Virus-Host Coevolution as a Tool for Controlling Bacterial Resistance to Phage Therapy

Bacterial resistance to antibiotics is a global public health concern. New treatments are needed to combat resistant strains, among which phage therapy is a promising option. Probably the main advantages of phage therapy are its high specificity as well as rapid viral adaptability, which in principle allows using phage evolution to overcome resistance. Here, we have performed serial coevolution passages between Escherichia coli and its phage T7 to investigate the ability of coevolved phages to reduce the emergence of resistances. We find that the initial bacterial population is less likely to undergo resistance when challenged with experimentally coevolved phages than when challenged with the wild-type phage. Hence, our findings suggest that coevolved phage preparations could be used to increase the efficacy of phage therapy

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

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

Vitronectin-based hydrogels recapitulate neuroblastoma growth conditions

The tumor microenvironment plays an important role in cancer development and the use of 3D in vitro systems that decouple different elements of this microenvironment is critical for the study of cancer progression. In neuroblastoma (NB), vitronectin (VN), an extracellular matrix protein, has been linked to poor prognosis and appears as a promising therapeutic target. Here, we developed hydrogels that incorporate VN into 3D polyethylene glycol (PEG) hydrogel networks to recapitulate the native NB microenvironment. The stiffness of the VN/PEG hydrogels was modulated to be comparable to the in vivo values reported for NB tissue samples. We used SK-N-BE (2) NB cells to demonstrate that PEGylated VN promotes cell adhesion as the native protein does. Furthermore, the PEGylation of VN allows its crosslinking into the hydrogel network, providing VN retention within the hydrogels that support viable cells in 3D. Confocal imaging and ELISA assays indicate that cells secrete VN also in the hydrogels and continue to reorganize their 3D environment. Overall, the 3D VN-based PEG hydrogels recapitulate the complexity of the native tumor extracellular matrix, showing that VN-cell interaction plays a key role in NB aggressiveness, and that VN could potentially be targeted in preclinical drug studies performed on the presented hydrogels

Vitronectin-based hydrogels recapitulate neuroblastoma growth conditions

The tumor microenvironment plays an important role in cancer development and the use of 3D in vitro systems that decouple different elements of this microenvironment is critical for the study of cancer progression. In neuroblastoma (NB), vitronectin (VN), an extracellular matrix protein, has been linked to poor prognosis and appears as a promising therapeutic target. Here, we developed hydrogels that incorporate VN into 3D polyethylene glycol (PEG) hydrogel networks to recapitulate the native NB microenvironment. The stiffness of the VN/PEG hydrogels was modulated to be comparable to the in vivo values reported for NB tissue samples. We used SK-N-BE (2) NB cells to demonstrate that PEGylated VN promotes cell adhesion as the native protein does. Furthermore, the PEGylation of VN allows its crosslinking into the hydrogel network, providing VN retention within the hydrogels that support viable cells in 3D. Confocal imaging and ELISA assays indicate that cells secrete VN also in the hydrogels and continue to reorganize their 3D environment. Overall, the 3D VN-based PEG hydrogels recapitulate the complexity of the native tumor extracellular matrix, showing that VN-cell interaction plays a key role in NB aggressiveness, and that VN could potentially be targeted in preclinical drug studies performed on the presented hydrogels

Integrated CGH/WES Analyses Advance Understanding of Aggressive Neuroblastoma Evolution: A Case Study

Neuroblastoma (NB) is the most common extra-cranial malignancy in preschool children. To portray the genetic landscape of an overly aggressive NB leading to a rapid clinical progression of the disease, tumor DNA collected pre- and post-treatment has been analyzed. Array comparative genomic hybridization (aCGH), whole-exome sequencing (WES), and pharmacogenetics approaches, respectively, have identified relevant copy number alterations (CNAs), single nucleotide variants (SNVs), and polymorphisms (SNPs) that were then combined into an integrated analysis. Spontaneously formed 3D tumoroids obtained from the recurrent mass have also been characterized. The results prove the power of combining CNAs, SNVs, and SNPs analyses to assess clonal evolution during the disease progression by evidencing multiple clones at disease onset and dynamic genomic alterations during therapy administration. The proposed molecular and cytogenetic integrated analysis empowers the disease follow-up and the prediction of tumor recurrence

Metabolic Classification and Intervention Opportunities for Tumor Energy Dysfunction

A comprehensive view of cell metabolism provides a new vision of cancer, conceptualized as tissue with cellular-altered metabolism and energetic dysfunction, which can shed light on pathophysiological mechanisms. Cancer is now considered a heterogeneous ecosystem, formed by tumor cells and the microenvironment, which is molecularly, phenotypically, and metabolically reprogrammable. A wealth of evidence confirms metabolic reprogramming activity as the minimum common denominator of cancer, grouping together a wide variety of aberrations that can affect any of the different metabolic pathways involved in cell physiology. This forms the basis for a new proposed classification of cancer according to the altered metabolic pathway(s) and degree of energy dysfunction. Enhanced understanding of the metabolic reprogramming pathways of fatty acids, amino acids, carbohydrates, hypoxia, and acidosis can bring about new therapeutic intervention possibilities from a metabolic perspective of cancer

Metabolic Classification and Intervention Opportunities for Tumor Energy Dysfunction.

A comprehensive view of cell metabolism provides a new vision of cancer, conceptualized as tissue with cellular-altered metabolism and energetic dysfunction, which can shed light on pathophysiological mechanisms. Cancer is now considered a heterogeneous ecosystem, formed by tumor cells and the microenvironment, which is molecularly, phenotypically, and metabolically reprogrammable. A wealth of evidence confirms metabolic reprogramming activity as the minimum common denominator of cancer, grouping together a wide variety of aberrations that can affect any of the different metabolic pathways involved in cell physiology. This forms the basis for a new proposed classification of cancer according to the altered metabolic pathway(s) and degree of energy dysfunction. Enhanced understanding of the metabolic reprogramming pathways of fatty acids, amino acids, carbohydrates, hypoxia, and acidosis can bring about new therapeutic intervention possibilities from a metabolic perspective of cancer

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

Immunometabolism Modulation in Therapy

The study of cancer biology should be based around a comprehensive vision of the entire tumor ecosystem, considering the functional, bioenergetic and metabolic state of tumor cells and those of their microenvironment, and placing particular importance on immune system cells. Enhanced understanding of the molecular bases that give rise to alterations of pathways related to tumor development can open up new therapeutic intervention opportunities, such as metabolic regulation applied to immunotherapy. This review outlines the role of various oncometabolites and immunometabolites, such as TCA intermediates, in shaping pro/anti-inflammatory activity of immune cells such as MDSCs, T lymphocytes, TAMs and DCs in cancer. We also discuss the extraordinary plasticity of the immune response and its implication in immunotherapy efficacy, and highlight different therapeutic intervention possibilities based on controlling the balanced systems of specific metabolites with antagonistic functions