Acetyl-CoA promotes glioblastoma cell adhesion and migration through Ca2+-NFAT signaling.

The metabolite acetyl-coenzyme A (acetyl-CoA) is the required acetyl donor for lysine acetylation and thereby links metabolism, signaling, and epigenetics. Nutrient availability alters acetyl-CoA levels in cancer cells, correlating with changes in global histone acetylation and gene expression. However, the specific molecular mechanisms through which acetyl-CoA production impacts gene expression and its functional roles in promoting malignant phenotypes are poorly understood. Here, using histone H3 Lys27 acetylation (H3K27ac) ChIP-seq (chromatin immunoprecipitation [ChIP] coupled with next-generation sequencing) with normalization to an exogenous reference genome (ChIP-Rx), we found that changes in acetyl-CoA abundance trigger site-specific regulation of H3K27ac, correlating with gene expression as opposed to uniformly modulating this mark at all genes. Genes involved in integrin signaling and cell adhesion were identified as acetyl-CoA-responsive in glioblastoma cells, and we demonstrate that ATP citrate lyase (ACLY)-dependent acetyl-CoA production promotes cell migration and adhesion to the extracellular matrix. Mechanistically, the transcription factor NFAT1 (nuclear factor of activated T cells 1) was found to mediate acetyl-CoA-dependent gene regulation and cell adhesion. This occurs through modulation of Ca2+ signals, triggering NFAT1 nuclear translocation when acetyl-CoA is abundant. The findings of this study thus establish that acetyl-CoA impacts H3K27ac at specific loci, correlating with gene expression, and that expression of cell adhesion genes are driven by acetyl-CoA in part through activation of Ca2+-NFAT signaling.

Heterogeneity of brain extracellular matrix and astrocyte activation.

From the blood brain barrier to the synaptic space, astrocytes provide structural, metabolic, ionic, and extracellular matrix (ECM) support across the brain. Astrocytes include a vast array of subtypes, their phenotypes and functions varying both regionally and temporally. Astrocytes' metabolic and regulatory functions poise them to be quick and sensitive responders to injury and disease in the brain as revealed by single cell sequencing. Far less is known about the influence of the local healthy and aging microenvironments on these astrocyte activation states. In this forward-looking review, we describe the known relationship between astrocytes and their local microenvironment, the remodeling of the microenvironment during disease and injury, and postulate how they may drive astrocyte activation. We suggest technology development to better understand the dynamic diversity of astrocyte activation states, and how basal and activation states depend on the ECM microenvironment. A deeper understanding of astrocyte response to stimuli in ECM-specific contexts (brain region, age, and sex of individual), paves the way to revolutionize how the field considers astrocyte-ECM interactions in brain injury and disease and opens routes to return astrocytes to a healthy quiescent state.

Strain-Stiffening Hydrogels with Dynamic, Secondary Cross-Linking.

Hydrogels are water-swollen, typically soft networks useful as biomaterials and in other fields of biotechnology. Hydrogel networks capable of sensing and responding to external perturbations, such as light, temperature, pH, or force, are useful across a wide range of applications requiring on-demand cross-linking or dynamic changes. Thus far, although mechanophores have been described as strain-sensitive reactive groups, embedding this type of force-responsiveness into hydrogels is unproven. Here, we synthesized multifunctional polymers that combine a hydrophilic zwitterion with permanently cross-linking alkenes, and dynamically cross-linking disulfides. From these polymers, we created hydrogels that contain irreversible and strong thiol-ene cross-links and reversible disulfide cross-links, and they stiffened in response to strain, increasing hundreds of kPa in modulus under compression. We examined variations in polymer composition and used a constitutive model to determine how to balance the number of thiol-ene vs disulfide cross-links to create maximally force-responsive networks. These strain-stiffening hydrogels represent potential biomaterials that benefit from the mechanoresponsive behavior needed for emerging applications in areas such as tissue engineering.

Cavitation induced fracture of intact brain tissue.

Nonpenetrating traumatic brain injuries (TBIs) are linked to cavitation. The structural organization of the brain makes it particularly susceptible to tears and fractures from these cavitation events, but limitations in existing characterization methods make it difficult to understand the relationship between fracture and cavitation in this tissue. More broadly, fracture energy is an important, yet often overlooked, mechanical property of all soft tissues. We combined needle-induced cavitation with hydraulic fracture models to induce and quantify fracture in intact brains at precise locations. We report here the first measurements of the fracture energy of intact brain tissue that range from 1.5 to 8.9 J/m2, depending on the location in the brain and the model applied. We observed that fracture consistently occurs along interfaces between regions of brain tissue. These fractures along interfaces allow cavitation-related damage to propagate several millimeters away from the initial injury site. Quantifying the forces necessary to fracture brain and other soft tissues is critical for understanding how impact and blast waves damage tissue in vivo and has implications for the design of protective gear and tissue engineering.

Materials-driven approaches to understand extrinsic drug resistance in cancer.

Metastatic cancer has a poor prognosis, because it is broadly disseminated and associated with both intrinsic and acquired drug resistance. Critical unmet needs in effectively killing drug resistant cancer cells include overcoming the drug desensitization characteristics of some metastatic cancers/lesions, and tailoring therapeutic regimens to both the tumor microenvironment and the genetic profiles of the resident cancer cells. Bioengineers and materials scientists are developing technologies to determine how metastatic sites exclude therapies, and how extracellular factors (including cells, proteins, metabolites, extracellular matrix, and abiotic factors) at metastatic sites significantly affect drug pharmacodynamics. Two looming challenges are determining which feature, or combination of features, from the tumor microenvironment drive drug resistance, and what the relative impact is of extracellular signals vs. intrinsic cell genetics in determining drug response. Sophisticated systems biology tools that can de-convolve a crowded network of signals and responses, as well as controllable microenvironments capable of providing discrete and tunable extracellular cues can help us begin to interrogate the high dimensional interactions governing drug resistance in patients.

Systems approaches to uncovering the contribution of environment-mediated drug resistance.

Cancer drug response is heavily influenced by the extracellular matrix (ECM) environment. Despite a clear appreciation that the ECM influences cancer drug response and progression, a unified view of how, where, and when environment-mediated drug resistance contributes to cancer progression has not coalesced. Here, we survey some specific ways in which the ECM contributes to cancer resistance with a focus on how materials development can coincide with systems biology approaches to better understand and perturb this contribution. We argue that part of the reason that environment-mediated resistance remains a perplexing problem is our lack of a wholistic view of the entire range of environments and their impacts on cell behavior. We cover a series of recent experimental and computational tools that will aid exploration of ECM reactions space, and how they might be synergistically integrated.

OrgDyn: feature- and model-based characterization of spatial and temporal organoid dynamics.

SUMMARY: Organoid model systems recapitulate key features of mammalian tissues and enable high throughput experiments. However, the impact of these experiments may be limited by manual, non-standardized, static or qualitative phenotypic analysis. OrgDyn is an open-source and modular pipeline to quantify organoid shape dynamics using a combination of feature- and model-based approaches on time series of 2D organoid contour images. Our pipeline consists of (i) geometrical and signal processing feature extraction, (ii) dimensionality reduction to differentiate dynamical paths, (iii) time series clustering to identify coherent groups of organoids and (iv) dynamical modeling using point distribution models to explain temporal shape variation. OrgDyn can characterize, cluster and model differences among unique dynamical paths that define diverse final shapes, thus enabling quantitative analysis of the molecular basis of tissue development and disease. AVAILABILITY AND IMPLEMENTATION: https://github.com/zakih/organoidDynamics (BSD 3-Clause License). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Seeded laser-induced cavitation for studying high-strain-rate irreversible deformation of soft materials.

Characterizing the high-strain-rate and high-strain mechanics of soft materials is critical to understanding the complex behavior of polymers and various dynamic injury mechanisms, including traumatic brain injury. However, their dynamic mechanical deformation under extreme conditions is technically difficult to quantify and often includes irreversible damage. To address such challenges, we investigate an experimental method, which allows quantification of the extreme mechanical properties of soft materials using ultrafast stroboscopic imaging of highly reproducible laser-induced cavitation events. As a reference material, we characterize variably cross-linked polydimethylsiloxane specimens using this method. The consistency of the laser-induced cavitation is achieved through the introduction of laser absorbing seed microspheres. Based on a simplified viscoelastic model, representative high-strain-rate shear moduli and viscosities of the soft specimens are quantified across different degrees of crosslinking. The quantified rheological parameters align well with the time-temperature superposition prediction of dynamic mechanical analysis. The presented method offers significant advantages with regard to quantifying high-strain rate, irreversible mechanical properties of soft materials and tissues, compared to other methods that rely upon the cyclic dynamics of cavitation. These advances are anticipated to aid in the understanding of how damage and injury develop in soft materials and tissues.

Tumor-stroma interactions differentially alter drug sensitivity based on the origin of stromal cells.

Due to tumor heterogeneity, most believe that effective treatments should be tailored to the features of an individual tumor or tumor subclass. It is still unclear, however, what information should be considered for optimal disease stratification, and most prior work focuses on tumor genomics. Here, we focus on the tumor microenvironment. Using a large-scale coculture assay optimized to measure drug-induced cell death, we identify tumor-stroma interactions that modulate drug sensitivity. Our data show that the chemo-insensitivity typically associated with aggressive subtypes of breast cancer is not observed if these cells are grown in 2D or 3D monoculture, but is manifested when these cells are cocultured with stromal cells, such as fibroblasts. Furthermore, we find that fibroblasts influence drug responses in two distinct and divergent manners, associated with the tissue from which the fibroblasts were harvested. These divergent phenotypes occur regardless of the drug tested and result from modulation of apoptotic priming within tumor cells. Our study highlights unexpected diversity in tumor-stroma interactions, and we reveal new principles that dictate how fibroblasts alter tumor drug responses.

Zwitterionic PEG-PC Hydrogels Modulate the Foreign Body Response in a Modulus-Dependent Manner.

Reducing the foreign body response (FBR) to implanted biomaterials will enhance their performance in tissue engineering. Poly(ethylene glycol) (PEG) hydrogels are increasingly popular for this application due to their low cost, ease of use, and the ability to tune their compliance via molecular weight and cross-linking densities. PEG hydrogels can elicit chronic inflammation in vivo, but recent evidence has suggested that extremely hydrophilic, zwitterionic materials and particles can evade the immune system. To combine the advantages of PEG-based hydrogels with the hydrophilicity of zwitterions, we synthesized hydrogels with comonomers PEG and the zwitterion phosphorylcholine (PC). Recent evidence suggests that stiff hydrogels elicit increased immune cell adhesion to hydrogels, which we attempted to reduce by increasing hydrogel hydrophilicity. Surprisingly, hydrogels with the highest amount of zwitterionic comonomer elicited the highest FBR. Lowering the hydrogel modulus (165 to 3 kPa), or PC content (20 to 0 wt %), mitigated this effect. A high density of macrophages was found at the surface of implants associated with a high FBR, and mass spectrometry analysis of the proteins adsorbed to these gels implicated extracellular matrix, immune response, and cell adhesion protein categories as drivers of macrophage recruitment. Overall, we show that modulus regulates macrophage adhesion to zwitterionic-PEG hydrogels, and demonstrate that chemical modifications to hydrogels should be studied in parallel with their physical properties to optimize implant design.

Biomaterials in Mechano-oncology: Means to Tune Materials to Study Cancer.

ECM stiffness is emerging as a prognostic marker of tumor aggression or potential for relapse. However, conflicting reports muddle the question of whether increasing or decreasing stiffness is associated with aggressive disease. This chapter discusses this controversy in more detail, but the fact that tumor stiffening plays a key role in cancer progression and in regulating cancer cell behaviors is clear. The impact of having in vitro biomaterial systems that could capture this stiffening during tumor evolution is very high. These cell culture platforms could help reveal the mechanistic underpinnings of this evolution, find new therapeutic targets to inhibit the cross talk between tumor development and ECM stiffening, and serve as better, more physiologically relevant platforms for drug screening.

Strain-stiffening gels based on latent crosslinking.

Gels represent an increasingly important class of soft materials with applications ranging from regenerative medicine to commodity materials. However, gels typically exhibit relative mechanical weakness, which worsens under repeated strain. Here we report a new class of responsive gels with latent crosslinking moieties that exhibit strain-stiffening behavior. This property results from the lability of disulfides, initially isolated in a protected state, then activated to crosslink on-demand. The thiol groups are induced to form inter-chain crosslinks when subjected to mechanical compression, resulting in a gel that strengthens under strain. Molecular shielding design elements regulate the strain-sensitivity and spontaneous crosslinking tendencies of the polymer network. These strain-responsive gels represent a rational design of new advanced materials with on-demand stiffening properties and potential applications in elastomers, adhesives, foams, films, and fibers.

Thermal-Responsive Behavior of a Cell Compatible Chitosan/Pectin Hydrogel.

Biopolymer hydrogels are important materials for wound healing and cell culture applications. While current synthetic polymer hydrogels have excellent biocompatibility and are nontoxic, they typically function as a passive matrix that does not supply any additional bioactivity. Chitosan (CS) and pectin (Pec) are natural polymers with active properties that are desirable for wound healing. Unfortunately, the synthesis of CS/Pec materials have previously been limited by harsh acidic synthesis conditions, which further restricted their use in biomedical applications. In this study, a zero-acid hydrogel has been synthesized from a mixture of chitosan and pectin at biologically compatible conditions. For the first time, we demonstrated that salt could be used to suppress long-range electrostatic interactions to generate a thermoreversible biopolymer hydrogel that has temperature-sensitive gelation. Both the hydrogel and the solution phases are highly elastic, with a power law index of close to -1. When dried hydrogels were placed into phosphate buffered saline solution, they rapidly rehydrated and swelled to incorporate 2.7× their weight. As a proof of concept, we removed the salt from our CS/Pec hydrogels, thus, creating thick and easy to cast polyelectrolyte complex hydrogels, which proved to be compatible with human marrow-derived stem cells. We suggest that our development of an acid-free CS/Pec hydrogel system that has excellent exudate uptake, holds potential for wound healing bandages.

Outlook and opportunities for engineered environments of breast cancer dormancy.

Dormant, disseminated breast cancer cells resist treatment and may relapse into malignant metastases after decades of quiescence. Identifying how and why these dormant breast cancer cells are triggered into outgrowth is a key unsolved step in treating latent, metastatic breast cancer. However, our understanding of breast cancer dormancy in vivo is limited by technical challenges and ethical concerns with triggering the activation of dormant breast cancer. In vitro models avoid many of these challenges by simulating breast cancer dormancy and activation in well-controlled, bench-top conditions, creating opportunities for fundamental insights into breast cancer biology that complement what can be achieved through animal and clinical studies. In this review, we address clinical and preclinical approaches to treating breast cancer dormancy, how precisely controlled artificial environments reveal key interactions that regulate breast cancer dormancy, and how future generations of biomaterials could answer further questions about breast cancer dormancy.

Acute and Chronic Neural and Glial Response to Mild Traumatic Brain Injury in the Hippocampus.

Traumatic brain injury (TBI) is an established risk factor for developing neurodegenerative disease. However, how TBI leads from acute injury to chronic neurodegeneration is limited to post-mortem models. There is a lack of connections between in vitro and in vivo TBI models that can relate injury forces to both macroscale tissue damage and brain function at the cellular level. Needle-induced cavitation (NIC) is a technique that can produce small cavitation bubbles in soft tissues, which allows us to relate small strains and strain rates in living tissue to ensuing acute and chronic cell death, tissue damage, and tissue remodeling. Here, we applied NIC to mouse brain slices to create a new model of TBI with high spatial and temporal resolution. We specifically targeted the hippocampus, which is a brain region critical for learning and memory and an area in which injury causes cognitive pathologies in humans and rodent models. By combining NIC with patch-clamp electrophysiology, we demonstrate that NIC in the Cornu Ammonis (CA)3 region of the hippocampus dynamically alters synaptic release onto CA1 pyramidal neurons in a cannabinoid 1 receptor (CB1R)-dependent manner. Further, we show that NIC induces an increase in extracellular matrix proteins associated with neural repair that is mitigated by CB1R antagonism. Together, these data lay the groundwork for advanced approaches in understanding how TBI impacts neural function at the cellular level, and the development of treatments that promote neural repair in response to brain injury.

Heparin decamer bridges a growth factor and an oligolysine by different charge-driven interactions.

Full-length heparin is widely used in tissue engineering applications due its multiple protein-binding sites that allow it to retain growth factor affinity while associating with oligopeptide components of the tissue scaffold. However, the extent to which oligopeptide coupling interferes with cognate protein binding is difficult to predict. To investigate such simultaneous interactions, we examined a well-defined ternary system comprised of acidic fibroblast growth factor (FGF), tetralysine (K4), with a heparin decamer (dp10) acting as a noncovalent coupler. Electrospray ionization mass spectrometry was used to assess binding affinities and complex stoichiometries as a function of ionic strength for dp10·K4 and FGF·dp10. The ionic strength dependence of K4·dp10 formation is qualitatively consistent with binding driven by the release of condensed counterions previously suggested for native heparin with divalent oligopeptides (Mascotti, D. P.; Lohman, T. M. Biochemistry 1995, 34, 2908-2915). On the other hand, FGF binding displays more complex ionic strength dependence, with higher salt resistance. Remarkably, dp10 that can bind two FGF molecules can only bind one tetralysine. The limited binding of K4 to dp10 suggests that the tetralysine might not block growth factor binding, and the 1:1:1 ternary complex is indeed observed. The analysis of mass distribution of the bound dp10 chains in FGF·dp10, FGF2·dp10, and FGF·dp10·K4 complexes indicated that higher degrees of dp10 sulfation promote the formation of FGF2·dp10 and FGF·dp10·K4. Thus, the selectivity of appropriately chosen short heparin chains could be used to modulate growth factor sequestration and release in a way not feasible with heterogeneous native heparin. In support of this, human hepatocellular carcinoma cells (HEP3Bs) treated with FGF·dp10·K4 were found to exhibit biological activity similar to cells treated with FGF.

PEG-phosphorylcholine hydrogels as tunable and versatile platforms for mechanobiology.

We report here the synthesis of a new class of hydrogels with an extremely wide range of mechanical properties suitable for cell studies. Mechanobiology has emerged as an important field in bioengineering, in part due to the development of synthetic polymer gels and fibrous protein biomaterials to control and quantify how cells sense and respond to mechanical forces in their microenvironment. To address the problem of limited availability of biomaterials, in terms of both mechanical range and optical clarity, we have prepared hydrogels that combine poly(ethylene glycol) (PEG) and phosphorylcholine (PC) zwitterions. Our goal was to create a hydrogel platform that exceeds the range of Young's moduli reported for similar hydrogels, while being simple to synthesize and manipulate. The Young's modulus of these "PEG-PC" hydrogels can be tuned over 4 orders of magnitude, much greater than commonly used hydrogels such as PEG-diacrylate, PEG-dimethacrylate, and polyacrylamide, with smaller average mesh sizes and optical clarity. We prepared PEG-PC hydrogels to study how substrate mechanical properties influence cell morphology, focal adhesion structure, and proliferation across multiple mammalian cell lines, as a proof of concept. These novel PEG-PC biomaterials represent a new and useful class of mechanically tunable hydrogels for mechanobiology.

Engineering the endometrium.

Endometrial tissue is a dynamic environment that regenerates alongside the menstrual cycle and in response to sex hormones. This month in Med, Gnecco and colleagues1 present an innovative, synthetic model of endometrial environment that supports long-term cultures, simulates a 28-day menstrual cycle, and could be employed to investigate reproductive pathophysiology.

Challenges and Opportunities Modeling the Dynamic Tumor Matrisome.

We need novel strategies to target the complexity of cancer and, particularly, of metastatic disease. As an example of this complexity, certain tissues are particularly hospitable environments for metastases, whereas others do not contain fertile microenvironments to support cancer cell growth. Continuing evidence that the extracellular matrix (ECM) of tissues is one of a host of factors necessary to support cancer cell growth at both primary and secondary tissue sites is emerging. Research on cancer metastasis has largely been focused on the molecular adaptations of tumor cells in various cytokine and growth factor environments on 2-dimensional tissue culture polystyrene plates. Intravital imaging, conversely, has transformed our ability to watch, in real time, tumor cell invasion, intravasation, extravasation, and growth. Because the interstitial ECM that supports all cells in the tumor microenvironment changes over time scales outside the possible window of typical intravital imaging, bioengineers are continuously developing both simple and sophisticated in vitro controlled environments to study tumor (and other) cell interactions with this matrix. In this perspective, we focus on the cellular unit responsible for upholding the pathologic homeostasis of tumor-bearing organs, cancer-associated fibroblasts (CAFs), and their self-generated ECM. The latter, together with tumoral and other cell secreted factors, constitute the "tumor matrisome". We share the challenges and opportunities for modeling this dynamic CAF/ECM unit, the tools and techniques available, and how the tumor matrisome is remodeled (e.g., via ECM proteases). We posit that increasing information on tumor matrisome dynamics may lead the field to alternative strategies for personalized medicine outside genomics.