Desarrollo de metodología ómicas para el estudio y diagnóstico precoz del cáncer de pulmón

El cáncer de pulmón (CP) constituye una de las muertes más comunes por neoplasia en el mundo provocando más de 1.300.000 muertes al año. La supervivencia a 5 años comprende un 65% cuando la enfermedad se diagnostica en estadios precoces, disminuyendo a un 10% cuando se encuentra en estados avanzados. Por ello, la búsqueda de biomarcadores capaces de detectar de forma precoz el CP, así como capaces de evaluar la progresión del mismo, constituye un importante reto en medicina. En este sentido, las metodologías ómicas son herramientas de análisis muy poderosas que permiten determinar un gran número de moléculas, como metabolitos, proteínas o metales unidos a proteínas. Estas biomoléculas pueden sufrir alteraciones en respuesta a una enfermedad, por lo que podrían servir como marcadores de diagnóstico. Así, en esta Tesis Doctoral se han aplicado tres metodologías ómicas (metabólomica, ionómica y metalómica), basadas en la espectrometría de masas, a muestras biológicas humanas de pacientes con cáncer de pulmón, con el objetivo de identificar biomoléculas alteradas en esta enfermedad que puedan servir como biomarcadores. Con el fin de abarcar una mayor cobertura de metabolitos se optimizó una plataforma metabolómica “no dirigida” basada en dos técnicas analíticas: la cromatografía de gases (GC-MS) y la infusión directa (DI-MS) acopladas a espectrometría de masas (MS). Esta plataforma fue aplicada a muestras de pacientes con CP, pacientes control y pacientes con diversas enfermedades pulmonares no cancerosas como la enfermedad pulmonar obstructiva crónica, EPOC. Las muestras empleadas en el estudio fueron muestras de suero sanguíneo, de orina, y como principal novedad del estudio, muestras de lavado broncoalveolar (LBA), de las que no había antecedentes en bibliografía. Para ello, los perfiles metabolómicos de pacientes con CP se compararon estadísticamente con los de personas sanas empleando el análisis multivariante de mínimos cuadrados parciales (PLS-DA) para encontrar metabolitos diferentes de forma significativa entre los grupos. El análisis PLSDA también fue aplicado para comparar la existencia de metabolitos alterados en pacientes con EPOC y evaluar su posible relación con el CP. Así mismo, se evaluó la diferenciación metabolómica entre estadios tempranos y avanzados de la enfermedad. Por otro lado, se desarrolló una metodología ionómica basada en ICPQQQ- MS para la determinación multielemental en muestras de suero, orina y LBA con el fin de evaluar la distribución de oligoelementos y metales tóxicos en CP. Además, a las muestras de suero sanguíneo se les aplicó un método de fraccionamiento para la separación de la fracción de alta (HMM) y baja (LMM) masa molecular y determinar la concentración de elementos en cada fracción. Finalmente, se desarrolló una metodología metalómica basada en la técnica de dilución isotópica por ICP-QQQ-MS para determinar la concentración de 3 selenoproteínas importantes presentes en suero: glutatión peroxidasa (eGPx), selenoproteína P (SELENOP) y selenoalbúmina (SeAlb). Esta metodología también permitió la separación de moléculas pequeñas de selenio. De esta forma, la aplicación de estas plataformas metabolómicas, ionómicas y metalómicas en diferentes muestras biológicas de pacientes con CP, han permitido estudiar la alteración de metabolitos, metales y biomoléculas de selenio asociadas al CP aportando nuevas contribuciones a la patología de esta enfermedad.Lung cancer (LC) is one of the most common deaths from neoplasia in the world, causing more than 1,300,000 deaths per year. The 5-year survival comprises 65% when the disease is diagnosed in early stages, decreasing to 10% in advanced stages. Therefore, the search for biomarkers capable of early detection of LC, as well as capable of evaluating its progression, is a challenge of great interest in medicine. In this sense, omics methodologies are very powerful analysis tools that allow determining a large number of molecules (metabolites, proteins, metals bound to proteins). These biomolecules can undergo alterations in response to a disease, so they could serve as diagnostic markers. Thus, in this Doctoral Thesis, three omics methodologies (metabolomics, ionomics and metallomics) based on mass spectrometry have been applied to human biological samples of patients with lung cancer in order to identify altered biomolecules in this disease that can serve as biomarkers. In order to achieve greater metabolite coverage, a "non-target" metabolomic platform was optimized based on two analytical techniques: gas chromatography (GC-MS) and direct infusion (DI-MS) coupled to mass spectrometry. This platform was applied to samples of patients with LC, control patients and patients with various non-cancerous lung diseases such as chronic obstructive pulmonary disease COPD. The samples used in the study were samples of blood serum, urine, and as the main novelty of the study, samples of bronchoalveolar lavage (BAL), of which there was no background in the literature. Therefore, the metabolic profiles of LC patients with those of healthy people were compared statistically by means of the multivariate analysis of partial least squares discriminant analysis (PLS-DA) in order to find significantly different metabolites between the groups. The PLS-DA analysis was also applied to compare the occurrence of altered metabolites in patients with COPD and evaluate their possible relationship with LC. Likewise, the metabolomic differentiation between early and advanced stages of the disease was evaluated. On the other hand, an ionomic methodology based on ICP-QQQ-MS was developed for multielemental determination in samples of serum, urine and BAL in order to evaluate the distribution of trace elements and toxic metals in LC. In addition, serum samples were subjected to a fractionation method for the separation of the high fraction (HMM) and low fraction (LMM) molecular mass and determine the concentration of elements in each fraction. Finally, a metallomic methodology based on the isotope dilution technique by ICP-QQQ-MS was developed to determine the concentration of 3 important selenoproteins present in serum: glutathione peroxidase (eGPx), selenoprotein P (SELENOP) and selenoalbumin (SeAlb). This methodology also allowed the separation of low molecular mass species of selenium. Thus, the application of these metabolomic, ionomic and metallomic platforms in different biological samples from patients with LC has allowed the study of the alteration of metal metabolites and selenium biomolecules associated with LC disease, giving place to new contributions to the pathology of this disease

Contribuciones a la caracterización del cuerpo humano como medio de transmisión en las técnicas de comunicaciones intracorporales.

Falta palabras claveLa necesidad de bio-dispositivos de reducido consumo, tamaño y costo ha motivado la investigación de nuevas tecnologías de comunicaciones inalámbricas de corto alcance, entre las que se encuentran las técnicas de comunicaciones intracorporales (IBC). Estas utilizan el cuerpo humano como medio de transmisión de señales eléctricas para la conexión de dispositivos colocados sobre la superficie corporal, cercanos a esta e implantados. Las ventajas de esta técnica residen en el uso de señales de baja frecuencia y amplitud que se acoplan mediante pequeños electrodos al cuerpo, favoreciendo la disminución del consumo y la minimización de las interferencias con otros dispositivos. Sin embargo, aún existen importantes retos científico-técnicos a solventar con el fin de obtener una metodología de diseño de los sistemas IBC sistemática y precisa. Para ello es fundamental avanzar en el conocimiento de los verdaderos mecanismos de transmisión del cuerpo humano como canal de comunicación. El objetivo de esta tesis doctoral es profundizar en la caracterización del cuerpo humano como medio de transmisión de señales tanto desde una perspectiva teórica como experimental. De esta forma, se han propuesto diferentes modelos de canal: un modelo de la piel como símil de línea de transmisión, un modelo computacional 3D del brazo humano y un modelo físico circuital phantom. También se han implementado montajes de medida armonizados para la determinación de la atenuación corporal para ambos tipos de acoplamiento galvánico y capacitivo, así como para analizar diferentes cuestiones técnicas de interés como estrategias de aislamiento de tierras, influencia de la resistencia de carga y efectos de cables y conectores. Los resultados abarcan la simulación y validación de los modelos propuestos, incluyendo atenuación, dispersión, tasa binaria y de error para diferentes esquemas de modulación digitales. Se han simulado también variables eléctricas como bioimpedancia, campo eléctrico y corriente a través de los tejidos. Por último, se ha realizado un conjunto exhaustivo de medidas de atenuación estudiándose variables como longitud de canal, distancia inter-electrodo, tipo de electrodo, diferentes partes del cuerpo, etc. Como principales conclusiones, se ha avanzado en el entendimiento de algunos de los mecanismos de transmisión a través del cuerpo humano y la piel, lo que a su vez nos ha permitido identificar el rango de operación de cada uno de los tipos de acoplamiento, así como definir un conjunto de especificaciones prácticas para la implementación de esquemas de medida IBC más precisos

Metal dyshomeostasis based biomarkers for lung cancer diagnosis using human biofluids

Lung cancer (LC) is one of the most common causes of cancer-related deaths in the world and it is well known that trace elements play important roles in the carcinogenic process activating and inhibiting enzymatic reactions and metalloproteins, in which they usually participate as cofactors. A cross-sectional study was conducted on 48 lung cancer patients and 39 controls (56 men and 31 women), aged 44-76 years between March 2011 and June 2012. Eleven elements have been included in the study: V, Cr, Mn, Fe, Co, Cu, Zn, Se, Mo, Cd, and Pb, some of them considered toxic (V, Cd, Cr and Pb), while others are essential (Co, Mo, Se, Fe and Zn), and they have been analyzed by ICP-QQQ-MS in serum, urine and for the first time in bronchoalveolar lavage fluid (BALF). In order to understand the involvement of metals in this process, an analytical metallomic approach based on non-denaturing precipitation of proteins (NDPP) has been optimized for the fractionation of high molecular mass (HMM) and low molecular mass (LMM) metal species, in order to distinguish between metal species that affect the biological activity and toxicological potential of the elements. In this work, the NDPP followed by the analysis of metals by ICP-QQQ-MS has been applied for the first time to serum, urine and BALF samples from lung cancer patients and controls in order to get metal-size molecule profiles (MSMP), which can be used as metal-based biomarkers of altered metabolic processes such as oxidative stress and homeostasis. In this sense, we have demonstrated that several metals are good biomarkers when they are related to labile complexes, complexed with low molecular mass ligands, or in the form of metalloproteins (i.e. V and Cr in HMM and Cu in LMM), which has been described for the first time. On the other hand, metal dyshomeostasis biomarkers are proposed using metal ratios and correlations. Finally, the ratios between elements were shown to be important biomarkers for lung cancer in serum (V/Mn, V/Pb, V/Zn, Cr/Pb), urine (Cr/Cd, Mn/Cd, V/Cd, Co/Cd, Cd/Pb) and BALF (V/Cu), which reflects the dyshomeostasis of metals in lung cancer. In this sense, several metals are correlated to others suggesting also the existence of an interconnected homeostasis in lung cancer.The authors thank the Spanish Ministry of Economy and Competitiveness (CTM2015-67902-C2-1-P) and Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government. P12-FQM-0442). B. Callejon-Leblic thanks the Ministerio de Educacion for a predoctoral scholarship FPU13/03615. Finally, the authors are grateful to FEDER (European Community) (UNHU13-1E-1611 and UNHU15-CE-3140)

Heteroatom-tagged proteomics of lung cancer and chronic obstructive pulmonary disease human serum reveal alterations in selenoproteins

Heteroatom-tagged proteomics allows the absolute quantification of selenoproteins using the heteroatom as a “tag” into a selective and sensitive atomic detector instead of a molecular one. Using this analytical method, about 90% of total selenium in human serum/plasma can be measured as selenoproteins and total selenometabolites and thus, the status of selenium can be determined. Herein, we determined the absolute concentration of selenoproteins in human serum patients with lung cancer (LC) and chronic obstructive pulmonary disease (COPD), a competing cause of morbidity and mortality in smokers as well as an independent risk factor for LC. We conducted an observational study of 154 human serum samples obtained from LC and COPD patients with varying severity of disease, including COPD patients who developed LC during follow-up and healthy controls (HC). Using heteroatom-tagged proteomics, we determined extracellular glutathione peroxidase (eGPx), selenoprotein P (SELENOP), and selenoalbumin (SeAlb). Associations between selenoproteins were also studied as potential biomarkers of disease. The concentration of eGPx was significantly higher in the all-inclusive COPD cohort compared to HC, COPD patients with LC, or those with mild obstructive lung disease, while SELENOP concentration was significantly decreased in LC patients compared to HC and COPD. We found an inverse correlation between SELENOP and SeAlb in HC, but also in LC patients, and especially in patients with COPD and LC. Moreover, we found that eGPx and selenometabolite concentrations were positively associated with LC human serum. Selenoprotein concentrations were altered in COPD and LC when compared to healthy controls suggesting a potential role of the selenoproteome in the diagnosis and/or treatment of these tobacco-related diseases.This work has been supported by the project “Heteroatom-tagged proteomics and metabolomics to study lung cancer. Influence of gut microbiota” (Ref.: PY20_00366). Project of Excellence. Regional Ministry of Economy, Knowledge, Business and University, Andalusia, Spain. The authors also thank the grants Ref. 651/2018 and 115/2020 from the Spanish Society of Pneumology and Surgery (SEPAR) and 08/2018 from the Association of Pneumology and Thoracic Surgery (Neumosur) that supported samples recruitment at the hospitals and biobank registration. The authors also thank Instituto de Salud Carlos III (AES16/01783) and unrestricted funding from Menarini Group and AstraZeneca“. Funding for open access charge: Universidad de Huelva / CBUA.Departamento de Química "Profesor José Carlos Vílchez Martín

The metallome of lung cancer and its potential use as biomarker

Carcinogenesis is a very complex process in which metals have been found to be critically involved. In this sense, a disturbed redox status and metal dyshomeostasis take place during the onset and progression of cancer, and it is well-known that trace elements participate in the activation or inhibition of enzymatic reactions and metalloproteins, in which they usually participate as cofactors. Until now, the role of metals in cancer have been studied as an effect, establishing that cancer onset and progression affects the disturbance of the natural chemical form of the essential elements in the metabolism. However, it has also been studied as a cause, giving insights related to the high exposure of metals giving a place to the carcinogenic process. On the other hand, the chemical species of the metal or metallobiomolecule is very important, since it finally affects the biological activity or the toxicological potential of the element and their mobility across different biological compartments. Moreover, the importance of metal homeostasis and metals interactions in biology has also been demonstrated, and the ratios between some elements were found to be different in cancer patients; however, the interplay of elements is rarely reported. This review focuses on the critical role of metals in lung cancer, which is one of the most insidious forms of cancer, with special attention to the analytical approaches and pitfalls to extract metals and their species from tissues and biofluids, determining the ratios of metals, obtaining classification profiles, and finally defining the metallome of lung cancer.This research was funded by the projects CTM2015-67902-C-1-P from the Spanish Ministry of Economy and Competitiveness, and P12-FQM-0442 from the Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government, Spain). Projects Neumosur (8/2012 and 9/2015) and SEPAR (124/2012 and 091/2016). Belén Callejón Leblic thanks the Ministry of Education, Culture and Sport for a predoctoral scholarship. Finally, authors are grateful to FEDER (European Community) for financial support, grants number UNHU13-1E-1611 and UNHU15-CE-3140

Mice brain metabolomics after the exposure to a “chemical cocktail” and selenium supplementation through the gut-brain axis

Several environmental pollutants have been shown to damage brain and affect gut microbiota. Limited evidence is available about the impact of “chemical cocktails” (CC) of xenobiotics on brain metabolome and their possible influence in the gut-brain crosstalk. To this end, BALB/c mice were exposed to heavy metals (As, Hg, Cd) and pharmaceuticals (diclofenac and flumequine) under regular rodent diet or supplemented with selenium (Se). Selenium, an antioxidant well-known for its antagonism against the neurotoxicity of several pollutants, modulated several brain metabolic impairments caused by CC (e.g., brain levels of the excitatory amino acid N-acetyl aspartic acid) by influencing mainly the metabolisms of purine, glycosylate and dicarboxylate, glutamate, glycerophospholipid, alanine and aspartate. Numerous associations were obtained between brain metabolites and gut microbes and they changed after Se-supplementation (e.g., Lactobacillus was positively associated with a brain ceramide, phosphoserine, phosphocholine, vitamin D3 derivative, fatty acids, malic acid, amino acids, and urea after the exposure, but not after Se-supplementation). Our results showed numerous evidences about the impact of CC on brain metabolome, the potential role of Se as an antagonist and their impact on the gut-brain axis. Further research is needed to understand the complex mechanism of action implied on CC-brain-microbiota interactions.This work was supported by the projects: PG2018–096608-B-C21 and PID2021-123073NB-C21 from the Spanish Ministry of Science and Innovation (MICIN). Generación del Conocimiento. MCI/AEI/ FEDER “Una manera de hacer Europa”, UHU-1256905 and UHU-202009 from the FEDER Andalusian Operative Program 2014–2020 (Ministry of Economy, Knowledge, Business and Universities, Regional Government of Andalusia, Spain). The authors are grateful to FEDER (European Community) for financial support, Grant UNHU13–1E-1611. CPM thanks MICIN for a predoctoral grant (ref. PRE2019–091650). Funding for open access charge: Universidad de Huelva / CBUA. The authors would like to acknowledge the support from The Ramón Areces Foundation (ref. CIVP19A5918)

Gut-gonad crosstalk in mice exposed to a “chemical cocktail” combining metabolomics and microbial profile by amplicon sequencing

Testes are very prone to be damaged by environmental pollutants, but there is a lack of information about the impact of “chemical cocktails” (CC) on the testicular metabolome and the possible influence in the gut-gonad crosstalk. For this, BALB/c mice were given flumequine and diclofenac orally in food and potentially toxic trace elements (Cd, Hg, As) in drinking water. A mice group was supplemented with selenium, a well-known antagonist against many pollutants. Our results revealed that the steroid 5-alpha-androstan-17-beta-ol propionate, suggested as a parameter of androgenicity independent of testosterone levels, proline that improves reproductive indicators in male rabbits affected by environmental stress) among others metabolites are only present after CC exposure with rodent and selenium supplemented diet. Selenium also antagonized the up-or down-regulation of anandamide (20:l, n-9) (p 0,05 and FC 0.74 of CC-Se vs C), that regulates gonadotropin-releasing hormones in mammals, 2,3-dinor-11b-PGF2a (p 0,05 and FC 0.34 of CC-Se vs C), which has been related with reproductive hormones, besides others testicular metabolites altered by the exposure to the CC and reversed the levels to control. Moreover, numerous significant associations between gut microbes and testicular metabolites indicated a possible impact of pollutants in the testes mediated by gut microbiota due to a gut-gonad crosstalk.This work was supported by the projects: PID2021-123073NB-C21 from the Spanish Ministry of Science and Innovation (MICIN). Generaci´ on del Conocimiento. MCI/AEI/FEDER “Una manera de hacer Europa”; UHU-1256905 and UHU-202009 from the FEDER Andalusian Operative Program 2014–2020 (Ministry of Economy, Knowledge, Business and Universities, Regional Government of Andalusia, Spain). The authors are grateful to FEDER (European Community) for financial support, Grant UNHU13-1E-1611. CPM thanks MICIN for a predoctoral grant (ref. PRE2019-091,650). MSR and MCC also acknowledge the award of the Spanish Government MCIN/AEI to the Institute of Agrochemistry and Food Technology (IATA-CSIC) as Centre of Excellence Severo Ochoa (CEX2021-001189-S MCIN/AEI / 10.13039/ 501100011033). The authors would like to acknowledge the support from The Ram´ on Areces Foundation (ref. CIVP19A5918). Funding for open access charge: Universidad de Huelva/CBUA.Departamento de Química "Profesor José Carlos Vílchez Martín

Untargeted Gut Metabolomics to Delve the Interplay between Selenium Supplementation and Gut Microbiota

El selenio (Se) es un oligoelemento con funciones importantes para la salud debido a las propiedades antioxidantes de las selenoproteínas. Para analizar la interacción entre Se y la microbiota intestinal, se determinaron los perfiles metabolómicos intestinales en ratones convencionales (C) y con microbiota empobrecida (Abx) después de la suplementación con Se (Abx-Se) mediante metabolómica no dirigida, utilizando una multiplataforma analítica basada en GC-MS y UHPLC-QTOF-MS (MassIVE ID MSV000087829). El perfil de la microbiota intestinal se realizó mediante la secuenciación del amplicón del gen 16S rRNA. Se encontraron diferencias significativas en los niveles de aproximadamente el 70 % de los metabolitos intestinales determinados, incluidos los acilos grasos, los glicerolípidos, los glicerofosfolípidos y los esteroides, en Abx-Se en comparación con Abx, y solo el 30 % fue diferente entre Abx-Se y C, lo que sugiere un efecto importante de la suplementación con Se en el metabolismo de los ratones Abx. A nivel de género, el análisis de correlación mostró fuertes asociaciones entre los metabolitos y los perfiles bacterianos intestinales. Asimismo, una mayor abundancia de Lactobacillus spp., un género potencialmente beneficioso enriquecido tras la suplementación con Se, se asoció con niveles más altos de lípidos de prenol, fosfatidilgliceroles (C-Se), esteroides y diterpenoides (Abx-Se), y también con niveles más bajos de ácidos grasos (Abx-Se). Por lo tanto, observamos una interacción crucial entre la ingesta de Se-microbiota-metabolitos, aunque se necesitan más estudios para aclarar los mecanismos específicos. Este es el primer estudio sobre la metabolómica intestinal no dirigida después del agotamiento de la microbiota y la suplementación con Se.Selenium (Se) is an essential trace element with important health roles due to the antioxidant properties of selenoproteins. To analyze the interplay between Se and gut microbiota, gut metabolomic profiles were determined in conventional (C) and microbiota depleted mice (Abx) after Se-supplementation (Abx-Se) by untargeted metabolomics, using an analytical multiplatform based on GC-MS and UHPLC-QTOF-MS (MassIVE ID MSV000087829). Gut microbiota profiling was performed by 16S rRNA gene amplicon sequencing. Significant differences in the levels of about 70% of the gut metabolites determined, including fatty acyls, glycerolipids, glycerophospholipids, and steroids, were found in Abx-Se compared to Abx, and only 30% were different between Abx-Se and C, suggesting an important effect of Se-supplementation on Abx mice metabolism. At genus level, the correlation analysis showed strong associations between metabolites and gut bacterial profiles. Likewise, higher abundance of Lactobacillus spp., a potentially beneficial genus enriched after Se-supplementation, was associated with higher levels of prenol lipids, phosphatidylglycerols (C-Se), steroids and diterpenoids (Abx-Se), and also with lower levels of fatty acids (Abx-Se). Thus, we observed a crucial interaction between Se intake–microbiota–metabolites, although further studies to clarify the specific mechanisms are needed. This is the first study about untargeted gut metabolomics after microbiota depletion and Se-supplementation.This work was supported by the projects PG2018-096608-B-C21 f from the Spanish Ministry of Science and innovation (MCIN). Generación del Conocimiento. MCIN/ AEI /10.13039/501100011033/ FEDER “Una manera de hacer Europa” and UHU-1256905 from the FEDER Andalusian Operative Program 2014–2020 (Ministry of Economy, Knowledge, Business and Universities, Regional Government of Andalusia, Spain). Authors would like to acknowledge the support from The Ramón Areces Foundation (ref CIVP19A5918). Authors are grateful to FEDER (European Community) for financial support, Grant UNHU13-1X10-1611. Funding for open access charge: Universidad de Huelva / CBU

Metallomic Signatures of Lung Cancer and Chronic Obstructive Pulmonary Disease

Lung cancer (LC) is the leading cause of cancer deaths, and chronic obstructive pulmonary disease (COPD) can increase LC risk. Metallomics may provide insights into both of these tobaccorelated diseases and their shared etiology. We conducted an observational study of 191 human serum samples, including those of healthy controls, LC patients, COPD patients, and patients with both COPD and LC.We found 18 elements (V, Al, As, Mn, Co, Cu, Zn, Cd, Se,W, Mo, Sb, Pb, Tl, Cr, Mg, Ni, and U) in these samples. In addition, we evaluated the elemental profiles of COPD cases of varying severity. The ratios and associations between the elements were also studied as possible signatures of the diseases. COPD severity and LC have a significant impact on the elemental composition of human serum. The severity of COPD was found to reduce the serum concentrations of As, Cd, and Tl and increased the serum concentrations of Mn and Sb compared with healthy control samples, while LC was found to increase Al, As, Mn, and Pb concentrations. This study provides new insights into the effects of LC and COPD on the human serum elemental profile that will pave the way for the potential use of elements as biomarkers for diagnosis and prognosis. It also sheds light on the potential link between the two diseases, i.e., the evolution of COPD to LC.This work has been supported by the project "Heteroatom-tagged proteomics and metabolomics to study lung cancer. Influence of gut microbiota” (Ref.: PY20_00366) (Project of Excellence, Regional Ministry of Economy, Knowledge, Business and University, Andalusia, Spain). The authors are also grateful for grants 651/2018 and 115/2020 from the Spanish Society of Pneumology and Surgery (SEPAR) and grant 08/2018 from the Association of Pneumology and Thoracic Surgery (Neumosur), which were used to facilitate recruitment at the hospitals and biobank registration. The authors also thank Instituto de Salud Carlos III (AES16/01783) and wish to express their gratitude for the unrestricted funding from the Menarini Group and AstraZeneca