Immune evolution from preneoplasia to invasive lung adenocarcinomas and underlying molecular features

The mechanism by which anti-cancer immunity shapes early carcinogenesis of lung adenocarcinoma (ADC) is unknown. In this study, we characterize the immune contexture of invasive lung ADC and its precursors by transcriptomic immune profiling, T cell receptor (TCR) sequencing and multiplex immunofluorescence (mIF). Our results demonstrate that anti-tumor immunity evolved as a continuum from lung preneoplasia, to preinvasive ADC, minimally-invasive ADC and frankly invasive lung ADC with a gradually less effective and more intensively regulated immune response including down-regulation of immune-activation pathways, up-regulation of immunosuppressive pathways, lower infiltration of cytotoxic T cells (CTLs) and anti-tumor helper T cells (Th), higher infiltration of regulatory T cells (Tregs), decreased T cell clonality, and lower frequencies of top T cell clones in later-stages. Driver mutations, chromosomal copy number aberrations (CNAs) and aberrant DNA methylation may collectively impinge host immune responses and facilitate immune evasion, promoting the outgrowth of fit subclones in preneoplasia into dominant clones in invasive ADC

Simultaneous evolutionary expansion and constraint of genomic heterogeneity in multifocal lung cancer.

Recent genomic analyses have revealed substantial tumor heterogeneity across various cancers. However, it remains unclear whether and how genomic heterogeneity is constrained during tumor evolution. Here, we sequence a unique cohort of multiple synchronous lung cancers (MSLCs) to determine the relative diversity and uniformity of genetic drivers upon identical germline and environmental background. We find that each multicentric primary tumor harbors distinct oncogenic alterations, including novel mutations that are experimentally demonstrated to be functional and therapeutically targetable. However, functional studies show a strikingly constrained tumorigenic pathway underlying heterogeneous genetic variants. These results suggest that although the mutation-specific routes that cells take during oncogenesis are stochastic, genetic trajectories may be constrained by selection for functional convergence on key signaling pathways. Our findings highlight the robust evolutionary pressures that simultaneously shape the expansion and constraint of genomic diversity, a principle that holds important implications for understanding tumor evolution and optimizing therapeutic strategies.Across cancer types tumor heterogeneity has been observed, but how this relates to tumor evolution is unclear. Here, the authors sequence multiple synchronous lung cancers, highlighting the evolutionary pressures that simultaneously shape the expansion and constraint of genomic heterogeneity

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

COMPUTATIONAL GENOMIC MODELS FOR SPATIO-TEMPORAL INVESTIGATION OF EARLY LUNG CANCER PATHOLOGY

Lung cancer, of which non-small cell lung cancer (NSCLC) is the most common form, is the second most prevalent cancer and the leading cause of cancer-related deaths. NSCLCs primarily comprise adenocarcinomas (LUAD) and squamous cell carcinomas (LUSC). Advances in early detection and prevention have been limited by the lack of early-stage biomarkers and targets. A comprehensive molecular characterization of premalignant lesions and tumor-adjacent normal tissue can aid in better understanding NSCLC pathogenesis. However, these investigations are further challenged by limited tissue availability and low cellular fractions of detectable somatic mutations. Therefore, there is a dearth of knowledge about the pathogenesis of premalignant lung lesions, especially for atypical adenomatous hyperplasia (AAH), the only known precursor to LUADs. We performed a cross-platform integrative analysis comprising targeted DNA sequencing, genotype array profiling and transcriptome sequencing of matched AAHs, LUADs and normal tissues from 23 early-stage patients. The study revealed potentially divergent pathways based on the mutation status of AAH (BRAF vs KRAS), recurrent chromosomal aberrations (17p loss) and the presence of immune deregulation early in the pathogenesis of AAHs. Molecular changes, characteristic of NSCLCs, might also occur in normal tissues, preceding identifiable premalignancy-associated morphological changes. We sought to comprehensively survey the somatic mutational architecture of the normal airway in early-stage NSCLCs. Targeted DNA sequencing allowed us to capture driver mutations at low cellular fractions, typical of these non-malignant tissues. Additionally, genotype array profiling helped characterize subtle chromosomal aberrations in these tissues. This multi-region study included tumor-adjacent and -distant airways, nasal epithelia and uninvolved normal lung (collectively cancerized field) along with matched multi-region NSCLCs and blood cells from 48 patients. Integrative computational analysis revealed genomic airway field carcinogenesis in 52% of cases. The airway field exhibited mutations in known drivers, that were present at lower frequencies compared to NSCLCs, suggestive of selection-driven clonal expansion. These driver events also comprised somatic “two-hit” alterations in matched airway field and NSCLCs. Our study design offers spatiotemporal insights into NSCLC development and suggests potential targets for early detection and treatment, in possibly less hostile environments of premalignancy. To validate and enhance the utility of the bioinformatic techniques devised and implemented for these investigations, I also provide methods to expand such analyses across multiple tumor sites

Histopathology-selective spatial oncogenic phenotypes in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) constitutes over 85% of lung cancer. Histologically, NSCLC can be broadly classified into adenocarcinoma (AC), squamous cell carcinoma (SCC), large cell carcinoma (LCC), and adenosquamous carcinoma (ASC). AC represents about 65% of all NSCLC cases, and it can be further subdivided based on tumor size and primary growth patterns, such as papillary, acinar, and mucinous. The formation of NSCLC histotypes is orchestrated by cells of origin, genetic alterations, and microenvironmental properties. Although NSCLC carries significant heterogeneity, some genetic mutations, functional phenotypes, and therapeutic responses are associated with specific NSCLC histotypes. Therefore, understanding histotype-selective etiology becomes essential for mechanistic studies and therapeutic applications in the NSCLC research field. Image-based tissue phenotyping has been commonly used for histological classification. It also allows the direct visualization of the distribution and expression of functional molecules. Quantifying such in situ phenotypes can be applied to hypothesis-based functional studies or data-driven correlative analyses. The first part of this thesis developed a spatial image analysis tool package. The making of Spa-RQ, an open-source tool package for image registration and quantification, reflected on the need to perform spatial phenotyping using serial tissue sections in a standardized laboratory workflow. Subsequently, we applied Spa-RQ to identify the histotype-selective, rather than genetically defined activation of MAPK, AKT, and mTOR signaling pathways in murine and human NSCLC samples. The diverse co-activation patterns between these pathways in different tissue compartments, measured by marker expression overlapping using Spa-RQ, may associate with heterogeneous responses towards combinatorial targeted therapies. The second part of this thesis work investigated the histotype-selective functions of a potential therapeutic target. The lung developmental transcription factor SOX9 is silenced in normal adult lung epithelia while it is re-expressed in NSCLC tissues. Its oncogenicity is widely acknowledged but has thus far not been confirmed in NSCLC subtypes. Analyzing the correlation between SOX9 expression and histotype-specific clinical staging, survival, and invasiveness revealed a clinical significance for increased SOX9 expression only in non-mucinous ACs, despite its broad expression in ASC, SCC, and mucinous AC. Supporting this, by comparing the histotype spectra in mouse models following Sox9 loss, we identified a critical role of SOX9 in promoting lung papillary AC progression. On the other hand, its expression was not required for developing squamous and mucinous structure tissues. Finally, using spatial phenotyping, we explained such opposing roles of SOX9 in NSCLC subtypes by the different cells of origin and microenvironmental properties: SOX9 expression was required to form advanced AC from the lung alveolar progenitor cells; on the contrary, its expression was dispensable for SCC development and even interfered with squamous metastasis. Therefore, this work exposed SOX9 as a potential drug target specific to a subgroup of lung AC. In summary, the identification of histotype-selective functional oncogenic phenotypes, as achieved in this thesis, contributes to understanding the heterogeneous nature of tumorigenesis, cancer progression, and drug sensitivities.Non-small cell lung cancer (NSCLC) constitutes over 85% of lung cancer. Histologically, NSCLC can be broadly classified into adenocarcinoma (AC), squamous cell carcinoma (SCC), large cell carcinoma (LCC), and adenosquamous carcinoma (ASC). AC represents about 65% of all NSCLC cases, and it can be further subdivided based on tumor size and primary growth patterns, such as papillary, acinar, and mucinous. The formation of NSCLC histotypes is orchestrated by cells of origin, genetic alterations, and microenvironmental properties. Although NSCLC carries significant heterogeneity, some genetic mutations, functional phenotypes, and therapeutic responses are associated with specific NSCLC histotypes. Therefore, understanding histotype-selective etiology becomes essential for mechanistic studies and therapeutic applications in the NSCLC research field. Image-based tissue phenotyping has been commonly used for histological classification. It also allows the direct visualization of the distribution and expression of functional molecules. Quantifying such in situ phenotypes can be applied to hypothesis-based functional studies or data-driven correlative analyses. The first part of this thesis developed a spatial image analysis tool package. The making of Spa-RQ, an open-source tool package for image registration and quantification, reflected on the need to perform spatial phenotyping using serial tissue sections in a standardized laboratory workflow. Subsequently, we applied Spa-RQ to identify the histotype-selective, rather than genetically defined activation of MAPK, AKT, and mTOR signaling pathways in murine and human NSCLC samples. The diverse co-activation patterns between these pathways in different tissue compartments, measured by marker expression overlapping using Spa-RQ, may associate with heterogeneous responses towards combinatorial targeted therapies. The second part of this thesis work investigated the histotype-selective functions of a potential therapeutic target. The lung developmental transcription factor SOX9 is silenced in normal adult lung epithelia while it is re-expressed in NSCLC tissues. Its oncogenicity is widely acknowledged but has thus far not been confirmed in NSCLC subtypes. Analyzing the correlation between SOX9 expression and histotype-specific clinical staging, survival, and invasiveness revealed a clinical significance for increased SOX9 expression only in non-mucinous ACs, despite its broad expression in ASC, SCC, and mucinous AC. Supporting this, by comparing the histotype spectra in mouse models following Sox9 loss, we identified a critical role of SOX9 in promoting lung papillary AC progression. On the other hand, its expression was not required for developing squamous and mucinous structure tissues. Finally, using spatial phenotyping, we explained such opposing roles of SOX9 in NSCLC subtypes by the different cells of origin and microenvironmental properties: SOX9 expression was required to form advanced AC from the lung alveolar progenitor cells; on the contrary, its expression was dispensable for SCC development and even interfered with squamous metastasis. Therefore, this work exposed SOX9 as a potential drug target specific to a subgroup of lung AC. In summary, the identification of histotype-selective functional oncogenic phenotypes, as achieved in this thesis, contributes to understanding the heterogeneous nature of tumorigenesis, cancer progression, and drug sensitivity