An in vitro model of cancer invasion with heterogeneous ECM created with droplet microfluidics.

Metastasis is a multi-step process that is critically affected by cues from the tumor micro-environment (TME), such as from the extracellular matrix (ECM). The role of the ECM in the onset of metastasis, invasion, is not yet fully understood. A further complicating factor is that the ECM in the TME is mostly heterogeneous, in particular presenting a basement membrane (BM) directly enveloping the tumor, which acts as a barrier to invasion into the surrounding stromal ECM. To systematically investigate the role of ECM in invasion, appropriate in vitro models with control over such ECM heterogeneity are essential. We present a novel high-throughput microfluidic approach to build such a model, which enables to capture the invasion of cancer cells from the tumor, through the BM and into the stromal tissue. We used a droplet-maker device to encapsulate cells in beads of a primary hydrogel mimicking BM, Matrigel, which were then embedded in a secondary hydrogel mimicking stromal ECM, collagen I. Our technology ultimately provides control over parameters such as tissue size, cell count and type, and ECM composition and stiffness. As a proof-of-principle, we carried out a comparative study with two breast cancer cell types, and we observed typical behavior consistent with previous studies. Highly invasive MDA-MB-231 cells showed single cell invasion behavior, whereas poorly invasive MCF-7 cells physically penetrated the surrounding matrix collectively. A comparative analysis conducted between our heterogeneous model and previous models employing a single type of hydrogel, either collagen I or Matrigel, has unveiled a substantial difference in terms of cancer cell invasion distance. Our in vitro model resembles an in vivo heterogeneous cancer microenvironment and can potentially be used for high throughput studies of cancer invasion.

Directing cellular responses in a nanocomposite 3D matrix for tissue regeneration with nanoparticle-mediated drug delivery.

Hydrogels play an important role in tissue engineering due to their native extracellular matrix-like characteristics, but they are insufficient in providing the necessary stimuli to support tissue formation. Efforts to integrate bioactive cues directly into hydrogels are hindered by incompatibility with hydrophobic drugs, issues of burst/uncontrolled release, and rapid degradation of the bioactive molecules. Skeletal muscle tissue repair requires internal stimuli and communication between cells for regeneration, and nanocomposite systems offer to improve the therapeutic effects in tissue regeneration. Here, the versatility of mesoporous silica nanoparticles (MSN) was leveraged to formulate a nanoparticle-hydrogel composite and to combine the benefits of controlled delivery of bioactive cues and cellular support. The tunable surface characteristics of MSNs were exploited to optimize homogeneity and intracellular drug delivery in a 3D matrix. Nanocomposite hydrogels formulated with acetylated or succinylated MSNs achieved high homogeneity in 3D distribution, with succinylated MSNs being rapidly internalized and acetylated MSNs exhibiting slower cellular uptake. MSN-hydrogel nanocomposites simultaneously allowed efficient local intracellular delivery of a hydrophobic model drug. To further study the efficiency of directing cell response, a Notch signaling inhibitor (DAPT) was incorporated into succinylated MSNs and incorporated into the hydrogel. MSN-hydrogel nanocomposites effectively downregulated the Notch signaling target genes, and accelerated and maintained the expression of myogenic markers. The current findings demonstrate a proof-of-concept in effective surface engineering strategies for MSN-based nanocomposites, suited for hydrophobic drug delivery in tissue regeneration with guided cues.

Mechanical regulation of the Notch signaling pathway.

The mechanical regulation of Notch signaling is an emerging area of interest in cell biology. Notch is essential in many physiological processes in which mechanical stress plays an important role. This review provides an overview of the mechanoregulation of Notch signaling in multiple steps of the pathway. First, we discuss the current knowledge on the direct mechanoregulation of Notch receptor maturation and localization to the membrane and the effect of mechanical stress on the Notch components. Next, we explore how ligand-receptor interactions and membrane dynamics are possible subjects to mechano-regulation, emphasizing the role of cytoskeletal interactions, membrane stiffness, and endocytic complex formation. We further delve into the necessity of tension generation for negative regulatory region (NRR) domain unfolding, facilitated by ligand endocytosis and other microforces. Additionally, we examine the indirect mechano-regulation of S2 and S3 cleavages. Finally, we discuss the mechanoregulation of the Notch intracellular domain (NICD) trafficking and nuclear entry and the impact of mechanical stress on heterochromatin dynamics and nuclear NICD interactions. This review aims to draw attention to the intricate interplay between mechanical cues and Notch signaling regulation, offering novel insights into the multifaceted nature of cellular mechanobiology.

SFAlab: image-based quantification of mechano-active ventral actin stress fibers in adherent cells.

Ventral actin stress fibers (SFs) are a subset of actin SFs that begin and terminate at focal adhesion (FA) complexes. Ventral SFs can transmit forces from and to the extracellular matrix and serve as a prominent mechanosensing and mechanotransduction machinery for cells. Therefore, quantitative analysis of ventral SFs can lead to deeper understanding of the dynamic mechanical interplay between cells and their extracellular matrix (mechanoreciprocity). However, the dynamic nature and organization of ventral SFs challenge their quantification, and current quantification tools mainly focus on all SFs present in cells and cannot discriminate between subsets. Here we present an image analysis-based computational toolbox, called SFAlab, to quantify the number of ventral SFs and the number of ventral SFs per FA, and provide spatial information about the locations of the identified ventral SFs. SFAlab is built as an all-in-one toolbox that besides analyzing ventral SFs also enables the identification and quantification of (the shape descriptors of) nuclei, cells, and FAs. We validated SFAlab for the quantification of ventral SFs in human fetal cardiac fibroblasts and demonstrated that SFAlab analysis i) yields accurate ventral SF detection in the presence of image imperfections often found in typical fluorescence microscopy images, and ii) is robust against user subjectivity and potential experimental artifacts. To demonstrate the usefulness of SFAlab in mechanobiology research, we modulated actin polymerization and showed that inhibition of Rho kinase led to a significant decrease in ventral SF formation and the number of ventral SFs per FA, shedding light on the importance of the RhoA pathway specifically in ventral SF formation. We present SFAlab as a powerful open source, easy to use image-based analytical tool to increase our understanding of mechanoreciprocity in adherent cells.

Environmental stiffness restores mechanical homeostasis in vimentin-depleted cells.

Recent experimental evidence indicates a role for the intermediate filament vimentin in regulating cellular mechanical homeostasis, but its precise contribution remains to be discovered. Mechanical homeostasis requires a balanced bi-directional interplay between the cell's microenvironment and the cellular morphological and mechanical state-this balance being regulated via processes of mechanotransduction and mechanoresponse, commonly referred to as mechanoreciprocity. Here, we systematically analyze vimentin-expressing and vimentin-depleted cells in a swatch of in vitro cellular microenvironments varying in stiffness and/or ECM density. We find that vimentin-expressing cells maintain mechanical homeostasis by adapting cellular morphology and mechanics to micromechanical changes in the microenvironment. However, vimentin-depleted cells lose this mechanoresponse ability on short timescales, only to reacquire it on longer time scales. Indeed, we find that the morphology and mechanics of vimentin-depleted cell in stiffened microenvironmental conditions can get restored to the homeostatic levels of vimentin-expressing cells. Additionally, we observed vimentin-depleted cells increasing collagen matrix synthesis and its crosslinking, a phenomenon which is known to increase matrix stiffness, and which we now hypothesize to be a cellular compensation mechanism for the loss of vimentin. Taken together, our findings provide further insight in the regulating role of intermediate filament vimentin in mediating mechanoreciprocity and mechanical homeostasis.

Shape-Morphing Photoresponsive Hydrogels Reveal Dynamic Topographical Conditioning of Fibroblasts.

The extracellular environment defines a physical boundary condition with which cells interact. However, to date, cell response to geometrical environmental cues is largely studied in static settings, which fails to capture the spatiotemporally varying cues cells receive in native tissues. Here, a photoresponsive spiropyran-based hydrogel is presented as a dynamic, cell-compatible, and reconfigurable substrate. Local stimulation with blue light (455 nm) alters hydrogel swelling, resulting in on-demand reversible micrometer-scale changes in surface topography within 15 min, allowing investigation into cell response to controlled geometry actuations. At short term (1 h after actuation), fibroblasts respond to multiple rounds of recurring topographical changes by reorganizing their nucleus and focal adhesions (FA). FAs form primarily at the dynamic regions of the hydrogel; however, this propensity is abolished when the topography is reconfigured from grooves to pits, demonstrating that topographical changes dynamically condition fibroblasts. Further, this dynamic conditioning is found to be associated with long-term (72 h) maintenance of focal adhesions and epigenetic modifications. Overall, this study offers a new approach to dissect the dynamic interplay between cells and their microenvironment and shines a new light on the cell's ability to adapt to topographical changes through FA-based mechanotransduction.

Organ-on-a-chip technologies for biomedical research and drug development: A focus on the vasculature.

Current biomedical models fail to replicate the complexity of human biology. Consequently, almost 90% of drug candidates fail during clinical trials after decades of research and billions of investments in drug development. Despite their physiological similarities, animal models often misrepresent human responses, and instead, trigger ethical and societal debates regarding their use. The overall aim across regulatory entities worldwide is to replace, reduce, and refine the use of animal experimentation, a concept known as the Three Rs principle. In response, researchers develop experimental alternatives to improve the biological relevance of in vitro models through interdisciplinary approaches. This article highlights the emerging organ-on-a-chip technologies, also known as microphysiological systems, with a focus on models of the vasculature. The cardiovascular system transports all necessary substances, including drugs, throughout the body while in charge of thermal regulation and communication between other organ systems. In addition, we discuss the benefits, limitations, and challenges in the widespread use of new biomedical models. Coupled with patient-derived induced pluripotent stem cells, organ-on-a-chip technologies are the future of drug discovery, development, and personalized medicine.

A multiscale computational model of arterial growth and remodeling including Notch signaling.

Blood vessels grow and remodel in response to mechanical stimuli. Many computational models capture this process phenomenologically, by assuming stress homeostasis, but this approach cannot unravel the underlying cellular mechanisms. Mechano-sensitive Notch signaling is well-known to be key in vascular development and homeostasis. Here, we present a multiscale framework coupling a constrained mixture model, capturing the mechanics and turnover of arterial constituents, to a cell-cell signaling model, describing Notch signaling dynamics among vascular smooth muscle cells (SMCs) as influenced by mechanical stimuli. Tissue turnover was regulated by both Notch activity, informed by in vitro data, and a phenomenological contribution, accounting for mechanisms other than Notch. This novel framework predicted changes in wall thickness and arterial composition in response to hypertension similar to previous in vivo data. The simulations suggested that Notch contributes to arterial growth in hypertension mainly by promoting SMC proliferation, while other mechanisms are needed to fully capture remodeling. The results also indicated that interventions to Notch, such as external Jagged ligands, can alter both the geometry and composition of hypertensive vessels, especially in the short term. Overall, our model enables a deeper analysis of the role of Notch and Notch interventions in arterial growth and remodeling and could be adopted to investigate therapeutic strategies and optimize vascular regeneration protocols.

Modulating epigenetic modifications for cancer therapy (Review).

Cancer is a global public health concern. Alterations in epigenetic processes are among the earliest genomic aberrations occurring during cancer development and are closely related to progression. Unlike genetic mutations, aberrations in epigenetic processes are reversible, which opens the possibility for novel pharmacological treatments. Non­coding RNAs (ncRNAs) represent an essential epigenetic mechanism, and emerging evidence links ncRNAs to carcinogenesis. Epigenetic drugs (epidrugs) are a group of promising target therapies for cancer treatment acting as coadjuvants to reverse drug resistance in cancer. The present review describes central epigenetic aberrations during malignant transformation and explains how epidrugs target DNA methylation, histone modifications and ncRNAs. Furthermore, clinical trials focused on evaluating the effect of these epidrugs alone or in combination with other anticancer therapies and other ncRNA­based therapies are discussed. The use of epidrugs promises to be an effective tool for reversing drug resistance in some patients with cancer.

An N-glycan on the C2 domain of JAGGED1 is important for Notch activation.

The canonical members of the Jagged/Serrate and Delta families of transmembrane ligands have an extracellular, amino-terminal C2 domain that binds to phospholipids and is required for optimal activation of the Notch receptor. Somatic mutations that cause amino substitutions in the C2 domain in human JAGGED1 (JAG1) have been identified in tumors. We found in reporter cell assays that mutations affecting an N-glycosylation site reduced the ligand's ability to activate Notch. This N-glycosylation site located in the C2 domain is conserved in the Jagged/Serrate family but is lacking in the Delta family. Site-specific glycan analysis of the JAG1 amino terminus demonstrated that occupancy of this site by either a complex-type or high-mannose N-glycan was required for full Notch activation in reporter cell assays. Similarly to JAG1 variants with defects in Notch binding, N-glycan removal, either by mutagenesis of the glycosylation site or by endoglycosidase treatment, reduced receptor activation. The N-glycan variants also reduced receptor activation in a Notch signaling-dependent vascular smooth muscle cell differentiation assay. Loss of the C2 N-glycan reduced JAG1 binding to liposomes to a similar extent as the loss of the entire C2 domain. Molecular dynamics simulations suggested that the presence of the N-glycan limits the orientation of JAG1 relative to the membrane, thus facilitating Notch binding. These data are consistent with a critical role for the N-glycan in promoting a lipid-binding conformation that is required to orient Jagged at the cell membrane for full Notch activation.

Reversible and bidirectional signaling of notch ligands.

The Notch signaling pathway is a direct cell-cell communication system involved in a wide variety of biological processes, and its disruption is observed in several pathologies. The pathway is comprised of a ligand-expressing (sender) cell and a receptor-expressing (receiver) cell. The canonical ligands are members of the Delta/Serrate/Lag-1 (DSL) family of proteins. Their binding to a Notch receptor in a neighboring cell induces a conformational change in the receptor, which will undergo regulated intramembrane proteolysis (RIP), liberating the Notch intracellular domain (NICD). The NICD is translocated to the nucleus and promotes gene transcription. It has been demonstrated that the ligands can also undergo RIP and nuclear translocation, suggesting a function for the ligands in the sender cell and possible bidirectionality of the Notch pathway. Although the complete mechanism of ligand processing is not entirely understood, and its dependence on Notch receptors has not been ruled out. Also, ligands have autonomous functions beyond Notch activation. Here we review the concepts of reverse and bidirectional signalization of DSL proteins and discuss the characteristics that make them more than just ligands of the Notch pathway.

Notch signaling regulates strain-mediated phenotypic switching of vascular smooth muscle cells.

Mechanical stimuli experienced by vascular smooth muscle cells (VSMCs) and mechanosensitive Notch signaling are important regulators of vascular growth and remodeling. However, the interplay between mechanical cues and Notch signaling, and its contribution to regulate the VSMC phenotype are still unclear. Here, we investigated the role of Notch signaling in regulating strain-mediated changes in VSMC phenotype. Synthetic and contractile VSMCs were cyclically stretched for 48 h to determine the temporal changes in phenotypic features. Different magnitudes of strain were applied to investigate its effect on Notch mechanosensitivity and the phenotypic regulation of VSMCs. In addition, Notch signaling was inhibited via DAPT treatment and activated with immobilized Jagged1 ligands to understand the role of Notch on strain-mediated phenotypic changes of VSMCs. Our data demonstrate that cyclic strain induces a decrease in Notch signaling along with a loss of VSMC contractile features. Accordingly, the activation of Notch signaling during cyclic stretching partially rescued the contractile features of VSMCs. These findings demonstrate that Notch signaling has an important role in regulating strain-mediated phenotypic switching of VSMCs.

Computational analysis of the role of mechanosensitive Notch signaling in arterial adaptation to hypertension.

Arteries grow and remodel in response to mechanical stimuli. Hypertension, for example, results in arterial wall thickening. Cell-cell Notch signaling between vascular smooth muscle cells (VSMCs) is known to be involved in this process, but the underlying mechanisms are still unclear. Here, we investigated whether Notch mechanosensitivity to strain may regulate arterial thickening in hypertension. We developed a multiscale computational framework by coupling a finite element model of arterial mechanics, including residual stress, to an agent-based model of mechanosensitive Notch signaling, to predict VSMC phenotypes as an indicator of growth and remodeling. Our simulations revealed that the sensitivity of Notch to strain at mean blood pressure may be a key mediator of arterial thickening in hypertensive arteries. Further simulations showed that loss of residual stress can have synergistic effects with hypertension, and that changes in the expression of Notch receptors, but not Jagged ligands, may be used to control arterial growth and remodeling and to intensify or counteract hypertensive thickening. Overall, we identify Notch mechanosensitivity as a potential mediator of vascular adaptation, and we present a computational framework that can facilitate the testing of new therapeutic and regenerative strategies.

Engineered patterns of Notch ligands Jag1 and Dll4 elicit differential spatial control of endothelial sprouting.

Spatial regulation of angiogenesis is important for the generation of functional engineered vasculature in regenerative medicine. The Notch ligands Jag1 and Dll4 show distinct expression patterns in endothelial cells and, respectively, promote and inhibit endothelial sprouting. Therefore, patterns of Notch ligands may be utilized to spatially control sprouting, but their potential and the underlying mechanisms of action are unclear. Here, we coupled in vitro and in silico models to analyze the ability of micropatterned Jag1 and Dll4 ligands to spatially control endothelial sprouting. Dll4 patterns, but not Jag1 patterns, elicited spatial control. Computational simulations of the underlying signaling dynamics suggest that different timing of Notch activation by Jag1 and Dll4 underlie their distinct ability to spatially control sprouting. Hence, Dll4 patterns efficiently direct the sprouts, whereas longer exposure to Jag1 patterns is required to achieve spatial control. These insights in sprouting regulation offer therapeutic handles for spatial regulation of angiogenesis.

Optogenetic control of NOTCH1 signaling.

The Notch signaling pathway is a crucial regulator of cell differentiation as well as tissue organization, whose deregulation is linked to the pathogenesis of different diseases. NOTCH1 plays a key role in breast cancer progression by increasing proliferation, maintenance of cancer stem cells, and impairment of cell death. NOTCH1 is a mechanosensitive receptor, where mechanical force is required to activate the proteolytic cleavage and release of the Notch intracellular domain (NICD). We circumvent this limitation by regulating Notch activity by light. To achieve this, we have engineered an optogenetic NOTCH1 receptor (optoNotch) to control the activation of NOTCH1 intracellular domain (N1ICD) and its downstream transcriptional activities. Using optoNotch we confirm that NOTCH1 activation increases cell proliferation in MCF7 and MDA-MB-468 breast cancer cells in 2D and spheroid 3D cultures, although causing distinct cell-type specific migratory phenotypes. Additionally, optoNotch activation induced chemoresistance on the same cell lines. OptoNotch allows the fine-tuning, ligand-independent, regulation of N1ICD activity and thus a better understanding of the spatiotemporal complexity of Notch signaling. Video Abstract.

GIT1 protects against breast cancer growth through negative regulation of Notch.

Hyperactive Notch signalling is frequently observed in breast cancer and correlates with poor prognosis. However, relatively few mutations in the core Notch signalling pathway have been identified in breast cancer, suggesting that as yet unknown mechanisms increase Notch activity. Here we show that increased expression levels of GIT1 correlate with high relapse-free survival in oestrogen receptor-negative (ER(-)) breast cancer patients and that GIT1 mediates negative regulation of Notch. GIT1 knockdown in ER(-) breast tumour cells increased signalling downstream of Notch and activity of aldehyde dehydrogenase, a predictor of poor clinical outcome. GIT1 interacts with the Notch intracellular domain (ICD) and influences signalling by inhibiting the cytoplasm-to-nucleus transport of the Notch ICD. In xenograft experiments, overexpression of GIT1 in ER(-) cells prevented or reduced Notch-driven tumour formation. These results identify GIT1 as a modulator of Notch signalling and a guardian against breast cancer growth.

Engineering tissue morphogenesis: taking it up a Notch.

Recreating functional tissues through bioengineering strategies requires steering of complex cell fate decisions. Notch, a juxtacrine signaling pathway, regulates cell fate and controls cellular organization with local precision. The engineering-friendly characteristics of the Notch pathway provide handles for engineering tissue patterning and morphogenesis. We discuss the physiological significance and mechanisms of Notch signaling with an emphasis on its potential use for engineering complex tissues. We highlight the current state of the art of Notch activation and provide a view on the design aspects, opportunities, and challenges in modulating Notch for tissue-engineering strategies. We propose that finely tuned control of Notch contributes to the generation of tissues with accurate form and functionality.

Mechano-regulated cell-cell signaling in the context of cardiovascular tissue engineering.

Cardiovascular tissue engineering (CVTE) aims to create living tissues, with the ability to grow and remodel, as replacements for diseased blood vessels and heart valves. Despite promising results, the (long-term) functionality of these engineered tissues still needs improvement to reach broad clinical application. The functionality of native tissues is ensured by their specific mechanical properties directly arising from tissue organization. We therefore hypothesize that establishing a native-like tissue organization is vital to overcome the limitations of current CVTE approaches. To achieve this aim, a better understanding of the growth and remodeling (G&R) mechanisms of cardiovascular tissues is necessary. Cells are the main mediators of tissue G&R, and their behavior is strongly influenced by both mechanical stimuli and cell-cell signaling. An increasing number of signaling pathways has also been identified as mechanosensitive. As such, they may have a key underlying role in regulating the G&R of tissues in response to mechanical stimuli. A more detailed understanding of mechano-regulated cell-cell signaling may thus be crucial to advance CVTE, as it could inspire new methods to control tissue G&R and improve the organization and functionality of engineered tissues, thereby accelerating clinical translation. In this review, we discuss the organization and biomechanics of native cardiovascular tissues; recent CVTE studies emphasizing the obtained engineered tissue organization; and the interplay between mechanical stimuli, cell behavior, and cell-cell signaling. In addition, we review past contributions of computational models in understanding and predicting mechano-regulated tissue G&R and cell-cell signaling to highlight their potential role in future CVTE strategies.

Sensitization of MCF7 Cells with High Notch1 Activity by Cisplatin and Histone Deacetylase Inhibitors Applied Together.

Histone deacetylase inhibitors (HDIs) are promising anti-cancer agents that inhibit proliferation of many types of cancer cells including breast carcinoma (BC) cells. In the present study, we investigated the influence of the Notch1 activity level on the pharmacological interaction between cisplatin (CDDP) and two HDIs, valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, vorinostat), in luminal-like BC cells. The type of drug-drug interaction between CDDP and HDIs was determined by isobolographic analysis. MCF7 cells were genetically modified to express differential levels of Notch1 activity. The cytotoxic effect of SAHA or VPA was higher on cells with decreased Notch1 activity and lower for cells with increased Notch1 activity than native BC cells. The isobolographic analysis demonstrated that combinations of CDDP with SAHA or VPA at a fixed ratio of 1:1 exerted additive or additive with tendency toward synergism interactions. Therefore, treatment of CDDP with HDIs could be used to optimize a combined therapy based on CDDP against Notch1-altered luminal BC. In conclusion, the combined therapy of HDIs and CDDP may be a promising therapeutic tool in the treatment of luminal-type BC with altered Notch1 activity.

PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion.

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.