Estudio del armazón arquitectónico y del sistema vascular de los tumores neuroblásticos

Los pacientes con tumores neuroblásticos presentan una evolución clínica heterogénea, desde la regresión espontánea hasta una alta propensión para la diseminación metastática generalizada. Aunque la aplicación de una clasificación de riesgo pre-tratamiento bien definida tiene un papel central en la mejora de la supervivencia durante los últimos años, han de llevarse a cabo más avances para mejorar la superviencia de los pacientes en general y específicamente el subgrupo de pacientes de alto riesgo. El estudio morfológico del tejido tumoral está contribuyendo a dicha mejora. La categoría histológica o el porcentaje de estroma tumoral, así como el grado de diferenciación de las células neuroblásticas, determinadas por el patólogo con el microscopio óptico, son factores con un papel importante en el diagnóstico y el pronóstico de los pacientes. Actualmente, dada la relevancia de la matriz extracelular tumoral en la biotensegridad y la mecanotransducción, su arquitectura y la topología de sus elementos, así como su interacción están siendo cada vez más considerados. Su cuantificación y caracterización con técnicas de imagen microscópicas empiezan a ser utilizadas. Nuestra hipótesis es que el destino de una célula tumoral neuroblástica es complejo y entre otros factores, está determinado por las características de un grupo de elementos estructurales no celulares de la matriz extracelular. Además pensamos que aplicando los patrones derivados del análisis morfométrico de estos elementos y asociandolos al impacto de los factores pronósticos conocidos, se mejorará la supervivencia de los pacientes. Nuestro objetivo es el desarrollo de técnicas morfométricas para caracterizar distintos elementos del andamiaje de la matriz extracelular y de la vascularización con el fin de encontrar usos potenciales como nuevos marcadores con valor pronóstico para mejorar la estratificación de los pacientes, o como dianas terapéuticas para ser capaces de remodelar los elementos aberrantes del andamiaje tisular, incluyendo la microvascularización. Hemos construido 19 micromatrices de tejido incluyendo más de 500 neuroblastomas, que fueron teñidos con alzul alcián a pH 2,5, Gomori, tricrómico de Masson, orceína y anti-CD31 para glicosaminoglicanos, fibras de reticulina, fibras de colágeno tipo I, fibras elásticas y vasos sanguíneos, respectivamente. Las laminillas fueron digitalizadas con un escáner de preparaciones y distintos algoritmos de análisis de imagen fueron diseñados o personalizados para detectar y caracterizar la cantidad, el tamaño y la forma de los distintos elementos estudiados de la matriz extracelular. Estos parámetros se relacionaron con los distintos subgrupos de neuroblastoma, teniendo en cuenta varias características clínicas, histopatológicas y genéticas. Los resultados obtenidos mostraron que las fibras de reticulina eran los componentes mayoritarios del andamiaje fibroso y que la abundancia y arquitectura de la microvascularización era relevante para el pronóstico de los niños con neuroblastoma. Una matriz extracelular rígida y poco porosa con vasos sanguíneos con luces irregulares se detectó principalmente en tumores pertenecientes a pacientes con pronóstico desfavorable. Un subgrupo de la cohorte de alto riesgo con muy mala supervivencia pudo ser definido por variables morfométricas de las fibras de reticulina y de los vasos sanguíneos. Concretamente, las muestras con un mayores áreas ocupadas tanto por fibras de reticulina formando grandes redes entrecruzadas, ramificadas y de organización compleja, como por vasos sanguíneos, junto con capilares y vasos tipo sinusoide de forma irregular y vénulas y arteriolas dilatas, estaban asociadas a un pronóstico muy desfavorable. En esta cohorte, las células con amplificación del gen MYCN conllevaron cambios topológicos detectables en relación a las fibras de reticulina y los vasos sanguíneos. Podemos concluir que es possible y conveniente cuantificar la sustancia fundamental, caracterizar el andamiaje fibroso y el sistema vascular de los tumors neuroblásticos gracias al análisis morfométrico de imágenes microscópicas. Algunas de las características morfométricas relaciondas con los distintos elementos de la matriz extracelular estudiados podrían ser usadas como ayuda diagnóstica del grupo de pacientes con riesgo ultra alto, tras estudiar una mayor cohorte. Los resultados obtenidos sugieren la necesidad de realizar trabajos multidisciplinarios para integrar de estos estudios a nivel internacional y que la información morfométrica de los elementos de la matriz extracelular, incluyendo el sistema vascular, pueda ser utilizada para una terapia basada en la mecanotransducción.Neuroblastic tumor patients present an heterogeneous clinical evolution, from spontaneous regression to a high propensity for widespread metastatic dissemination. Although the application of a well-defined pre-treatment risk classification plays a central role in the improvement of survival during the last years, more efforts must be done to improve patient’s survival in general and specifically in the subgroup of high risk patients. The morphological study of the tumoral tissue is contributing to such improvement. The histological category or the percentage of tumoral stroma, as well as the degree of differentiation of neuroblastic cells, evaluated by the pathologist with light microscopy, are factors that play a role in the diagnosis and prognosis of the patients. Given the role of tumoral extracellular matrix in biotensegrity and mechanotransduction, its architecture and the topology of its elements, as well as their interaction are being increasingly considered. Its quantification and characterization with microscopic image techniques start to be used. We hypothesize that the destiny of a neuroblastic tumor cell is complex and, is in part directed by characteristics of a set of non-cellular extracellular matrix structural elements. Additionally, we think that the application of the patterns derived from the morphometric analysis of such elements and their association with the impact of the known prognostic factors, patient’s survival will be improved. We aim to develop morphometric techniques to characterize different extracellular matrix scaffolding and vascular elements to find out potential uses as new prognostic markers for a better pre-treatment stratification of the patients or as therapeutic targets to be able to remodel the aberrant elements of the tissue scaffolding, including microvascularization. We constructed 19 tissue microarrays including more than 500 neuroblastomas which were stained with alcian blue pH 2.5, Gomori, Masson’s trichrome, orcein and anti-CD31 for glycosaminoglycans, reticulin fibers, collagen type I fibers, elastic fibers and blood vessels, respectively. The slides were digitized with a whole-slide scanner and different image-analysis algorithms were designed or customized to specifically detect and characterize the amount, the size and the shape of the different extracellular matrix elements studied. These parameters were related to different neuroblastoma subgroups, taking into account several clinical, histopathological and genetic features. The results obtained showed that reticulin fibers were the main components of the fibrous scaffolding and that microvasculature amount and architecture were relevant in the prognosis of neuroblastoma patients. A stiff and poorly porous extracellular matrix with irregularly-shaped vascular lumens was mainly detected in tumors belonging to patients with unfavorable prognosis. A subgroup of the high risk cohort with very poor survival could be defined by morphometric variables of reticulin fibers and blood vessels. Specificallly, those samples with high stained areas occupied by reticulin fibers forming large, crosslinking, branching and disorganized networks and by blood vessels, as well as with irregularly-shaped capillaries and sinusoid-like vessels and dilated venules, presented a very unfavorable survival. In this cohort, cells with MYCN gene amplification led to detectable topological changes regarding reticulin fibers and bood vessels. We can conclude that it is possible and convenient to quantify the fundamental substance and characterize the architecture of the fibrous scaffolding and the vascular system of neuroblastic tumors by means of the morphometric analysis of microscopic images. Some of the morphometric features related to the different extracellular matrix elements studied could be used as a diagnostic support for the ultra-high risk group of patients, after studying a larger cohort. The obtained results suggest the need of developing multidisciplinary efforts for an international integration of these studies, and that the morphometric information of the elements of the extracellular matrix, including the vascular system, could be used for a therapy based on mechanotransduction

Tumor Microenvironment Heterogeneity: A Review of the Biology Masterpiece, Evaluation Systems, and Therapeutic Implications

A tumor can be considered as a highly heterogeneous functional tissue, connected and dependent on the microenvironment, which sends and receives signals to and from the tumor tissue itself. Tumor cells alter the mechanical properties of the microenvironment in order to create favorable conditions for their proliferation. Stromal cells and non‐cellular elements of the extracellular matrix (ECM), including the host immune system, the fibrous scaffolding, the fundamental substance, and blood vascularization can determine tumoral cell morphologies, functions, aggressiveness, and response to treatment, as well as an accurate assessment of prognosis of the patients. Robust morphometric digital pathology techniques that are able to standardize measurements and analyse whole sets of immunohistochemical images are called for to identify, describe, and quantify the elements of the ECM. The computer‐automated segmentation algorithms are therefore required to increase the knowledge on the tumor microenvironment heterogeneity and to provide new therapeutic targets

TMA Vessel Segmentation Based on Color and Morphological Features: Application to Angiogenesis Research

Given that angiogenesis and lymphangiogenesis are strongly related to prognosis in neoplastic and other pathologies and that many methods exist that provide different results, we aim to construct a morphometric tool allowing us to measure different aspects of the shape and size of vascular vessels in a complete and accurate way. The developed tool presented is based on vessel closing which is an essential property to properly characterize the size and the shape of vascular and lymphatic vessels. The method is fast and accurate improving existing tools for angiogenesis analysis. The tool also improves the accuracy of vascular density measurements, since the set of endothelial cells forming a vessel is considered as a single object

Digital morphometric analysis of skin elements.

Objetivo: Proponer el uso de la infraestructura digital morfométrica para la cuantificación microscópica objetiva de elementos celulares y de la matriz extracelular en heridas y cicatrizaciones de la piel. Descripción de posibles usos, ventajas e inconvenientes. Presentación de una propuesta en diversos cortes histológicos del sistema tegumentario. Material y métodos: En el ejemplo empleado se utilizaron cortes de piel fina sana teñidos con técnicas histoquímicas e inmunohistoquímicas. Las muestras se digitalizaron con el escáner Panoramic MIDI (3D Histech Ltd.) y las imágenes obtenidas se analizaron con el software Panoramic Viewer v.1.15 (3D Histech Ltd) para cuantificar elementos celulares y con el software Image Pro-plus v.6.0 (Media Cybernetics) para cuantificar y caracterizar elementos fibrosos de la matriz extracelular. Resultados: Por un lado cuantificamos las células CD1a+ de la epidermis y la dermis detectando 164 elementos positivos por mm2 y 2.039 elementos negativos por mm2 ; representando el 7,46% de las células cutáneas y, por tanto, las células de Langerhans epidérmicas y macrófagos dérmicos. También valoramos fibras de la matriz extracelular en las capas papilar y reticular de la dermis. En relación con las fibras elásticas, se cuantificó un número mayor por unidad de área con mayor porcentaje de área teñida en la dermis profunda o capa reticular. Mientras que, para las fibras de colágeno, se detectó mayor cantidad por unidad de área con menor porcentaje de área teñida en la dermis superficial o capa papilar. Con respecto a las características de las fibras elásticas en las dos capas, distinguimos que formaban redes más ovoides, estaban dispuestas de forma más oblicua a la epidermis y eran más cortas, estrechas y rectilíneas en la dermis papilar. Para las fibras de colágeno detectamos que formaban haces más redondeados, estaban dispuestos de forma más perpendicular a la epidermis y eran más cortos, estrechos y ondulados en la dermis papilar. Conclusiones: La cuantificación objetiva de células y fibras de la matriz extracelular en imágenes microscópicas digitalizadas proporciona una herramienta rápida, fiable y discriminativa que puede ser útil para numerosos estudios cutáneos. Palabras Clave: Herida - Cicatrización - Estudio Histológico - Piel

Extracellular matrix, biotensegrity and tumor microenvironment. An update and overview

The extracellular matrix (ECM) constitutes a three-dimensional network that surrounds all cells, organs and tissues in the body. It forms a biophysical filter for protection, nutrition and cell innervation, as well as the medium for facilitating immune response, angiogenesis, fibrosis and tissue regeneration. It is the mechanism by which mechanical forces are transmitted to the basement membrane which, through the integrins, supports the tensegrity system and activates the epigenetic mechanisms of the cell. A review and update on current knowledge on this topic reveals how disturbance of the ECM leads to a loss of efficient filtering, nutrition, elimination, and cell denervation functions, in addition to loss of regeneration capacity and disorders in mechanotransduction. Furthermore, such disturbance results in a loss of substrate, and with it the ability to provide a proper immune response against tumor, toxic and infectious agents. Reciprocal communication between ECM stromal and parenchymatous cells directs gene expression. The oncogenic capacity of the stroma derives from the associated cells as well as from the tumor cells, the angiogenic microenvironment and from an alteration in tensegrity; all of which are dependent on the ECM. It has been shown that the malignant phenotype is reversible by correction of the altered cues of the ECM

Psychosocial and Diet-Related Lifestyle Clusters in Overweight and Obesity

This study explored intraindividual multidimensional profiles integrating psychosocial factors, namely, body image and satisfaction, weight-related self-stigma, positivity, and happiness, and behavioural-lifestyle factors, namely, adherence to a healthy diet, among Spanish adults with overweight or obesity. We further aimed to investigate the association of excess weight (i.e., measured body mass index, BMI) with the abovementioned multidimensional configurations. A convenience sample of 100 adult individuals (60% females) with excessive weight (69% overweight; 31% obesity) was recruited. They completed self-reports regarding the study variables, and their weight and height were measured. With a perspective centered on the individual, a cluster analysis was performed. Three distinct intraindividual psychosocial and diet-related profiles were identified: a group of healthy individuals with excess weight (46%); a group of individuals who were negatively affected by their excessive weight and showed the most distressed profile (18%); and a group of dysfunctional individuals who seemed to be excessively unrealistic and optimistic regarding their excessive weight and unhealthy lifestyles, but were troubled by their weight (36%). Furthermore, individuals in the affected cluster had higher obesity (mean BMI ± SD = 32.1 ± 3.7) than those in the clusters of healthy (28.0 ± 3.0) and dysfunctional individuals (28.1 ± 3.3) (p < 0.05). The results showed that there are specific psychosocial and lifestyle profiles in the adult population with excess weight and that there are relationships among psychological, behavioural, and body-composition factors. For clinical application purposes, it is important to account for the heterogeneity within individuals who are obese and to individualize the interventions, with a focus from weight change to the individual’s overall well-being.“Psicología de la Salud/Medicina Conductual” Research Group (CTS-267)“Psicología del Ejercicio, el Deporte y la Salud” Research Group (CTS-980) by the Junta de Andalucía (Spain)Research Project “Quality of life and body image in adults with obesity” (PIVA Projects, Ref. ICB2) by the Universidad Autónoma Ciudad Juárez (Mexico

1p36 deletion results in a decrease in glycosaminoglycans which is associated with aggressiveness in neuroblastic tumors

Despite our deep understanding of neuroblastic tumors, some patients still suffer treatment failure, so pre-treatment risk stratification still requires improvement and the search for new therapeutic targets must continue. Here we correlated prognostic clinical and biological features of neuroblastic tumors with the density of extracellular matrix glycosaminoglycans (the main components of the extracellular matrix ‘ground substance’), in nearly 400 primary samples. We also studied the relationship between the density of extracellular matrix glycosaminoglycans and the expression of B3GALT6, an enzyme required for their synthesis. We associated a decrease in glycosaminoglycans with neuroblastomas that were histopathologically poorly-differentiated or undifferentiated, as well as with metastatic disease, and 1p36 deleted tumors. This decrease in glycosaminoglycans was also related to abnormal nuclear B3GALT6 expression in neuroblastic cells. These findings point towards the importance of the ground substance in the aggressiveness of neuroblastic tumors, which should therefore be considered when developing novel therapies for treating neuroblastomas

Neuroblastoma after Childhood: Prognostic Relevance of Segmental Chromosome Aberrations, ATRX Protein Status, and Immune Cell Infiltration

Neuroblastoma (NB) is a common malignancy in children but rarely occurs during adolescence or adulthood. This subgroup is characterized by an indolent disease course, almost uniformly fatal, yet little is known about the biologic characteristics. The aim of this study was to identify differential features regarding DNA copy number alterations, α-thalassemia/mental retardation syndrome X-linked (ATRX) protein expression, and the presence of tumor-associated inflammatory cells. Thirty-one NB patients older than 10 years who were included in the Spanish NB Registry were considered for the current study; seven young and middle-aged adult patients (range 18-60 years) formed part of the cohort. We performed single nucleotide polymorphism arrays, immunohistochemistry for immune markers (CD4, CD8, CD20, CD11b, CD11c, and CD68), and ATRX protein expression. Assorted genetic profiles were found with a predominant presence of a segmental chromosome aberration (SCA) profile. Preadolescent and adolescent NB tumors showed a higher number of SCA, including 17q gain and 11q deletion. There was also a marked infiltration of immune cells, mainly high and heterogeneous, in young and middle-aged adult tumors. ATRX negative expression was present in the tumors. The characteristics of preadolescent, adolescent, young adult, and middle-aged adult NB tumors are different, not only from childhood NB tumors but also from each other. Similar examinations of a larger number of such tumor tissues from cooperative groups should lead to a better older age–dependent tumor pattern and to innovative, individual risk-adapted therapeutic approaches for these patients

Vascular patterns provide therapeutic targets in aggressive neuroblastic tumors.

Angiogenesis is essential for tumor growth and metastasis, nevertheless, in NB, results between different studies on angiogenesis have yielded contradictory results. An image analysis tool was developed to characterize the density, size and shape of total blood vessels and vascular segments in 458 primary neuroblastic tumors contained in tissue microarrays. The results were correlated with clinical and biological features of known prognostic value and with risk of progression to establish histological vascular patterns associated with different degrees of malignancy. Total blood vessels were larger, more abundant and more irregularly-shaped in tumors of patients with associated poor prognostic factors than in the favorable cohort. Tumor capillaries were less abundant and sinusoids more abundant in the patient cohort with unfavorable prognostic factors. Additionally, size of post-capillaries & metarterioles as well as higher sinusoid density can be included as predictive factors for survival. These patterns may therefore help to provide more accurate pre-treatment risk stratification, and could provide candidate targets for novel therapies