3D Matrices Research Articles

Multiplexed Detection of Molecular Interactions with DNA Origami Engineered Cells in 3D Collagen Matrices.

The interactions of cells with signaling molecules present in their local microenvironment maintain cell proliferation, differentiation, and spatial organization and mediate progression of diseases such as metabolic disorders and cancer. Real-time monitoring of the interactions between cells and their extracellular ligands in a three-dimensional (3D) microenvironment can inform detection and understanding of cell processes and the development of effective therapeutic agents. DNA origami technology allows for the design and fabrication of biocompatible and 3D functional nanodevices via molecular self-assembly for various applications including molecular sensing. Here, we report a robust method to monitor live cell interactions with molecules in their surrounding environment in a 3D tissue model using a microfluidic device. We used a DNA origami cell sensing platform (CSP) to detect two specific nucleic acid sequences on the membrane of B cells and dendritic cells. We further demonstrated real-time detection of biomolecules with the DNA sensing platform on the surface of dendritic cells in a 3D microfluidic tissue model. Our results establish the integration of live cells with membranes engineered with DNA nanodevices into microfluidic chips as a highly capable biosensor approach to investigate subcellular interactions in physiologically relevant 3D environments under controlled biomolecular transport.

Axioms of Decision Criteria for 3D Matrix Games and Their Applications

In this paper, we define characteristic axioms for 3D matrix games and extend the definitions of the decision criteria under uncertainty to three dimensions in order to investigate the simultaneous effect of two different states on the decision process. We first redefine the Laplace, Wald, Hurwicz, and Savage criteria in 3D. We present a new definition depending on only the ∞-norm of the 3D payoff matrix for the Laplace criterion in 3D. Then, we demonstrate that the Laplace criterion in 3D explicitly satisfies all the proposed axioms, as well as the other three criteria. Moreover, we illustrate a fundamental example for a three-dimensional matrix with 3D figures and show the usage of each criterion in detail. In the second example, we model a decision process during the COVID-19 pandemic for South Korea to show the applicability of the 3D decision criteria using real data with two different states of nature for individuals’ actions for the quarantine. Additionally, we present an agricultural insurance problem and analyze the effects of the hailstorm and different speeds of wind on the harvest by the 3D criteria. To the best of our knowledge, this is the first study that brings 3D matrices in decision and game theories together.

Collagen-Based Biomimetic Systems to Study the Biophysical Tumour Microenvironment.

The extracellular matrix (ECM) is a pericellular network of proteins and other molecules that provides mechanical support to organs and tissues. ECM biophysical properties such as topography, elasticity and porosity strongly influence cell proliferation, differentiation and migration. The cell's perception of the biophysical microenvironment (mechanosensing) leads to altered gene expression or contractility status (mechanotransduction). Mechanosensing and mechanotransduction have profound implications in both tissue homeostasis and cancer. Many solid tumours are surrounded by a dense and aberrant ECM that disturbs normal cell functions and makes certain areas of the tumour inaccessible to therapeutic drugs. Understanding the cell-ECM interplay may therefore lead to novel and more effective therapies. Controllable and reproducible cell culturing systems mimicking the ECM enable detailed investigation of mechanosensing and mechanotransduction pathways. Here, we discuss ECM biomimetic systems. Mainly focusing on collagen, we compare and contrast structural and molecular complexity as well as biophysical properties of simple 2D substrates, 3D fibrillar collagen gels, cell-derived matrices and complex decellularized organs. Finally, we emphasize how the integration of advanced methodologies and computational methods with collagen-based biomimetics will improve the design of novel therapies aimed at targeting the biophysical and mechanical features of the tumour ECM to increase therapy efficacy.

Single-Walled Carbon Nanotubes as Fluorescent Probes for Monitoring the Self-Assembly and Morphology of Peptide/Polymer Hybrid Hydrogels.

Hydrogels formed via supramolecular self-assembly of fluorenylmethyloxycarbonyl (Fmoc)-conjugated amino acids provide excellent scaffolds for 3D cell culture, tissue engineering, and tissue recovery matrices. Such hydrogels are usually characterized by rheology or electron microscopy, which are invasive and cannot provide real-time information. Here, we incorporate near-infrared fluorescent single-walled carbon nanotubes (SWCNTs) into Fmoc-diphenylalanine hydrogels as fluorescent probes, reporting in real-time on the morphology and time-dependent structural changes of the self-assembled hydrogels in the transparency window of biological tissue. We further demonstrate that the gelation process and structural changes upon the addition of cross-linking ions are transduced into spectral modulations of the SWCNT-fluorescence. Moreover, morphological differences of the hydrogels induced by polymer additives are manifested in unique features in fluorescence images of the incorporated SWCNTs. SWCNTs can thus serve as optical probes for noninvasive, long-term monitoring of the self-assembly gelation process and the fate of the resulting peptide hydrogel during long-term usage.

Scaffold-mediated switching of lymphoma metabolism in culture

BackgroundDiffuse large B cell lymphoma (DLBCL) is an aggressive subtype of non-Hodgkin lymphoma (NHL) and accounts for about a third of all NHL cases. A significant proportion (~40%) of treated DLBCL patients develop refractory or relapsed disease due to drug resistance which can be attributed to metabolomic and genetic variations amongst diverse DLBCL subtypes. An assay platform that reproduces metabolic patterns of DLBCL in vivo could serve as a useful model for DLBCL.MethodsThis report investigated metabolic functions in 2D and 3D cell cultures using parental and drug-resistant DLBCL cell lines as compared to patient biopsy tissue.ResultsA 3D culture model controlled the proliferation of parental and drug-resistant DLBCL cell lines, SUDHL-10, SUDHL-10 RR (rituximab resistant), and SUDHL-10 OR (obinutuzumab resistant), as well as retained differential sensitivity to CHOP. The results from metabolic profiling and isotope tracer studies with d-glucose-13C6 indicated metabolic switching in 3D culture when compared with a 2D environment. Analysis of DLBCL patient tumor tissue revealed that the metabolic changes in 3D grown cells were shifted towards that of clinical specimens.Conclusion3D culture restrained DLBCL cell line growth and modulated metabolic pathways that trend towards the biological characteristics of patient tumors. Counter-intuitively, this research thereby contends that 3D matrices can be a tool to control tumor function towards a slower growing and metabolically dormant state that better reflects in vivo tumor physiology.

Fast spatial structure constrained 3D prestack inversion based on the Sylvester equation

Structurally constrained inversion (SCI) is a multitrace processing technology that can effectively reduce the complexity of prestack seismic inversion caused by complex geology such as faults and steeply dipping structures. Nevertheless, several factors limit its wide application in 3D prestack inversion, the most significant of which is the high computational cost. In addition, the existing SCI is mostly applied in 2D profiles, which can only consider structural features in one direction, making it challenging to ensure spatial continuity. We have developed a fast spatial structure constrained inversion for 3D prestack seismic data. First, based on the conventional SCI, we develop a new way to describe the spatial structural characteristics of the stratum by estimating the dip angles along two perpendicular directions. Second, we introduce the spatial structure constraints through the Hadamard product operator and construct a computationally well-behaved objective function with spatial structural constrained. Using our matrix operators, which are symbols representing the multiplication of 2D and 3D matrices, the minimization of the objective function can be simplified to solve a specific Sylvester equation. Moreover, we justify the rationality of our operator symbols and derive a fast algorithm for solving 3D problems with the Sylvester equation. Finally, the stability and computational efficiency of our method are compared with the 2D SCI algorithm on the 3D overthrust model and field prestack seismic data.

Random-forest model for drug–target interaction prediction via Kullback–Leibler divergence

Virtual screening has significantly improved the success rate of early stage drug discovery. Recent virtual screening methods have improved owing to advances in machine learning and chemical information. Among these advances, the creative extraction of drug features is important for predicting drug–target interaction (DTI), which is a large-scale virtual screening of known drugs. Herein, we report Kullback–Leibler divergence (KLD) as a DTI feature and the feature-driven classification model applicable to DTI prediction. For the purpose, E3FP three-dimensional (3D) molecular fingerprints of drugs as a molecular representation allow the computation of 3D similarities between ligands within each target (Q–Q matrix) to identify the uniqueness of pharmacological targets and those between a query and a ligand (Q–L vector) in DTIs. The 3D similarity matrices are transformed into probability density functions via kernel density estimation as a nonparametric estimation. Each density model can exploit the characteristics of each pharmacological target and measure the quasi-distance between the ligands. Furthermore, we developed a random forest model from the KLD feature vectors to successfully predict DTIs for representative 17 targets (mean accuracy: 0.882, out-of-bag score estimate: 0.876, ROC AUC: 0.990). The method is applicable for 2D chemical similarity.

Growth Factor-Free Vascularization of Marine-Origin Collagen Sponges Using Cryopreserved Stromal Vascular Fractions from Human Adipose Tissue.

The successful integration of transplanted three-dimensional tissue engineering (TE) constructs depends greatly on their rapid vascularization. Therefore, it is essential to address this vascularization issue in the initial design of constructs for perfused tissues. Two of the most important variables in this regard are scaffold composition and cell sourcing. Collagens with marine origins overcome some issues associated with mammal-derived collagen while maintaining their advantages in terms of biocompatibility. Concurrently, the freshly isolated stromal vascular fraction (SVF) of adipose tissue has been proposed as an advantageous cell fraction for vascularization purposes due to its highly angiogenic properties, allowing extrinsic angiogenic growth factor-free vascularization strategies for TE applications. In this study, we aimed at understanding whether marine collagen 3D matrices could support cryopreserved human SVF in maintaining intrinsic angiogenic properties observed for fresh SVF. For this, cryopreserved human SVF was seeded on blue shark collagen sponges and cultured up to 7 days in a basal medium. The secretome profile of several angiogenesis-related factors was studied throughout culture times and correlated with the expression pattern of CD31 and CD146, which showed the formation of a prevascular network. Upon in ovo implantation, increased vessel recruitment was observed in prevascularized sponges when compared with sponges without SVF cells. Immunohistochemistry for CD31 demonstrated the improved integration of prevascularized sponges within chick chorioalantoic membrane (CAM) tissues, while in situ hybridization showed human cells lining blood vessels. These results demonstrate the potential of using cryopreserved SVF combined with marine collagen as a streamlined approach to improve the vascularization of TE constructs.

Discovery and Development of Tumor Angiogenesis Assays.

The angiogenesis process was described in its basic concepts in the works of the Scottish surgeon John Hunter and terminologically assessed in the early twentieth century. An aberrant angiogenesis is a prerequisite for cancer cells in solid tumors to grow and metastasize. The sprouting of new blood vessels is one of the major characteristics of cancer and represents a gateway for tumor cells to enter both the blood and lymphatic circulation systems. In vivo, ex vivo, and in vitro models of angiogenesis have provided essential tools for cancer research and antiangiogenic drug screening. Several in vivo studies have been performed to investigate the various steps of tumor angiogenesis and in vitro experiments contributed to dissecting the molecular bases of this phenomenon. Moreover, coculture of cancer and endothelial cells in 2D and 3D matrices have contributed to improve the recapitulation of the complex process of tumor angiogenesis, including the peculiar conditions of tumor microenvironment.

Integrin-specific hydrogels for growth factor-free vasculogenesis

Integrin-binding biomaterials have been extensively evaluated for their capacity to enable de novo formation of capillary-like structures/vessels, ultimately supporting neovascularization in vivo. Yet, the role of integrins as vascular initiators in engineered materials is still not well understood. Here, we show that αvβ3 integrin-specific 3D matrices were able to retain PECAM1+ cells from the stromal vascular fraction (SVF) of adipose tissue, triggering vasculogenesis in vitro in the absence of extrinsic growth factors. Our results suggest that αvβ3-RGD-driven signaling in the formation of capillary-like structures prevents the activation of the caspase 8 pathway and activates the FAK/paxillin pathway, both responsible for endothelial cells (ECs) survival and migration. We also show that prevascularized αvβ3 integrin-specific constructs inosculate with the host vascular system fostering in vivo neovascularization. Overall, this work demonstrates the ability of the biomaterial to trigger vasculogenesis in an integrin-specific manner, by activating essential pathways for EC survival and migration within a self-regulatory growth factor microenvironment. This strategy represents an improvement to current vascularization routes for Tissue Engineering constructs, potentially enhancing their clinical applicability.

Injectable nanoporous microgels generate vascularized constructs and support bone regeneration in critical-sized defects

Large and aberrant bone fractures require ossification and concomitant vascularization for proper healing. Evidence indicates that osteogenesis and vessel growth are coupled in bone fractures. Although the synergistic role of endothelial cells has been recognized, vascularizing large bone grafts remains a challenge and has apprehended the clinical translation of engineered bone constructs. Here, we describe a facile method to fabricate vascularized constructs using chitosan and gelatin-based microgels that promote osteogenesis of human mesenchymal stromal cells (MSC) while supporting endothelial sprouting and network formation. The microgels are enzymatically degradable and had a high hydration rate with a volume swelling ratio of ~ 493% and a polymer density of ~ 431 mg/cm3, which is comparable to that of native skeletal tissues. AFM indentation of the surface showed an average Young’s modulus of 189 kPa, falling in a range that is conducive to both osteogenesis and vasculogenesis. The osteogenic microgel containing chitosan, gelatin, and hydroxyapatite, mimicking the bone matrix, supported robust attachment, proliferation, and differentiation of MSC. On the other hand, the vasculogenic microgels containing only gelatin, enriched endothelial phenotype and enabled vascular networks formation when embedded in 3D matrices. Combining the two types of microgels created a hybrid construct that sustained the functions of both osteogenic and vasculogenic microgels and enhanced one another. Using a murine model, we also show that the osteogenic microgels regenerate bone in a critical-sized defect with > 95% defect closure by week 12. These multifunctional microgels can be administered minimally invasively and can conformally fill large bone defects. This work lays the foundation to establish principles of designing multiphasic scaffolds with tissue-specific biophysical and biochemical properties for regenerating vascularized and interfacial tissues.

A Computational Template for Three-Dimensional Modeling of the Vascular Scaffold of the Human Thyroid Gland.

We recently designed an innovative scaffold-bioreactor unit for the bioengineering of a three-dimensional (3D) bioartificial human thyroid gland or its miniaturized replica as a part of a microfluidic chip test system. This device is based on the evidence that the 3D geometry of the intraglandular stromal/vascular scaffold (SVS; i.e., the fibrous and vascular matrix) of mammalian viscera plays a key role in guiding growth and differentiation of in vitro seeded cells. Therefore, we initiated a research program focused on computer-aided reconstruction of the 2nd to 4th order intralobar arterial network (IAN) of the human thyroid gland as a reliable surrogate for its 3D SVS, to be used as an input for rapid prototyping of a biomaterial replica. To this end, we developed a computational template that works within the Mathematica environment, giving rise to a quasi-fractal growth of the IAN distribution, constrained within an approximation of the thyroid lobe shape as a closed surface. Starting from edge detection of planar images of real human thyroid lobes acquired by in vivo real-time ultrasonography, we performed data approximation of the lobar profiles based on splines and Bezier curves, providing 3D lobar shapes as geometric boundaries for vessel growth by a diffusion-limited aggregation model. Our numerical procedures allowed for a robust connection between development of lobar arterial trees and thyroid lobe shape, led to a vascular self-similarity consistent with that of a cadaveric lobar arterial cast, and reproduced arterial vessels in a proportion not statistically different from that described for the real human thyroid gland. We conclude that our algorithmic template offers a reliable reproduction of the extremely complex IAN of the adult human thyroid lobe, potentially useful as a computational guidance for bioprinting of thyroid lobe matrix replicas. In addition, due to the simplicity and limited number of morphometrical parameters required by our system, we predict its application to the design of a number of patient-tailored human bioartificial organs and organs-on-chip, including parenchymal viscera and bones. Impact statement The study introduces the computer simulation of the three-dimensional (3D) intrinsic vascular matrix of the human thyroid gland, offering a general concept applicable to a number of other human viscera. Indeed, it provides a flexible software tool for reproduction of a 3D surrogate of the organ's 3D stromal matrix, suitable for eventual 3D bioprinting with biomaterials, and recellularization with organ-specific stem cells/progenitors. The final expectation is the design of patient-tailored 3D organ's matrices upon clinical request.

Establishment of a human 3D pancreatic adenocarcinoma model based on a patient-derived extracellular matrix scaffold

Pancreatic cancer is likely to become one of the leading causes of cancer-related death in many countries within the next decade. Surgery is the potentially curative treatment for pancreatic ductal adenocarcinoma (PDAC), although only 10%–20% of patients have a resectable disease after diagnosis. Despite recent advances in curative surgery the current prognosis ranges from 6% to 10% globally. One of the main issues at the pre-clinical level is the lacking of model which simultaneously reflects the tumour microenvironment (TME) at both structural and cellular levels. Here we describe an innovative tissue engineering approach applied to PDAC starting from decellularized human biopsies in order to generate an organotypic 3D in vitro model. This in vitro 3D system recapitulates the ultrastructural environment of native tissue as demonstrated by histology, immunohistochemistry, immunofluorescence, mechanical analysis, and scanning electron microscopy. Mass spectrometry confirmed a different extracellular matrix (ECM) composition between decellularized healthy pancreas and PDAC by identifying a total of 110 non-redundant differently expressed proteins. Immunofluorescence analyses after 7 days of scaffold recellularization with PANC-1 and AsPC-1 pancreatic cell lines, were performed to assess the biocompatibility of 3D matrices to sustain engraftment, localization and infiltration. Finally, both PANC-1 and AsPC-1 cells cultured in 3D matrices showed a reduced response to treatment with FOLFIRINOX if compared to conventional bi-dimensional culture. Our 3D culture system with patient-derived tissue-specific decellularized ECM better recapitulates the pancreatic cancer microenvironment compared to conventional 2D culture conditions and represents a relevant approach for the study of pancreatic cancer response to chemotherapy agents.

IPSCs in Neurodegenerative Disorders: A Unique Platform for Clinical Research and Personalized Medicine.

In the past, several animal disease models were developed to study the molecular mechanism of neurological diseases and discover new therapies, but the lack of equivalent animal models has minimized the success rate. A number of critical issues remain unresolved, such as high costs for developing animal models, ethical issues, and lack of resemblance with human disease. Due to poor initial screening and assessment of the molecules, more than 90% of drugs fail during the final step of the human clinical trial. To overcome these limitations, a new approach has been developed based on induced pluripotent stem cells (iPSCs). The discovery of iPSCs has provided a new roadmap for clinical translation research and regeneration therapy. In this article, we discuss the potential role of patient-derived iPSCs in neurological diseases and their contribution to scientific and clinical research for developing disease models and for developing a roadmap for future medicine. The contribution of humaniPSCs in the most common neurodegenerative diseases (e.g., Parkinson’s disease and Alzheimer’s disease, diabetic neuropathy, stroke, and spinal cord injury) were examined and ranked as per their published literature on PUBMED. We have observed that Parkinson’s disease scored highest, followed by Alzheimer’s disease. Furthermore, we also explored recent advancements in the field of personalized medicine, such as the patient-on-a-chip concept, where iPSCs can be grown on 3D matrices inside microfluidic devices to create an in vitro disease model for personalized medicine.

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment.

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (<40 µm in thickness) of COL1 fibers that extend over distances less than 500 µm and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 µm in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

Numerical Simulation of Vibrational Sum Frequency Generation Intensity for Non-Centrosymmetric Domains Interspersed in an Amorphous Matrix: A Case Study for Cellulose in Plant Cell Wall.

Vibrational sum frequency generation (SFG) spectroscopy can specifically probe molecular species non-centrosymmetrically arranged in a centrosymmetric or isotropic medium. This capability has been extensively utilized to detect and study molecular species present at the two-dimensional (2D) interface at which the centrosymmetry or isotropy of bulk phases is naturally broken. The same principle has been demonstrated to be very effective for the selective detection of non-centrosymmetric crystalline nanodomains interspersed in three-dimensional (3D) amorphous phases. However, the full spectral interpretation of SFG features has been difficult due to the complexity associated with the theoretical calculation of SFG responses of such 3D systems. This paper describes a numerical method to predict the relative SFG intensities of non-centrosymmetric nanodomains in 3D systems as functions of their size and concentration as well as their assembly patterns, i.e., the distributions of tilt, azimuth, and rotation angles with respect to the lab coordinate. We applied the developed method to predict changes in the CH and OH stretch modes characteristic to crystalline cellulose microfibrils distributed with various orders, which are relevant to plant cell wall structures. The same algorithm can also be applied to any SFG-active nanodomains interspersed in 3D amorphous matrices.

Molecular basis for pericyte-induced capillary tube network assembly and maturation.

Here we address the functional importance and role of pericytes in capillary tube network assembly, an essential process that is required for vascularized tissue development, maintenance, and health. Healthy capillaries may be directly capable of suppressing human disease. Considerable advances have occurred in our understanding of the molecular and signaling requirements controlling EC lumen and tube formation in 3D extracellular matrices. A combination of SCF, IL-3, SDF-1α, FGF-2 and insulin (“Factors”) in conjunction with integrin- and MT1-MMP-induced signaling are required for EC sprouting behavior and tube formation under serum-free defined conditions. Pericyte recruitment to the abluminal EC tube surface results in elongated and narrow tube diameters and deposition of the vascular basement membrane. In contrast, EC tubes in the absence of pericytes continue to widen and shorten over time and fail to deposit basement membranes. Pericyte invasion, recruitment and proliferation in 3D matrices requires the presence of ECs. A detailed analysis identified that EC-derived PDGF-BB, PDGF-DD, ET-1, HB-EGF, and TGFβ1 are necessary for pericyte recruitment, proliferation, and basement membrane deposition. Blockade of these individual factors causes significant pericyte inhibition, but combined blockade profoundly interferes with these events, resulting in markedly widened EC tubes without basement membranes, like when pericytes are absent.

Macroporous Aligned Hydrogel Microstrands for 3D Cell Guidance.

Tissue engineering strongly relies on the use of hydrogels as highly hydrated 3D matrices to support the maturation of laden cells. However, because of the lack of microarchitecture and sufficient porosity, common hydrogel systems do not provide physical cell-instructive guidance cues and efficient transport of nutrients and oxygen to the inner part of the construct. A controlled, organized cellular alignment and resulting alignment of secreted ECM are hallmarks of muscle, tendons, and nerves and play an important role in determining their functional properties. Although several strategies to induce cellular alignment have been investigated in 2D systems, the generation of cell-instructive 3D hydrogels remains a challenge. Here, we report on the development of a simple and scalable method to efficiently generate highly macroporous constructs featuring aligned guidance cues. A precross-linked bulk hydrogel is pressed through a grid with variable opening sizes, thus deconstructing it into an array of aligned, high aspect ratio microgels (microstrands) with tunable diameter that are eventually stabilized by a second photoclick cross-linking step. This method has been investigated and optimized both in silico and in vitro, thereby leading to conditions with excellent viability and organized cellular alignment. Finally, as proof of concept, the method has been shown to direct aligned muscle tissue maturation. These findings demonstrate the 3D physical guidance potential of our system, which can be used for a variety of anisotropic tissues and applications.

ARHGEF9 regulates melanoma morphogenesis in environments with diverse geometry and elasticity by promoting filopodial-driven adhesion

SummaryRho GTP Exchange Factors (RhoGEFs) and Rho GTPase Activating Proteins (RhoGAPs) are large families of molecules that regulate shape determination in all eukaryotes. In pathologies such as melanoma, RhoGEF and RhoGAP activity underpins the ability of cells to invade tissues of varying elasticity. To identify RhoGEFs and RhoGAPs that regulate melanoma cell shape on soft and/or stiff materials, we performed genetic screens, in tandem with single-cell quantitative morphological analysis. We show that ARHGEF9/Collybistin (Cb) is essential for cell shape determination on both soft and stiff materials, and in cells embedded in 3D soft hydrogel. ARHGEF9 is required for melanoma cells to invade 3D matrices. Depletion of ARHGEF9 results in loss of tension at focal adhesions decreased cell-wide contractility, and the inability to stabilize protrusions. Taken together we show that ARHGEF9 promotes the formation of actin-rich filopodia, which serves to establish and stabilize adhesions and determine melanoma cell shape.

Increased Stiffness Downregulates Focal Adhesion Kinase Expression in Pancreatic Cancer Cells Cultured in 3D Self-Assembling Peptide Scaffolds.

The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that participates in integrin-mediated signal transduction and contributes to different biological processes, such as cell migration, survival, proliferation and angiogenesis. Moreover, FAK can be activated by autophosphorylation at position Y397 and trigger different signaling pathways in response to increased extracellular matrix stiffness. In addition, FAK is overexpressed and/or hyperactivated in many epithelial cancers, and its expression correlates with tumor malignancy and invasion potential. One of the characteristics of solid tumors is an over deposition of ECM components, which generates a stiff microenvironment that promotes, among other features, sustained cell proliferation and survival. Researchers are, therefore, increasingly developing cell culture models to mimic the increased stiffness associated with these kinds of tumors. In the present work, we have developed a new 3D in vitro model to study the effect of matrix stiffness in pancreatic ductal adenocarcinoma (PDAC) cells as this kind of tumor is characterized by a desmoplastic stroma and an increased stiffness compared to its normal counterpart. For that, we have used a synthetic self-assembling peptide nanofiber matrix, RAD16-I, which does not suffer a significant degradation in vitro, thus allowing to maintain the same local stiffness along culture time. We show that increased matrix stiffness in synthetic 3D RAD16-I gels, but not in collagen type I scaffolds, promotes FAK downregulation at a protein level in all the cell lines analyzed. Moreover, even though it has classically been described that stiff 3D matrices promote an increase in pFAKY397/FAK proteins, we found that this ratio in soft and stiff RAD16-I gels is cell-type-dependent. This study highlights how cell response to increased matrix stiffness greatly depends on the nature of the matrix used for 3D culture.