Simultaneous Mechanical and Topographical Characterization of Alginate Capsules

We used Atomic Force Microscopy (AFM) in order to characterize alginate microcapsules as for topography and stiffness simultaneously. Thus, we intend to identify those which are theoretically most biocompatible by this technique, reducing the need for animal testing

Mechanical and Topographical Characterization of Alginate Microcapsules

We used Atomic Force Microscopy (AFM) in order to characterize alginate microcapsules as for topography and stiffness. Thus, we intend to identify those which are theoretically most biocompatible by this technique, avoiding the need for animal testing

Development of microfluidic tools to reproduce and characterize the tumor microenvironment

A pesar de que la incidencia del cáncer está en aumento, sobre todo en los países desarrollados, el desarrollo de nuevos fármacos contra esta enfermedad es cada vez menos efectivo. Para revertir esta tendencia, aparece la necesidad de desarrollar mejores herramientas para reproducir y caracterizar el microentorno tumoral. Una de ellas son modelos in vitro más precisos.En este contexto, la microfluídica se presenta como una potente alternativa para el desarrollo de estos nuevos modelos in vitro más precisos, que puedan emplearse para un desarrollo y selección de fármacos más racional y efectivo. No obstante, se trata de un conjunto de técnicas poco extendido en los laboratorios de biología molecular. Así, en la presente tesis se desarrollan dos modelos microfluídicos del microentorno tumoral para tumores sólidos, junto a las herramientas necesarias para su caracterización, todo ello de fácil uso para tratar de generalizar la aplicación de los mismos.En el capítulo 1 se realiza una revisión del estado de la cuestión en lo referente a modelos de cáncer in vitro y su caracterización. En el capítulo 2 se desarrolla un modelo microfluídico de co-cultivo que permite estudiar las interacciones endotelio-tumor, así como la capacidad de penetración y erradicación de células tumorales de nuevos fármacos. En el capítulo 3 se presenta una herramienta para caracterizar los niveles de oxígeno molecular en cualquier punto de un cultivo in vitro 3D. En el capítulo 4 se presenta un modelo de tumor centrado en la generación y caracterización de gradientes biológicos, así como su adaptación a las técnicas tradicionales de biología molecular para el análisis del perfil genético del microentorno tumoral a lo largo del tiempo. Para generar los sistemas microfluídicos descritos anteriormente, se emplearon dispositivos fabricados mediante distintas técnicas y materiales. En los dispositivos se sembraron distintas poblaciones celulares, intentando así reproducir la estructura y organización de los tejidos biológicos. Mediante diferentes técnicas de microscopía (óptica, fluorescencia, confocal, imagen en tiempo real) y sondas fluorescentes se monitorizó la evolución y comportamiento celular. La caracterización del hidrogel sensible al oxígeno se realizó a través de las técnicas ya citadas, así como espectrofotometría, microscopía de fuerza atómica y electrónica de barrido en condiciones ambientales. Finalmente, la extracción de las células de los hidrogeles se realizó por medio de degradaciones enzimáticas, y la cuantificación de la expresión génica mediante extracción de RNA, retrotranscripción y reacción en cadena de la polimerasa cuantitativa.La conclusión general de la tesis, es que la utilización de modelos biomiméticos cambia dramáticamente el resultado de los ensayos realizados in vitro, por lo que su uso es necesario para obtener resultados relevantes y trasladables a la clínica. Asimismo, el desarrollo de sistemas biomiméticos in vitro del microentorno tumoral de uso generalizado es posible mediante el desarrollo de dispositivos de fácil uso, así como del establecimiento de métodos robustos de caracterización de los mismos, tanto in situ como “aguas abajo” del establecimiento de los modelos. Bibliografía: 1. Balkwill FR, Capasso M, Hagemann T (2012) The tumor microenvironment at a glance. J Cell Sci 125: 5591-5596.2. Junttila MR, de Sauvage FJ (2013) Influence of tumour micro-environment heterogeneity on therapeutic response. Nature 501: 346-354.3. Scannell JW, Blanckley A, Boldon H, Warrington B (2012) Diagnosing the decline in pharmaceutical R&D efficiency. Nat Rev Drug Discov 11: 191-200.4. Adriani G, Pavesi A, Tan AT, Bertoletti A, Thiery JP, et al. (2016) Microfluidic models for adoptive cell-mediated cancer immunotherapies. Drug Discov Today 21: 1472-1478.5. Ayuso JM, Virumbrales-Munoz M, Lacueva A, Lanuza PM, Checa-Chavarria E, et al. (2016) Development and characterization of a microfluidic model of the tumour microenvironment. Sci Rep 6: 36086.6. Ayuso JM, Monge R, Martínez-González A, Virumbrales-Muñoz M, Llamazares GA, et al. (2017) Glioblastoma on a microfluidic chip: Generating pseudopalisades and enhancing aggressiveness through blood vessel obstruction events. Neuro-Oncology: now230.<br /

Force spectroscopy-based simultaneous topographical and mechanical characterization to study polymer-to-polymer interactions in coated alginate microspheres

Cell-laden hydrogel microspheres have shown encouraging outcomes in the fields of drug delivery, tissue engineering or regenerative medicine. Beyond the classical single coating with polycations, many other different coating designs have been reported with the aim of improving mechanical properties and in vivo performance of the microspheres. Among the most common strategies are the inclusion of additional polycation coatings and the covalent bonding of the semi-permeable membranes with biocompatible crosslinkers such as genipin. However, it remains challenging to characterize the effects of the interactions between the polycations and the hydrogel microspheres over time in vitro. Here we use a force spectroscopy-based simultaneous topographical and mechanical characterization to study polymer-to-polymer interactions in alginate microspheres with different coating designs, maintaining the hydrogels in liquid. In addition to classical topography parameters, we explored, for the first time, the evolution of peak/valley features along the z axis via thresholding analysis and the cross-correlation between topography and stiffness profiles with resolution down to tens of nanometers. Thus, we demonstrated the importance of genipin crosslinking to avoid membrane detachment in alginate microspheres with double polycation coatings. Overall, this methodology could improve hydrogel design rationale and expedite in vitro characterization, therefore facilitating clinical translation of hydrogel-based technologies

Modulation of Antioxidant Potential with Coenzyme Q10 Suppressed Invasion of Temozolomide-Resistant Rat Glioma In Vitro and In Vivo.

The main reasons for the inefficiency of standard glioblastoma (GBM) therapy are the occurrence of chemoresistance and the invasion of GBM cells into surrounding brain tissues. New therapeutic approaches obstructing these processes may provide substantial survival improvements. The purpose of this study was to assess the potential of lipophilic antioxidant coenzyme Q10 (CoQ10) as a scavenger of reactive oxygen species (ROS) to increase sensitivity to temozolomide (TMZ) and suppress glioma cell invasion. To that end, we used a previously established TMZ-resistant RC6 rat glioma cell line, characterized by increased production of ROS, altered antioxidative capacity, and high invasion potential. CoQ10 in combination with TMZ exerted a synergistic antiproliferative effect. These results were confirmed in a 3D model of microfluidic devices showing that the CoQ10 and TMZ combination is more cytotoxic to RC6 cells than TMZ monotherapy. In addition, cotreatment with TMZ increased expression of mitochondrial antioxidant enzymes in RC6 cells. The anti-invasive potential of the combined treatment was shown by gelatin degradation, Matrigel invasion, and 3D spheroid invasion assays as well as in animal models. Inhibition of MMP9 gene expression as well as decreased N-cadherin and vimentin protein expression implied that CoQ10 can suppress invasiveness and the epithelial to mesenchymal transition in RC6 cells. Therefore, our data provide evidences in favor of CoQ10 supplementation to standard GBM treatment due to its potential to inhibit GBM invasion through modulation of the antioxidant capacity

Development of a Microfluidic Array to Study Drug Response in Breast Cancer

Luminal geometries are common structures in biology, which are challenging to mimic using conventional in vitro techniques based on the use of Petri dishes. In this context, microfluidic systems can mimic the lumen geometry, enabling a large variety of studies. However, most microfluidic models still rely on polydimethylsiloxane (PDMS), a material that is not amenable for high-throughput fabrication and presents some limitations compared with other materials such as polystyrene. Thus, we have developed a microfluidic device array to generate multiple bio-relevant luminal structures utilizing polystyrene and micro-milling. This platform offers a scalable alternative to conventional microfluidic devices designed in PDMS. Additionally, the use of polystyrene has well described advantages, such as lower permeability to hydrophobic molecules compared with PDMS, while maintaining excellent viability and optical properties. Breast cancer cells cultured in the devices exhibited high cell viability similar to PDMS-based microdevices. Further, co-culture experiments with different breast cell types showed the potential of the model to study breast cancer invasion. Finally, we demonstrated the potential of the microfluidic array for drug screening, testing chemotherapy drugs and photodynamic therapy agents for breast cancer

Microfluidic Tumor-on-a-Chip Model to Study Tumor Metabolic Vulnerability

Tumor-specific metabolic adaptations offer an interesting therapeutic opportunity to selectively destroy cancer cells. However, solid tumors also present gradients of nutrients and waste products across the tumor mass, forcing tumor cells to adapt their metabolism depending on nutrient availability in the surrounding microenvironment. Thus, solid tumors display a heterogenous metabolic phenotype across the tumor mass, which complicates the design of effective therapies that target all the tumor populations present. In this work, we used a microfluidic device to study tumor metabolic vulnerability to several metabolic inhibitors. The microdevice included a central chamber to culture tumor cells in a three-dimensional (3D) matrix, and a lumen in one of the chamber flanks. This design created an asymmetric nutrient distribution across the central chamber, generating gradients of cell viability. The results revealed that tumor cells located in a nutrient-enriched environment showed low to no sensitivity to metabolic inhibitors targeting glycolysis, fatty acid oxidation, or oxidative phosphorylation. Conversely, when cell density inside of the model was increased, compromising nutrient supply, the addition of these metabolic inhibitors disrupted cellular redox balance and led to tumor cell death

Patient-specific organotypic blood vessels as an in vitro model for anti-angiogenic drug response testing in renal cell carcinomaResearch in context

Background: Anti-angiogenic treatment failure is often attributed to drug resistance, unsuccessful drug delivery, and tumor heterogeneity. Recent studies have speculated that anti-angiogenic treatments may fail due to characteristics inherent to tumor-associated blood vessels. Tumor-associated blood vessels are phenotypically different from their normal counterparts, having defective or permeable endothelial monolayers, abnormal sprouts, and abnormal vessel hierarchy. Therefore, to predict the efficacy of anti-angiogenic therapies in an individual patient, in vitro models that mirror individual patient's tumor vascular biology and response to anti-angiogenic treatment are needed. Methods: We used a microfluidic in vitro organotypic model to create patient-specific biomimetic blood vessels from primary patient-specific tumor endothelial cells (TEnCs) and normal endothelial cells (NEnC). We assessed number of sprouts and vessel organization via microscopy imaging and image analysis. We characterized NEnC and TEnC vessel secretions via multiplex bead-based ELISA. Findings: Using this model, we found that TEnC vessels exhibited more angiogenic sprouts than NEnC vessels. We also found a more disorganized and gap-filled endothelial monolayer. NEnCs and TEnC vessels exhibited heterogeneous functional drug responses across the five patients screened, as described in the clinic. Interpretation: Our model recapitulated hallmarks of TEnCs and NEnCs found in vivo and captured the functional and structural differences between TEnC and NEnC vessels. This model enables a platform for therapeutic drug screening and assessing patient-specific responses with great potential to inform personalized medicine approaches. Funding: NIH grants R01 EB010039, R33 CA225281, R01CA186134 University of Wisconsin Carbone Cancer Center (CA014520), and University of Wisconsin Hematology training grant T32 HL07899. Keywords: Organotypic, Lumen, Model, Anti-angiogenic, Renal, Carcinom

Microphysiological systems for solid tumor immunotherapy: opportunities and challenges

Immunotherapy remains more effective for hematologic tumors than for solid tumors. One of the main challenges to immunotherapy of solid tumors is the immunosuppressive microenvironment these tumors generate, which limits the cytotoxic capabilities of immune effector cells (e.g., cytotoxic T and natural killer cells). This microenvironment is characterized by hypoxia, nutrient starvation, accumulated waste products, and acidic pH. Tumor-hijacked cells, such as fibroblasts, macrophages, and T regulatory cells, also contribute to this inhospitable microenvironment for immune cells by secreting immunosuppressive cytokines that suppress the antitumor immune response and lead to immune evasion. Thus, there is a strong interest in developing new drugs and cell formulations that modulate the tumor microenvironment and reduce tumor cell immune evasion. Microphysiological systems (MPSs) are versatile tools that may accelerate the development and evaluation of these therapies, although specific examples showcasing the potential of MPSs remain rare. Advances in microtechnologies have led to the development of sophisticated microfluidic devices used to recapitulate tumor complexity. The resulting models, also known as microphysiological systems (MPSs), are versatile tools with which to decipher the molecular mechanisms driving immune cell antitumor cytotoxicity, immune cell exhaustion, and immune cell exclusion and to evaluate new targeted immunotherapies. Here, we review existing microphysiological platforms to study immuno-oncological applications and discuss challenges and opportunities in the field

Alkynyl gold(I) complex triggers necroptosis via ROS generation in colorectal carcinoma cells

Given the rise of apoptosis-resistant tumors, there exist a growing interest in developing new drugs capable of inducing different types of cell death to reduce colorectal cancer-related death rates. As apoptosis and necroptosis do not share cellular machinery, necroptosis induction may have a great therapeutic potential on those apoptosis-resistant cancers, despite the inflammatory effects associated with it. We have synthesized an alkynyl gold(I) complex [Au(CC-2-NC5H4)(PTA)] whose anticancer effect was tested on the colorectal adenocarcinoma Caco-2 cell line. With regard to its mechanism of action, this gold complex enters the mitochondria and disrupts its normal function, leading to an increase in ROS production, which triggers necroptosis. Necroptosis induction has been found dependent of TNF-α (Tumor necrosisfactor α) and TNFR1(Tumor necrosisfactor receptor 1) binding, RIP1(Receptor-Interacting Protein 1) activation and NF-κB (Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells) signaling. Moreover, the antitumor potential of [Au(CC-2-NC5H4)(PTA)] has also been confirmed on the 3D cancer model spheroid. Overall, the obtained data show firstly that gold complexes might have the ability of inducing necroptosis, and secondarily that our compound [Au(CC-2-NC5H4)(PTA)] is an interesting alternative to current chemotherapy drugs in cases of apoptosis resistance.We thank The Ministerio de Economia y Competitividad (CTQ2016-75816-C2-1-P and SAF2016-75441-R) and DGA (A-32 and E104) for financial support. MVM acknowledges the Ministerio de Educación, Cultura y Deportes (FPU 12/05640) of the Spanish Government for financial support.Peer reviewe