Búsqueda de dianas terapéuticas en los puntos de contacto de la célula tumoral con su matriz extracelular en tumores neuroblásticos

El neuroblastoma es un tumor embrionario del sistema nervioso simpático que representa el 15% de las muertes relacionadas con cáncer en la infancia. Se caracteriza por un amplio espectro de comportamientos clínicos derivados de su gran heterogeneidad en la presentación clínica y en los rasgos biológicos y genéticos. La clasificación de riesgo pre-tratamiento desempeña un papel central en la mejora de la supervivencia en estos pacientes, sin embargo, el subgrupo de pacientes de alto riesgo continúa teniendo una tasa de mortalidad particularmente alta, destacando la necesidad de identificar y validar nuevas terapias, modelos preclínicos y marcadores de respuesta terapéutica. Por este motivo, es crucial tener en cuenta el concepto de que un tumor es un tejido funcional e interconectado, donde las células tumorales proliferan sin control en una relación dependiente del macro-microambiente tumoral, estableciendo así un diálogo continuo y recíproco a través de la transmisión de señales que es esencial para la supervivencia e invasión. La complejidad biológica del neuroblastoma justifica claramente el uso cada vez mayor de estudios cuantitativos para obtener un mayor conocimiento de la biotensegridad, mecanotransducción, arquitectura, topología e interacción de sus elementos. Nuestra hipótesis es que las señales de tensión transferidas de la matriz extracelular a las células tumorales influyen en el crecimiento, diferenciación y migración de estas células, lo que exige el uso de diferentes enfoques para buscar dianas en los puntos de contacto entre estos elementos. La disposición de los patrones derivados del análisis morfométrico y topológico de muestras tumorales humanas, así como los derivados de modelos in vitro e in vivo, asociándolos con el impacto de factores pronósticos clínicos y biológicos conocidos, pueden mejorar la supervivencia en pacientes afectados por neuroblastoma, especialmente aquellos considerados de alto riesgo. El objetivo general de esta investigación es demostrar que la vitronectina presente en el microambiente tumoral del neuroblastoma es un conector crucial dentro de los elementos de la matriz extracelular, modulando las señales físicas y químicas entre las células tumorales y sus elementos circundantes para facilitar la migración. Los objetivos específicos de estudio en muestras de tumor de neuroblastoma humano son: a) caracterización morfométrica y topológica de la expresión de vitronectina a través del diseño de varios algoritmos; b) la correlación de la expresión de vitronectina y sus ligandos con las características clínicas de los pacientes y otras características biológicas del tumor con valor pronóstico conocido, para determinar los patrones histológicos de vitronectina con diferentes grados de malignidad; y c) la asociación de la expresión y distribución de vitronectina con otros elementos biotensegrales tumorales tales como: fibras de reticulina, colágeno tipo I, glucosaminoglucanos, vasos sanguíneos/linfáticos y células inmunes, para establecer un patrón de matriz extracelular biotensegral agresivo. Los objetivos específicos del estudio en líneas celulares de neuroblastoma son: a) identificación y caracterización de vitronectina y sus ligandos de unión tales como: integrina ανβ3, uPAR y PAI-1; y b) el establecimiento de los patrones de vitronectina y sus ligandos de unión en líneas celulares de neuroblastoma en comparación con muestras de neuroblastoma humano. Los objetivos definidos de estudio en los modelos in vivo de neuroblastoma son: a) generar xenoinjertos de tumor ortotópico en ratones inmunodeficientes y con deficiencia de vitronectina, utilizando la inoculación inicial de líneas celulares de neuroblastoma y la implantación posterior de sus fragmentos tumorales, para determinar su homología clínico-biológica con pacientes con neuroblastoma de alto riesgo; b) la caracterización de varios elementos del microambiente tumoral, como la vitronectina y sus ligandos, fibras reticulares, fibras de colágeno tipo I, glucosaminoglucanos, vasos sanguíneos y linfáticos, para determinar los patrones histológicos en este modelo; y c) describir las características genéticas de los tumores obtenidos para conocer el impacto de las propiedades de la matriz extracelular en la heterogeneidad genómica del neuroblastoma, particularmente la contribución de la vitronectina como elemento del macroambiente tumoral. La presente tesis doctoral se presenta como un compendio de tres publicaciones y datos relacionados, recientemente obtenidos, que proporcionan información sobre las interacciones mecánicas de las células neuroblásticas y los elementos extracelulares que las rodean, considerando la glucoproteína de adhesión, vitronectina, como un punto de contacto clave para facilitar la migración. Los artículos que constituyen el compendio son los siguientes: I. The tumour microenvironment as an integrated framework to understand cancer biology. Burgos-Panadero R*, Lucantoni F*, Gamero-Sandemetrio E, Cruz-Merino L, Álvaro T, Noguera R Cancer Lett. 2019. 1; 461:112-122. Factor de impacto a 5 años: 6.232. El objetivo de esta revisión es enfatizar la importancia de identificar biomarcadores que capturen las interacciones que ocurren en el microambiente tumoral y que estén relacionados con la agresividad del tumor, el pronóstico del paciente y la respuesta al tratamiento. El punto esencial en el presente trabajo, es la clasificación propuesta del estroma tumoral en tres grados, asociada con la implicación clínica y terapéutica, basada fundamentalmente en datos derivados de los estudios en neuroblastoma. II. Vitronectin as a molecular player of the tumor microenvironment in neuroblastoma. Burgos-Panadero R, Noguera I, Cañete A, Navarro S, Noguera R BMC Cancer. 2019. 22;19(1):479. Factor de impacto a 5 años: 3.424 El propósito de este estudio es caracterizar la vitronectina, como una molécula diana de la matriz extracelular, en una cohorte de tumores neuroblásticos elegidos por criterios de inestabilidad genética. El estudio inicial en modelos in vitro e in vivo de la proteína vitronectina y sus ligandos asociado con factores clínico-biológicos pronósticos conocidos, se ha ampliado durante un mayor período de tiempo. III. The topology of vitronectin: A complementary feature for neuroblastoma risk classification based on computer-aided detection. Vicente-Munuera P*, Burgos-Panadero R*, Noguera I, Navarro S, Noguera R, Escudero LM. Int J Cancer. 2020 Jan 15; 146(2):553-565. Factor de impacto a 5 años: 6.210 El objetivo de esta investigación es detectar patrones de distribución de vitronectina en el estroma tumoral que reflejen el comportamiento de las células neuroblásticas en una cohorte de tumores de neuroblastoma en el momento del diagnóstico, elegido por criterios de inestabilidad genética. Nuestra discusión se centra en el análisis comparativo entre características no topológicas y topológicas, así como su asociación con el comportamiento tumoral agresivo. Después de aplicar enfoques multidisciplinares utilizados en nuestros estudios, fundamentalmente técnicas de análisis de imagen en muestras tumorales de pacientes complementadas con modelos preclínicos, concluimos que es posible y útil caracterizar la arquitectura estromal y parenquimal de los tumores para avanzar en la comprensión de nuevas dianas terapéuticas, promoviendo a su vez estrategias de colaboración y propuestas de ensayos clínicos adecuadas.Neuroblastoma is an embryonic tumor of the sympathetic nervous system which accounts for 15% of childhood cancer-related deaths. It is characterized by a wide spectrum of clinical behaviors derived from its great heterogeneity in clinical presentation and biological and genetic traits. Pre-treatment risk classification plays a central role in improving survival in these patients, however, yet the high-risk patient subgroup continues to have a particularly high mortality rate, highlighting a need to identify and validate new therapies, preclinical models and markers of therapeutic response. For this reason, it is crucial to keep in mind the concept that a tumor is a functional and interconnected tissue, where tumor cells proliferate uncontrollably in a dependent relationship with tumor macro and microenvironment, thus establishing a continuous and reciprocal dialogue through signaling, which is essential for survival and invasion. The biological complexity of neuroblastoma, clearly justifies the increasing use of quantitative studies to gain greater knowledge of biotensegrity, mechanotransduction, architecture, topology and interaction of its elements. We hypothesize that tension signals transferred from the extracellular matrix to tumor cells influence growth, differentiation and migration of these cells, necessitating the use of different approaches to search for targets at the contact points between these elements. The arrangement of patterns derived from morphometric and topological analysis in human tumor samples, as well as those derived from in vitro and in vivo models, and their relationship with the impact of known clinical and biological prognostic factors, may improve survival in patients affected by neuroblastoma, especially those considered high risk. The general aim of this research is to demonstrate that the vitronectin present in the neuroblastoma tumor microenvironment is a crucial connector within the elements of the extracellular matrix, modulating physical and chemical signaling between tumor cells and their surrounding elements to facilitate migration. The specific objectives of the study in human neuroblastoma tumor samples are: a) morphometric and topological characterization of vitronectin expression through the design of various algorithms; b) correlating expression of vitronectin and its ligands with patient clinical features and other tumor biological characteristics with known prognostic value, to determine the histological patterns of vitronectin with different degrees of malignancy; and c) associating vitronectin expression and distribution with other tumor biotensegral elements such as reticular fibers, type I collagen, glycosaminoglycans, blood/lymphatic vessels and immune cells, to establish a pattern of aggressive biotensegral extracellular matrix. The specific objectives of neuroblastoma cell lines study are: a) identification and characterization of vitronectin and its binding ligands such as ανβ3 integrin, uPAR, and PAI-1; and b) establishing vitronectin and binding ligands patterns of neuroblastoma cell lines compared to human neuroblastoma samples. The defined study aims of the in vivo neuroblastoma models are to: a) generate orthotopic tumor xenografts in vitronectin-deficient and immunodeficient mice using the initial inoculation of neuroblastoma cell lines and the subsequent implantation of their tumor fragments, to determine their clinical-biological homology with high-risk neuroblastoma patients; b) characterize various elements of the tumor microenvironment such as vitronectin and its ligands, reticular fibers, type I collagen fibers, glycosaminoglycans, blood and lymphatic vessels, to determine histological patterns in this model; and c) describe the genetic characteristics of the tumors obtained to uncover the impact of extracellular matrix properties on the genomic heterogeneity of neuroblastoma, particularly the contribution of vitronectin as an element of the tumor macroenvironment. The present doctoral thesis is presented as a compendium of three publications and newly obtained related data providing insight into the mechanical interactions of neuroblastic cells and the extracellular elements that surround them, considering vitronectin adhesion glycoprotein as a key contact point to facilitate migration. The following articles make up this compendium: I. The tumor microenvironment as an integrated framework to understand cancer biology. Burgos-Panadero R*, Lucantoni F*, Gamero-Sandemetrio E, Cruz-Merino L, Álvaro T, Noguera R Cancer Lett. 2019. 1; 461:112-122. 5-year impact factor: 6.232. The purpose of this review is to emphasize the importance of identifying biomarkers that capture interactions occuring in the tumor microenvironment and that are related to the tumor aggressiveness, patient prognosis, and treatment response. A key highlight of the present work is the proposed classification of the tumor stroma into three grades, associated with clinical and therapeutic involvement, based primarily on data derived from neuroblastoma studies. II. Vitronectin as a molecular player of the tumor microenvironment in neuroblastoma. Burgos-Panadero R, Noguera I, Cañete A, Navarro S, Noguera R BMC Cancer. 2019. 22;19(1):479. 5-year impact factor: 3.424 The objective of this study is to characterize vitronectin as an extracellular matrix target molecule, in a cohort of neuroblastic tumors chosen by genetic instability criteria. The initial study in in vitro and in vivo models of the vitronectin protein and its ligands associated with known clinical-biological prognostic factors has been expanded over a greater time period. III. The topology of vitronectin: A complementary feature for neuroblastoma risk classification based on computer-aided detection. Vicente-Munuera P*, Burgos-Panadero R*, Noguera I, Navarro S, Noguera R, Escudero LM. Int J Cancer. 2020 Jan 15; 146(2):553-565. 5-year impact factor: 6.210. The goal of this research is to detect vitronectin distribution patterns in the tumor stroma that reflect neuroblastic cell behavior in a cohort of neuroblastoma tumors at the time of diagnosis chosen by criteria of genetic instability. Our discussion focuses on the comparative analysis between non-topological and topological characteristics and their association with aggressive tumor behavior. After applying multidisciplinary approaches used in our studies, fundamentally image analysis techniques from patient tumor samples complemented by preclinical models, we conclude that it is both possible and useful to characterize the stromal and parenchymal architecture of tumors to advance understanding of new therapeutic targets, while also promoting collaboration strategies and suitable clinical trial proposals

A three-dimensional bioprinted model to evaluate the effect of stiffness on neuroblastoma cell cluster dynamics and behavior

Three-dimensional (3D) bioprinted culture systems allow to accurately control microenvironment components and analyze their effects at cellular and tissue levels. The main objective of this study was to identify, quantify and localize the effects of physical-chemical communication signals between tumor cells and the surrounding biomaterial stiffness over time, defining how aggressiveness increases in SK-N-BE(2) neuroblastoma (NB) cell line. Biomimetic hydrogels with SK-N-BE(2) cells, methacrylated gelatin and increasing concentrations of methacrylated alginate (AlgMA 0%, 1% and 2%) were used. Young's modulus was used to define the stiffness of bioprinted hydrogels and NB tumors. Stained sections of paraffin-embedded hydrogels were digitally quantified. Human NB and 1% AlgMA hydrogels presented similar Young´s modulus mean, and orthotopic NB mice tumors were equally similar to 0% and 1% AlgMA hydrogels. Porosity increased over time; cell cluster density decreased over time and with stiffness, and cell cluster occupancy generally increased with time and decreased with stiffness. In addition, cell proliferation, mRNA metabolism and antiapoptotic activity advanced over time and with stiffness. Together, this rheological, optical and digital data show the potential of the 3D in vitro cell model described herein to infer how intercellular space stiffness patterns drive the clinical behavior associated with NB patients

The topology of vitronectin: A complementary feature for neuroblastoma risk classification based on computer‐aided detection

Tumors are complex networks of constantly interacting elements: tumor cells, stromal cells, immune and stem cells, blood/lympathic vessels, nerve fibers and extracellular matrix components. These elements can influence their microenvironment through mechanical and physical signals to promote tumor cell growth. To get a better understanding of tumor biology, cooperation between multidisciplinary fields is needed. Diverse mathematic computations and algorithms have been designed to find prognostic targets and enhance diagnostic assessment. In this work, we use computational digital tools to study the topology of vitronectin, a glycoprotein of the extracellular matrix. Vitronectin is linked to angiogenesis and migration, two processes closely related to tumor cell spread. Here, we investigate whether the distribution of this molecule in the tumor stroma may confer mechanical properties affecting neuroblastoma aggressiveness. Combining image analysis and graph theory, we analyze different topological features that capture the organizational cues of vitronectin in histopathological images taken from human samples. We find that the Euler number and the branching of territorial vitronectin, two topological features, could allow for a more precise pretreatment risk stratification to guide treatment strategies in neuroblastoma patients. A large amount of recently synthesized VN would create migration tracks, pinpointed by both topological features, for malignant neuroblasts, so that dramatic change in the extracellular matrix would increase tumor aggressiveness and worsen patient outcomes.L.M.E. is supported by the Ramón y Cajal program (PI13/01347). The work of L.M.E. and P. V.‐M. is funded by the Ministry of Economy, Industry, and Competitiveness grant BFU2016‐74975‐P cofunded by FEDER funds. P.V.‐M. and R.B.‐P. are supported by a contract by the Asociación Fundación Española contra el Cáncer. P.V.‐M. contract is also supported by Seville University (V plan propio). This work was funded by the FAECC (contract 2015, FAECC2015/006), CIBERONC (CB16/12/00484) and FIS (PI17/01558, Institute of Health Carlos III, Madrid/ERDF), NEN Association (Nico contra el cancer infantil 2017)

Integrating the Tumor Microenvironment into Cancer Therapy

© 2020 by the authors.Tumor progression is mediated by reciprocal interaction between tumor cells and their surrounding tumor microenvironment (TME), which among other factors encompasses the extracellular milieu, immune cells, fibroblasts, and the vascular system. However, the complexity of cancer goes beyond the local interaction of tumor cells with their microenvironment. We are on the path to understanding cancer from a systemic viewpoint where the host macroenvironment also plays a crucial role in determining tumor progression. Indeed, growing evidence is emerging on the impact of the gut microbiota, metabolism, biomechanics, and the neuroimmunological axis on cancer. Thus, external factors capable of influencing the entire body system, such as emotional stress, surgery, or psychosocial factors, must be taken into consideration for enhanced management and treatment of cancer patients. In this article, we review prognostic and predictive biomarkers, as well as their potential evaluation and quantitative analysis. Our overarching aim is to open up new fields of study and intervention possibilities, within the framework of an integral vision of cancer as a functional tissue with the capacity to respond to different non-cytotoxic factors, hormonal, immunological, and mechanical forces, and others inducing stroma and tumor reprogramming.This work was supported by grants from ISCIII (FIS) and FEDER (European Regional Development Fund) PI17/01558; CIBERONC (contract CB16/12/00484); Fundación Científica de la Asociación Española contra el Cáncer (FAECC2015/006) and NEN Association (Nico contra el cancer infantil 2017—PVR00157)

The tumour microenvironment as an integrated framework to understand cancer biology.

Cancer cells all share the feature of being immersed in a complex environment with altered cell-cell/cell-extracellular element communication, physicochemical information, and tissue functions. The so-called tumour microenvironment (TME) is becoming recognised as a key factor in the genesis, progression and treatment of cancer lesions. Beyond genetic mutations, the existence of a malignant microenvironment forms the basis for a new perspective in cancer biology where connections at the system level are fundamental. From this standpoint, different aspects of tumour lesions such as morphology, aggressiveness, prognosis and treatment response can be considered under an integrated vision, giving rise to a new field of study and clinical management. Nowadays, somatic mutation theory is complemented with study of TME components such as the extracellular matrix, immune compartment, stromal cells, metabolism and biophysical forces. In this review we examine recent studies in this area and complement them with our own research data to propose a classification of stromal changes. Exploring these avenues and gaining insight into malignant phenotype remodelling, could reveal better ways to characterize this disease and its potential treatment

Digital Image Analysis Applied to Tumor Cell Proliferation, Aggressiveness, and Migration-Related Protein Synthesis in Neuroblastoma 3D Models

Patient-derived cancer 3D models are a promising tool that will revolutionize personalized cancer therapy but that require previous knowledge of optimal cell growth conditions and the most advantageous parameters to evaluate biomimetic relevance and monitor therapy efficacy. This study aims to establish general guidelines on 3D model characterization phenomena, focusing on neuroblastoma. We generated gelatin-based scaffolds with different stiffness and performed SK-N-BE(2) and SH-SY5Y aggressive neuroblastoma cell cultures, also performing co-cultures with mouse stromal Schwann cell line (SW10). Model characterization by digital image analysis at different time points revealed that cell proliferation, vitronectin production, and migration-related gene expression depend on growing conditions and are specific to the tumor cell line. Morphometric data show that 3D in vitro models can help generate optimal patient-derived cancer models, by creating, identifying, and choosing patterns of clinically relevant artificial microenvironments to predict patient tumor cell behavior and therapeutic responses

Unraveling the extracellular matrix-tumor cell interactions to aid better targeted therapies for neuroblastoma

Treatment in children with high-risk neuroblastoma remains largely unsuccessful due to the development of metastases and drug resistance. The biological complexity of these tumors and their microenvironment represent one of the many challenges to face. Matrix glycoproteins such as vitronectin act as bridge elements between extracellular matrix and tumor cells and can promote tumor cell spreading. In this study, we established through a clinical cohort and preclinical models that the interaction of vitronectin and its ligands, such as αv integrins, are related to the stiffness of the extracellular matrix in high-risk neuroblastoma. These marked alterations found in the matrix led us to specifically target tumor cells within these altered matrices by employing nanomedicine and combination therapy. Loading the conventional cytotoxic drug etoposide into nanoparticles significantly increased its efficacy in neuroblastoma cells. We noted high synergy between etoposide and cilengitide, a high-affinity cyclic pentapeptide αv integrin antagonist. The results of this study highlight the need to characterize cell-extracellular matrix interactions, to improve patient care in high-risk neuroblastoma.This work was supported by grants from ISCIII and ERDF [PI17/01558] and [PI20/01107]; CIBERONC [contract CB16/12/00484]; Asociación Española contra el Cáncer [FAECC2015/006]; NEN Association [Nico contra el cancer infantil 2017 – PVR00157] and the Neuroblastoma Foundation [PVR00166]

Global loss of cellular m 6 A RNA methylation following infection with different SARS-CoV-2 variants

International audienceInsights into host–virus interactions during SARS-CoV-2 infection are needed to understand COVID-19 pathogenesis and may help to guide the design of novel antiviral therapeutics. N 6 -Methyladenosine modification (m 6 A), one of the most abundant cellular RNA modifications, regulates key processes in RNA metabolism during stress response. Gene expression profiles observed postinfection with different SARS-CoV-2 variants show changes in the expression of genes related to RNA catabolism, including m 6 A readers and erasers. We found that infection with SARS-CoV-2 variants causes a loss of m 6 A in cellular RNAs, whereas m 6 A is detected abundantly in viral RNA. METTL3, the m 6 A methyltransferase, shows an unusual cytoplasmic localization postinfection. The B.1.351 variant has a less-pronounced effect on METTL3 localization and loss of m 6 A than did the B.1 and B.1.1.7 variants. We also observed a loss of m 6 A upon SARS-CoV-2 infection in air/liquid interface cultures of human airway epithelia, confirming that m 6 A loss is characteristic of SARS-CoV-2-infected cells. Further, transcripts with m 6 A modification are preferentially down-regulated postinfection. Inhibition of the export protein XPO1 results in the restoration of METTL3 localization, recovery of m 6 A on cellular RNA, and increased mRNA expression. Stress granule formation, which is compromised by SARS-CoV-2 infection, is restored by XPO1 inhibition and accompanied by a reduced viral infection in vitro. Together, our study elucidates how SARS-CoV-2 inhibits the stress response and perturbs cellular gene expression in an m 6 A-dependent manner