediante técnicas mínimamente invasivas.[CA] A pesar dels avanços recents en el tractament del càncer de pulmó, les seues taxes d'incidència i mortalitat continuen sent altes a nivell mundial. Concretament, el càncer de pulmó de cèl·lules no petites (CPNM) representa gairebé el 85% de tots els càncers de pulmó, amb una taxa de supervivència a 5 anys molt limitada. Donat aquest escenari, l'objectiu principal d'aquest estudi és caracteritzar de manera exhaustiva els exosomes secretats per les cèl·lules de CPNM. Aquestes microvesícules estan involucrades en nombrosos processos tumorals i poden contenir una gran quantitat d'informació sobre les característiques moleculars de la malaltia. Per aconseguir-ho, es van utilitzar cultius primaris i línies cel·lulars (cultiu en diferents condicions), juntament amb mostres de sang perifèrica obtingudes de pacients amb CPNM. Un cribratge inicial en exosomes secrets in vitro va permetre identificar una quantitat significativa de mARNs i miARNs relacionats amb diversos processos biològics i vies de senyalització. A més, alguns gens com FDFT1 i SNAI1 van destacar per la seua sobreexpressió en exosomes derivats de cèl·lules crescuts en formació de tumorsferes (models 3D), que estan enriquides en poblacions de cèl·lules mare tumorals. A més, s'han trobat marcadors en aquestes microvesícules associats amb dos dels subtipus histològics més comuns: adenocarcinoma (LUAD) i carcinoma escamós (LUSC). Posteriorment, per validar els resultats obtinguts en exosomes, es van analitzar in silico els marcadors més significatius en una cohort de teixit de CPNM de la base de dades TCGA. Aquests resultats van revelar una associació entre l'expressió del gen SNAI1 i la supervivència dels pacients (OS i RFS, p <0,05). A més, l'expressió dels gens XAGE1B, SEPP1 i TTF-1 (prèviament identificats en exosomes) va mantenir una relació significativa amb el grup LUAD, mentre que CABYR, RIOK3 i CAPRIN1 van continuar sobreexpressats en els pacients de LUSC (prova de Mann-Whitney, p <0,05). Aquests marcadors també es van analitzar en una cohort de 186 pacients amb CPNM de l'Hospital General Universitari de València, on es va confirmar l'associació de l'expressió de SNAI1 i la supervivència dels pacients en estadi precoç (RFS en pacients de LUAD, p <0,05), així com la sobreexpressió de CABYR i RIOK3 en pacients de LUSC, i de XAGE1B i TTF-1 en LUAD. D'altra banda, els exosomes presents en mostres de sang de la cohort d'estadis avançats van permetre la identificació d'altres biomarcadors associats a característiques clíniques rellevants dels pacients. A més, la càrrega exosomàtica també es va utilitzar per detectar mutacions genètiques relacionades amb el tractament clínic del CPNM. En resum, els resultats obtinguts en aquesta tesi destaquen el potencial dels exosomes com a font de biomarcadors per a l'estudi de les diferents etapes del desenvolupament del CPNM. Aquestes microvesícules ofereixen una visió completa i en temps real de les característiques moleculars de la malaltia i poden ser obtingudes de manera repetida i amb una mínima invasió.[EN] Despite recent advancements in lung cancer treatment, its incidence and mortality rates remain high worldwide. Specifically, non-small cell lung cancer (NSCLC) accounts for nearly 85% of all lung cancers, with a 5-year survival rate of 20%. Given this scenario, the primary objective of this study is to comprehensively characterize the exosomes secreted by NSCLC cells. These microvesicles are known to be involved in numerous tumoral processes, potentially containing a wealth of information about the molecular characteristics of the disease. To achieve this, primary cultures and cell lines, along with peripheral blood samples obtained from NSCLC patients were used. An initial screening in exosomes secreted in vitro allowed the identification of a significant number of mRNAs and miRNAs, related to various biological processes and signaling pathways. Moreover, some genes such as FDFT1 and SNAI1 stood out due to their overexpression in exosomes derived from cells grown in tumorspheres formation (3D models), which are enriched in cancer stem cell population. Additionally, markers found within these microvesicles were associated with two of the most common histological subtypes: adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Subsequently, to validate the findings seen in exosomes, the most significant markers were analyzed in silico in an NSCLC tissue cohort from the TCGA database. These results revealed an association between the expression of SNAI1 and patient survival (OS and RFS, p<0.05). Furthermore, XAGE1B, SEPP1, and TTF-1 expression (previously identified in exosomes) maintained a significant relationship with the LUAD group, while CABYR, RIOK3, and CAPRIN1 remained overexpressed in LUSC patients (Mann-Whitney test, p<0.05). These markers were also analyzed in a cohort of 186 NSCLC patients from the University General Hospital of Valencia. The association of SNAI1 expression and the survival of early-stage patients (RFS in LUAD patients, p<0.05) was confirmed, as well as the overexpression of CABYR and RIOK3 in LUSC patients, and of XAGE1B and TTF-1 in LUAD. Furthermore, exosomes present in blood samples of the advanced-stage cohort, allowed the identification of other biomarkers associated with clinically relevant characteristics of the patients. Moreover, exosomal cargo was also used to detect gene mutations related to the clinical management of NSCLC. In summary, the results obtained in this thesis highlight the potential of exosomes as a source of biomarkers for the study of the different stages of NSCLC development. These microvesicles offer a comprehensive and real-time view of the disease's molecular features and can be obtained repeatedly and in a minimally invasive way.Duréndez Sáez, ME. (2024). Liquid Biopsy in non-small cell lung cancer: exosomes as a tool for the study of biomarkers [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/20343

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

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&rsquo; 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

Extracellular Vesicles As miRNA Nano-Shuttles: Dual Role in Tumor Progression

[EN] Tumor-derived extracellular vesicles (EVs) have a pleiotropic role in cancer, interacting with target cells of the tumor microenvironment, such as fibroblasts, immune and endothelial cells. EVs can modulate tumor progression, angiogenic switch, metastasis, and immune escape. These vesicles are nano-shuttles containing a wide spectrum of miRNAs that contribute to tumor progression. MiRNAs contained in extracellular vesicles (EV-miRNAs) are disseminated in the extracellular space and are able to influence the expression of target genes with either tumor suppressor or oncogenic functions, depending on both parental and target cells. Metastatic cancer cells can balance their oncogenic potential by expressing miRNAs with oncogenic function, whilst exporting miRNAs with tumor suppressor roles out of the cells. 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