uPARAP/Endo180: a multifaceted protein of mesenchymal cells.

peer reviewedThe urokinase plasminogen activator receptor-associated protein (uPARAP/Endo180) is already known to be a key collagen receptor involved in collagen internalization and degradation in mesenchymal cells and some macrophages. It is one of the four members of the mannose receptor family along with a macrophage mannose receptor (MMR), a phospholipase lipase receptor (PLA2R), and a dendritic receptor (DEC-205). As a clathrin-dependent endocytic receptor for collagen or large collagen fragments as well as through its association with urokinase (uPA) and its receptor (uPAR), uPARAP/Endo180 takes part in extracellular matrix (ECM) remodeling, cell chemotaxis and migration under physiological (tissue homeostasis and repair) and pathological (fibrosis, cancer) conditions. Recent advances that have shown an expanded contribution of this multifunctional protein across a broader range of biological processes, including vascular biology and innate immunity, are summarized in this paper. It has previously been demonstrated that uPARAP/Endo180 assists in lymphangiogenesis through its capacity to regulate the heterodimerization of vascular endothelial growth factor receptors (VEGFR-2 and VEGFR-3). Moreover, recent findings have demonstrated that it is also involved in the clearance of collectins and the regulation of the immune system, something which is currently being studied as a biomarker and a therapeutic target in a number of cancers

To form and function: on the role of basement membrane mechanics in tissue development, homeostasis and disease.

The basement membrane (BM) is a special type of extracellular matrix that lines the basal side of epithelial and endothelial tissues. Functionally, the BM is important for providing physical and biochemical cues to the overlying cells, sculpting the tissue into its correct size and shape. In this review, we focus on recent studies that have unveiled the complex mechanical properties of the BM. We discuss how these properties can change during development, homeostasis and disease via different molecular mechanisms, and the subsequent impact on tissue form and function in a variety of organisms. We also explore how better characterization of BM mechanics can contribute to disease diagnosis and treatment, as well as development of better in silico and in vitro models that not only impact the fields of tissue engineering and regenerative medicine, but can also reduce the use of animals in research

An In Vitro 3D Model to Evaluate Behaviour of Breast Cancer Cells and Response to Treatment

The field of 3D culture models of disease has started to move towards systems that aim to recapitulate the complexity of human tissues. However, despite recent improvements, current 3D systems remain overly simplistic, lacking the biophysical characteristics and diverse structures found in most organs. In this project, the cellular behaviour of breast cancer and their responsiveness to chemotherapeutic agents were evaluated under different 3D cell culture conditions. MDA-MB231 and SKBR3 cells were prepared as spheroids using ultra-low attachment plates and as 'artificial cancer masses' (ACM) by embedding cells in a dense collagen type-I. The ACMs were maintained under flow (150 μL/min) and flow/pressure (550 μL/min, ~19 mmHg) conditions. A significant reduction in cell viability was observed when cancer cells were grown as ACM compared to 2D culture. Cell viability also declined significantly when ACMs were maintained in flow/pressure condition compared to static condition. Similarly, an increase in the expression levels of markers of EMT was observed when cells were cultured as ACM. However, compared to static 3D incorporation of flow and pressure was associated with decreased expression levels of vimentin, HIF1-α, whilst MMP14 expression increased and snail remained unchanged. HER2 levels were increased in SKBR3 when the cells were cultured under flow/pressure (1.5 fold) compared to static condition. Overall, cells cultured as ACMs exhibited reduced responsiveness to doxorubicin compared to those grown in the conventional 2D culture. A decrease sensitivity was also observed in 3D/flow/pressure and 3D/flow compared to 3D/static condition. The results obtained in this study show that cancer cell behaviour and their response to therapeutic agents are affected by different microenvironments. Therefore, a new generation of 3D in vitro models need to be developed as pre-clinical drug testing platforms

Nicastrin and the gamma‐secretase complex in breast cancer

γ‐secretase is a membrane‐bound proteolytic complex, formed by nicastrin, APH1, presenilin and pen‐2, which cleaves over sixty known substrates, including Notch and Ecadherin, thus regulating cellular processes such as proliferation and adhesion. High levels of nicastrin have been demonstrated in 47.3 % (n=1050) high tumour grade breast tissues, whereas it is absent in 100% normal human breast (n=40). Although the mechanism for nicastrin up‐regulation in breast cancer is unknown, preliminary data suggests posttranscriptional regulation. Therefore, γ‐secretase is an important therapeutic target in breast cancer. GSI1, a commercial γ‐secretase inhibitor is cytotoxic exclusively for breast cancer cell lines, whereas non‐tumourigenic breast cells are not affected. GSI1 triggers G2/M arrest, culminating in apoptosis through down‐regulation of XIAP, Bcl‐2, Bax and Bcl‐XL in breast cancer cells. In addition, similar cytotoxicity has been found in a panel of cell lines derived from several types of cancer. We discuss whether the cytotoxic effect of GSI1 in breast cancer cell lines is mediated through inhibition of γ‐secretase or the proteasome. RNA interference of individual γ‐secretase components indicates that NCSTN knock‐down elicits 55% cell growth reduction, whereas knock‐down of the other complex components does not inhibit cell growth to the same extent. In addition, NCSTN siRNA reduces invasion in breast cancer cells. Disseminated and circulating tumour cells (CTCs) are clinically used as indicators of metastasis. Nicastrin has been found to be expressed by a rare population of cells in bone marrow aspirates and in CTCs from breast cancer patients with high risk of relapse. Thus, from the pharmacological point of view, nicastrin represents a novel therapeutic target. In addition, it is a novel biomarker for breast cancer with potential diagnostic applications

Transmisión de señales mecánicas en células indiferenciadas neuroblásticas. Estudios biológicos y preclínicos en modelos in vitro 3D

La biotensegridad es un principio físico de autoequilibrio en el que las fuerzas de tensión y compresión sostienen las estructuras biológicas y permiten la mecanotransducción de señales durante las interacciones célula-célula y célula-entorno. De esta manera, las células y los tejidos responden a las condiciones de su entorno y se adaptan a él. En este sentido, el cáncer muestra sus propios sistemas biotensegrales aberrantes que impulsan la carcinogénesis y la evolución de la enfermedad. La biotensegridad tumoral se define por la intercomunicación entre las poblaciones de células tumorales, los elementos de la matriz extracelular y las estructuras y moléculas del microambiente tumoral. En los últimos años, la inclusión de la patología digital y la inteligencia artificial en la medicina han permitido el estudio de las interacciones biotensegrales en la oncología traslacional. Tradicionalmente, la complejidad de las muestras humanas ha dirigido la investigación del cáncer hacia el uso de modelos experimentales, generalmente basados en modelos murinos in vivo o cultivos de células en monocapa in vitro. Actualmente, la ingeniería de tejidos permite desarrollar nuevos modelos experimentales, basados en plataformas tridimensionales ajustables in vitro, que posibilitan reproducir la biotensegridad tumoral de forma sencilla, controlable y precisa. Siguiendo la experiencia de nuestro laboratorio en el estudio de la matriz extracelular del neuroblastoma, el tumor sólido extracraneal más común en la infancia, esta tesis inicia una línea de investigación mediante la aplicación de análisis de imágenes digitales en modelos tridimensionales in vitro de neuroblastoma para evaluar el efecto biotensegral del microambiente tumoral sobre la agresividad del neuroblastoma. Este trabajo se presenta como un compendio de las siguientes tres publicaciones científicas y otros datos complementarios, donde se desarrollan cultivos celulares y cocultivos relevantes para neuroblastoma en hidrogeles: I. A three-dimensional bioprinted model to evaluate the effect of stiffness on neuroblastoma cell cluster dynamics and behavior. Monferrer E*, Martín-Vañó S*, Carretero A*, García-Lizarribar A, Burgos-Panadero R, Navarro S, Samitier J, Noguera R. Sci Rep. 2020 Apr 14;10(1):6370. II. Digital Image Analysis Applied to Tumor Cell Proliferation, Aggressiveness, and Migration-Related Protein Synthesis in Neuroblastoma 3D Models. Monferrer E*, Sanegre S*, Martín-Vañó S, García-Lizarribar A, Burgos-Panadero R, López-Carrasco A, Navarro S, Samitier J, Noguera R. Int J Mol Sci. 2020 Nov 17;21(22):8676. III. Vitronectin-based hydrogels recapitulate neuroblastoma growth conditions. Monferrer E*, Dobre O*, Trujillo S, González Oliva MA, Trubert-Paneli A, Acevedo-León D, Noguera R, Salmerón-Sánchez M. Front. Cell Dev. Biol. 2022 Oct 11;10:988699. En ellos, destacamos la necesidad de las condiciones 3D para el cultivo celular y el papel de la rigidez de la matriz extracelular para recapitular las señales mecánicas que desencadenan la respuesta de adaptación celular a lo largo del tiempo, como los cambios en la proliferación y la actividad del metabolismo del ARNm. Al evaluar la síntesis de proteínas relacionadas con la migración, evidenciamos un comportamiento distintivo de líneas celulares de neuroblastoma agresivas según sus características genéticas, así como de la presencia de células estromales en los modelos. Finalmente, reproducimos parte de la matriz extracelular del neuroblastoma de alto riesgo mediante la incorporación de vitronectina en los hidrogeles y buscamos caracterizar el impacto de este microambiente específico en las células del neuroblastoma. Concluimos que los cambios de comportamiento del tumor impulsados por la mecanotransducción de señales biotensegrales evolucionan de manera diferente dependiendo de las características del sistema, haciéndolos extremadamente complejos y difíciles de predecir in vivo. En consecuencia, es necesario desarrollar modelos tumorales básicos que recreen aspectos biotensegrales antes de generar plataformas completamente fisiopatológicas, ya que únicamente los primeros proporcionarán un conocimiento preciso de los mecanismos específicos de biotensegridad que regulan el comportamiento tumoral que podrían ser objeto terapéutico en estudios preclínicos realizados en estas plataformas.Biotensegrity is a physical self-balancing principle in which tension and compression forces sustain biological structures and allow signal mechanotransduction during cell-cell and cell-environment interactions. In this way, cells and tissues respond to their environment conditions, and subsequently adapt to it. In this sense, cancer diseases display their own aberrant biotensegral systems that drive carcinogenesis and disease evolution. Tumor biotensegrity is defined by the intercommunication between tumor cell populations, extracellular matrix elements, and tumor microenvironment structures and molecules. In recent years, digital pathology and artificial intelligence inclusion in medicine have allowed the study of biotensegral interactions in translational oncology. However, the complexity of human samples has shifted cancer research toward the use of experimental models, typically based on in vivo murine models or in vitro monolayer cell cultures. Alternatively, tissue engineering has allowed developing new experimental models, based on in vitro three-dimensional adjustable platforms, which allow reproducing tumor biotensegrity in a simple, controllable, and precise way. Following the expertise of our lab in the study of the extracellular matrix of neuroblastoma, the most common extracranial solid tumor in childhood, this thesis initiates a research line by applying digital image analysis in neuroblastoma in vitro three-dimensional models to evaluate the biotensegral effect of tumor microenvironment on neuroblastoma aggressiveness. Thus, this work is presented as a compendium of the following three scientific publications and complementary data, where cell cultures and cocultures relevant to neuroblastoma are developed in hydrogels: I. A three-dimensional bioprinted model to evaluate the effect of stiffness on neuroblastoma cell cluster dynamics and behavior. Monferrer E*, Martín-Vañó S*, Carretero A*, García-Lizarribar A, Burgos-Panadero R, Navarro S, Samitier J, Noguera R. Sci Rep. 2020 Apr 14;10(1):6370. II. Digital Image Analysis Applied to Tumor Cell Proliferation, Aggressiveness, and Migration-Related Protein Synthesis in Neuroblastoma 3D Models. Monferrer E*, Sanegre S*, Martín-Vañó S, García-Lizarribar A, Burgos-Panadero R, López-Carrasco A, Navarro S, Samitier J, Noguera R. Int J Mol Sci. 2020 Nov 17;21(22):8676. III. Vitronectin-based hydrogels recapitulate neuroblastoma growth conditions. Monferrer E*, Dobre O*, Trujillo S, González Oliva MA, Trubert-Paneli A, Acevedo-León D, Noguera R, Salmerón-Sánchez M. Front. Cell Dev. Biol. 2022 Oct 11;10:988699. We highlight the need of 3D conditions for cell culture and the role of the extracellular matrix stiffness when recapitulating the mechanical cues that trigger cell adaptation response over time, such as changes in proliferation and mRNA metabolism activity. Evaluating migration-related protein synthesis, we evince distinctive behavior of aggressive neuroblastoma cell lines depending on their genetic characteristics, as well as on the presence of stromal cells. Finally, we reproduce part of the high-risk neuroblastoma extracellular matrix by incorporating full-length vitronectin within the hydrogels and seek to characterize the impact of this specific microenvironment on neuroblastoma cells. We conclude that tumor behavior changes driven by biotensegrity mechanotransduction evolve differently regarding the system characteristics, making them extremely complex and difficult to predict in vivo. Accordingly, basic cancer models recreating biotensegral aspects need to be developed before generating fully pathophysiological platforms, since the former will provide accurate knowledge of specific biotensegrity mechanisms regulating tumor behavior that could be therapeutically targeted in preclinical studies performed on these platforms

Extracellular Matrix in Development and Disease

The extracellular matrix in development and disease deals with the molecular and cellular aspects of development and disease. Cells exist in three-dimensional scaffolding called the extracellular matrix. The matrix holds together the millions of cells that make up our blood vessels, organs, skin, and all tissues of the body. The matrix serves as a reservoir of signaling molecules as well. In bacterial cultures, biofilms form as an extracellular matrix and play essential roles in disease and drug resistance. Topics such as matrix structure and function, cell attachment and cell surface proteins mediating cell-matrix interactions, synthesis, regulation, composition, structure, assembly, remodeling, and function of the matrix are included. A common thread uniting the topics is the essential nature that the matrix plays in normal development and pathophysiology. Providing new knowledge will lead us to improved diagnostics, the preventions of disease progression, and therapeutic strategies for the repair and regeneration of tissues. Topics such as the extracellular matrix in hereditary diseases, reproduction, cancer, muscle, and tissue engineering applications, and diverse roles for integrins, are included in this collection

Current Frontiers and Perspectives in Cell Biology

A numerous internationally renowned authors in the pages of this book present the views of the fields of cell biology and their own research results or review of current knowledge. Chapters are divided into five sections that are dedicated to cell structures and functions, genetic material, regulatory mechanisms, cellular biomedicine and new methods in cell biology. Multidisciplinary and often quite versatile approach by many authors have imposed restrictions of this classification, so it is certain that many chapters could belong to the other sections of this book. The current frontiers, on the manner in which they described in the book, can be a good inspiration to many readers for further improving, and perspectives which are highlighted can be seen in many areas of fundamental biology, biomedicine, biotechnology and other applications of knowledge of cell biology. The book will be very useful for beginners to gain insight into new area, as well as experts to find new facts and expanding horizons