Peptide-Protein Coassemblies into Hierarchical and Bioactive Tubular Membranes.

Multicomponent self-assembly offers opportunities for the design of complex and functional biomaterials with tunable properties. Here, we demonstrate how minor modifications in the molecular structures of peptide amphiphiles (PAs) and elastin-like recombinamers (ELs) can be used to generate coassembling tubular membranes with distinct structures, properties, and bioactivity. First, by introducing minor modifications in the charge density of PA molecules (PAK2, PAK3, PAK4), different diffusion-reaction processes can be triggered, resulting in distinct membrane microstructures. Second, by combining different types of these PAs prior to their coassembly with ELs, further modifications can be achieved, tuning the structures and properties of the tubular membranes. Finally, by introducing the cell adhesive peptide RGDS in either the PA or EL molecules, it is possible to harness the different diffusion-reaction processes to generate tubular membranes with distinct bioactivities. The study demonstrates the possibility to trigger and achieve minor but crucial differences in coassembling processes and tune material structure and bioactivity. The study demonstrates the possibility to use minor, yet crucial, differences in coassembling processes to tune material structure and bioactivity.

Engineering the cellular mechanical microenvironment - from bulk mechanics to the nanoscale.

The field of mechanobiology studies how mechanical properties of the extracellular matrix (ECM), such as stiffness, and other mechanical stimuli regulate cell behaviour. Recent advancements in the field and the development of novel biomaterials and nanofabrication techniques have enabled researchers to recapitulate the mechanical properties of the microenvironment with an increasing degree of complexity on more biologically relevant dimensions and time scales. In this Review, we discuss different strategies to engineer substrates that mimic the mechanical properties of the ECM and outline how these substrates have been applied to gain further insight into the biomechanical interaction between the cell and its microenvironment.

Syndecan-4 tunes cell mechanics by activating the kindlin-integrin-RhoA pathway.

Extensive research over the past decades has identified integrins to be the primary transmembrane receptors that enable cells to respond to external mechanical cues. We reveal here a mechanism whereby syndecan-4 tunes cell mechanics in response to localized tension via a coordinated mechanochemical signalling response that involves activation of two other receptors: epidermal growth factor receptor and ß1 integrin. Tension on syndecan-4 induces cell-wide activation of the kindlin-2/ß1 integrin/RhoA axis in a PI3K-dependent manner. Furthermore, syndecan-4-mediated tension at the cell-extracellular matrix interface is required for yes-associated protein activation. Extracellular tension on syndecan-4 triggers a conformational change in the cytoplasmic domain, the variable region of which is indispensable for the mechanical adaptation to force, facilitating the assembly of a syndecan-4/α-actinin/F-actin molecular scaffold at the bead adhesion. This mechanotransduction pathway for syndecan-4 should have immediate implications for the broader field of mechanobiology.

GPER is a mechanoregulator of pancreatic stellate cells and the tumor microenvironment.

The mechanical properties of the tumor microenvironment are emerging as attractive targets for the development of therapies. Tamoxifen, an agonist of the G protein-coupled estrogen receptor (GPER), is widely used to treat estrogen-positive breast cancer. Here, we show that tamoxifen mechanically reprograms the tumor microenvironment through a newly identified GPER-mediated mechanism. Tamoxifen inhibits the myofibroblastic differentiation of pancreatic stellate cells (PSCs) in the tumor microenvironment of pancreatic cancer in an acto-myosin-dependent manner via RhoA-mediated contractility, YAP deactivation, and GPER signaling. This hampers the ability of PSCs to remodel the extracellular matrix and to promote cancer cell invasion. Tamoxifen also reduces the recruitment and polarization to the M2 phenotype of tumor-associated macrophages. Our results highlight GPER as a mechanical regulator of the tumor microenvironment that targets the three hallmarks of pancreatic cancer: desmoplasia, inflammation, and immune suppression. The well-established safety of tamoxifen in clinics may offer the possibility to redirect the singular focus of tamoxifen on the cancer cells to the greater tumor microenvironment and lead a new strategy of drug repurposing.

Tamoxifen mechanically reprograms the tumor microenvironment via HIF-1A and reduces cancer cell survival.

The tumor microenvironment is fundamental to cancer progression, and the influence of its mechanical properties is increasingly being appreciated. Tamoxifen has been used for many years to treat estrogen-positive breast cancer. Here we report that tamoxifen regulates the level and activity of collagen cross-linking and degradative enzymes, and hence the organization of the extracellular matrix, via a mechanism involving both the G protein-coupled estrogen receptor (GPER) and hypoxia-inducible factor-1 alpha (HIF-1A). We show that tamoxifen reduces HIF-1A levels by suppressing myosin-dependent contractility and matrix stiffness mechanosensing. Tamoxifen also downregulates hypoxia-regulated genes and increases vascularization in PDAC tissues. Our findings implicate the GPER/HIF-1A axis as a master regulator of peri-tumoral stromal remodeling and the fibrovascular tumor microenvironment and offer a paradigm shift for tamoxifen from a well-established drug in breast cancer hormonal therapy to an alternative candidate for stromal targeting strategies in PDAC and possibly other cancers.

Retinoic Acid Receptor-ß Is Downregulated in Hepatocellular Carcinoma and Cirrhosis and Its Expression Inhibits Myosin-Driven Activation and Durotaxis in Hepatic Stellate Cells.

Hepatic stellate cells (HSCs) are essential perisinusoidal cells in both healthy and diseased liver. HSCs modulate extracellular matrix (ECM) homeostasis when quiescent, but in liver fibrosis, HSCs become activated and promote excess deposition of ECM molecules and tissue stiffening via force generation and mechanosensing. In hepatocellular carcinoma (HCC), activated HSCs infiltrate the stroma and migrate to the tumor core to facilitate paracrine signaling with cancer cells. Because the function of HSCs is known to be modulated by retinoids, we investigated the expression profile of retinoic acid receptor beta (RAR-ß) in patients with cirrhosis and HCC, as well as the effects of RAR-ß activation in HSCs. We found that RAR-ß expression is significantly reduced in cirrhotic and HCC tissues. Using a comprehensive set of biophysical methods combined with cellular and molecular biology, we have elucidated the biomechanical mechanism by which all trans-retinoic acid promotes HSC deactivation via RAR-ß-dependent transcriptional downregulation of myosin light chain 2 expression. Furthermore, this also abrogated mechanically driven migration toward stiffer substrates. Conclusion: Targeting mechanotransduction in HSCs at the transcriptional level may offer therapeutic options for a range of liver diseases.

FAK controls the mechanical activation of YAP, a transcriptional regulator required for durotaxis.

Focal adhesion kinase (FAK) is a key molecule in focal adhesions and regulates fundamental processes in cells such as growth, survival, and migration. FAK is one of the first molecules recruited to focal adhesions in response to external mechanical stimuli and therefore is a pivotal mediator of cell mechanosignaling, and relays these stimuli to other mechanotransducers within the cytoplasm. Yes-associated protein (YAP) has been identified recently as one of these core mechanotransducers. YAP translocates to the nucleus following changes in cell mechanics to promote the expression of genes implicated in motility, apoptosis, proliferation, and organ growth. Here, we show that FAK controls the nuclear translocation and activation of YAP in response to mechanical activation and submit that the YAP-dependent process of durotaxis requires a cell with an asymmetric distribution of active and inactive FAK molecules.-Lachowski, D., Cortes, E., Robinson, B., Rice, A., Rombouts, K., Del Río Hernández, A. E. FAK controls the mechanical activation of YAP, a transcriptional regulator required for durotaxis.

Mechanical and Functional Responses in Astrocytes under Alternating Deformation Modes Using Magneto-Active Substrates.

This work introduces NeoMag, a system designed to enhance cell mechanics assays in substrate deformation studies. NeoMag uses multidomain magneto-active materials to mechanically actuate the substrate, transmitting reversible mechanical cues to cells. The system boasts full flexibility in alternating loading substrate deformation modes, seamlessly adapting to both upright and inverted microscopes. The multidomain substrates facilitate mechanobiology assays on 2D and 3D cultures. The integration of the system with nanoindenters allows for precise evaluation of cellular mechanical properties under varying substrate deformation modes. The system is used to study the impact of substrate deformation on astrocytes, simulating mechanical conditions akin to traumatic brain injury and ischemic stroke. The results reveal local heterogeneous changes in astrocyte stiffness, influenced by the orientation of subcellular regions relative to substrate strain. These stiffness variations, exceeding 50% in stiffening and softening, and local deformations significantly alter calcium dynamics. Furthermore, sustained deformations induce actin network reorganization and activate Piezo1 channels, leading to an initial increase followed by a long-term inhibition of calcium events. Conversely, fast and dynamic deformations transiently activate Piezo1 channels and disrupt the actin network, causing long-term cell softening. These findings unveil mechanical and functional alterations in astrocytes during substrate deformation, illustrating the multiple opportunities this technology offers.

Retinoic acid receptor ß modulates mechanosensing and invasion in pancreatic cancer cells via myosin light chain 2.

Pancreatic ductal adenocarcinoma (PDAC) is the most common and lethal form of pancreatic cancer, characterised by stromal remodelling, elevated matrix stiffness and high metastatic rate. Retinoids, compounds derived from vitamin A, have a history of clinical use in cancer for their anti-proliferative and differentiation effects, and more recently have been explored as anti-stromal therapies in PDAC for their ability to induce mechanical quiescence in cancer associated fibroblasts. Here, we demonstrate that retinoic acid receptor ß (RAR-ß) transcriptionally represses myosin light chain 2 (MLC-2) expression in pancreatic cancer cells. As a key regulatory component of the contractile actomyosin machinery, MLC-2 downregulation results in decreased cytoskeletal stiffness and traction force generation, impaired response to mechanical stimuli via mechanosensing and reduced ability to invade through the basement membrane. This work highlights the potential of retinoids to target the mechanical drivers of pancreatic cancer.

RGD density along with substrate stiffness regulate hPSC hepatocyte functionality through YAP signalling.

Human pluripotent stem cell-derived hepatocytes (hPSC-Heps) may be suitable for treating liver diseases, but differentiation protocols often fail to yield adult-like cells. We hypothesised that replicating healthy liver niche biochemical and biophysical cues would produce hepatocytes with desired metabolic functionality. Using 2D synthetic hydrogels which independently control mechanical properties and biochemical cues, we found that culturing hPSC-Heps on surfaces matching the stiffness of fibrotic liver tissue upregulated expression of genes for RGD-binding integrins, and increased expression of YAP/TAZ and their transcriptional targets. Alternatively, culture on soft, healthy liver-like substrates drove increases in cytochrome p450 activity and ureagenesis. Knockdown of ITGB1 or reducing RGD-motif-containing peptide concentration in stiff hydrogels reduced YAP activity and improved metabolic functionality; however, on soft substrates, reducing RGD concentration had the opposite effect. Furthermore, targeting YAP activity with verteporfin or forskolin increased cytochrome p450 activity, with forskolin dramatically enhancing urea synthesis. hPSC-Heps could also be successfully encapsulated within RGD peptide-containing hydrogels without negatively impacting hepatic functionality, and compared to 2D cultures, 3D cultured hPSC-Heps secreted significantly less fetal liver-associated alpha-fetoprotein, suggesting furthered differentiation. Our platform overcomes technical hurdles in replicating the liver niche, and allowed us to identify a role for YAP/TAZ-mediated mechanosensing in hPSC-Hep differentiation.

Substrate Stiffness-Driven Membrane Tension Modulates Vesicular Trafficking via Caveolin-1.

Liver fibrosis, a condition characterized by extensive deposition and cross-linking of extracellular matrix (ECM) proteins, is idiosyncratic in cases of chronic liver injury. The dysregulation of ECM remodeling by hepatic stellate cells (HSCs), the main mediators of fibrosis, results in an elevated ECM stiffness that drives the development of chronic liver disease such as cirrhosis and hepatocellular carcinoma. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is a key element in the regulation of ECM remodeling, which modulates the degradation and turnover of ECM components. We have previously reported that a rigid, fibrotic-like substrate can impact TIMP-1 expression at the protein level in HSCs without altering its mRNA expression. While HSCs are known to be highly susceptible to mechanical stimuli, the mechanisms through which mechanical cues regulate TIMP-1 at the post-translational level remain unclear. Here, we show a mechanism of regulation of plasma membrane tension by matrix stiffness. We found that this effect is orchestrated by the ß1 integrin/RhoA axis and results in elevated exocytosis and secretion of TIMP-1 in a caveolin-1- and dynamin-2-dependent manner. We then show that TIMP-1 and caveolin-1 expression increases in cirrhosis and hepatocellular carcinoma. These conditions are associated with fibrosis, and this effect can be recapitulated in 3D fibrosis models consisting of hepatic stellate cells encapsulated in a self-assembling polypeptide hydrogel. This work positions stiffness-dependent membrane tension as a key regulator of enzyme secretion and function and a potential target for therapeutic strategies that aim at modulating ECM remodeling in chronic liver disease.

Self-Assembling Polypeptide Hydrogels as a Platform to Recapitulate the Tumor Microenvironment.

The tumor microenvironment plays a critical role in modulating cancer cell migration, metabolism, and malignancy, thus, highlighting the need to develop in vitro culture systems that can recapitulate its abnormal properties. While a variety of stiffness-tunable biomaterials, reviewed here, have been developed to mimic the rigidity of the tumor extracellular matrix, culture systems that can recapitulate the broader extracellular context of the tumor microenvironment (including pH and temperature) remain comparably unexplored, partially due to the difficulty in independently tuning these parameters. Here, we investigate a self-assembled polypeptide network hydrogel as a cell culture platform and demonstrate that the culture parameters, including the substrate stiffness, extracellular pH and temperature, can be independently controlled. We then use this biomaterial as a cell culture substrate to assess the effect of stiffness, pH and temperature on Suit2 cells, a pancreatic cancer cell line, and demonstrate that these microenvironmental factors can regulate two critical transcription factors in cancer: yes-associated protein 1 (YAP) and hypoxia inducible factor (HIF-1A).

G Protein-Coupled Estrogen Receptor Regulates Actin Cytoskeleton Dynamics to Impair Cell Polarization.

Mechanical forces regulate cell functions through multiple pathways. G protein-coupled estrogen receptor (GPER) is a seven-transmembrane receptor that is ubiquitously expressed across tissues and mediates the acute cellular response to estrogens. Here, we demonstrate an unidentified role of GPER as a cellular mechanoregulator. G protein-coupled estrogen receptor signaling controls the assembly of stress fibers, the dynamics of the associated focal adhesions, and cell polarization via RhoA GTPase (RhoA). G protein-coupled estrogen receptor activation inhibits F-actin polymerization and subsequently triggers a negative feedback that transcriptionally suppresses the expression of monomeric G-actin. Given the broad expression of GPER and the range of cytoskeletal changes modulated by this receptor, our findings position GPER as a key player in mechanotransduction.

The Planar Polarity Component VANGL2 Is a Key Regulator of Mechanosignaling.

VANGL2 is a component of the planar cell polarity (PCP) pathway, which regulates tissue polarity and patterning. The Vangl2 Lp mutation causes lung branching defects due to dysfunctional actomyosin-driven morphogenesis. Since the actomyosin network regulates cell mechanics, we speculated that mechanosignaling could be impaired when VANGL2 is disrupted. Here, we used live-imaging of precision-cut lung slices (PCLS) from Vangl2 Lp/+ mice to determine that alveologenesis is attenuated as a result of impaired epithelial cell migration. Vangl2 Lp/+ tracheal epithelial cells (TECs) and alveolar epithelial cells (AECs) exhibited highly disrupted actomyosin networks and focal adhesions (FAs). Functional assessment of cellular forces confirmed impaired traction force generation in Vangl2 Lp/+ TECs. YAP signaling in Vangl2 Lp airway epithelium was reduced, consistent with a role for VANGL2 in mechanotransduction. Furthermore, activation of RhoA signaling restored actomyosin organization in Vangl2 Lp/+ , confirming RhoA as an effector of VANGL2. This study identifies a pivotal role for VANGL2 in mechanosignaling, which underlies the key role of the PCP pathway in tissue morphogenesis.

GPER Activation Inhibits Cancer Cell Mechanotransduction and Basement Membrane Invasion via RhoA.

The invasive properties of cancer cells are intimately linked to their mechanical phenotype, which can be regulated by intracellular biochemical signalling. Cell contractility, induced by mechanotransduction of a stiff fibrotic matrix, and the epithelial-mesenchymal transition (EMT) promote invasion. Metastasis involves cells pushing through the basement membrane into the stroma-both of which are altered in composition with cancer progression. Agonists of the G protein-coupled oestrogen receptor (GPER), such as tamoxifen, have been largely used in the clinic, and interest in GPER, which is abundantly expressed in tissues, has greatly increased despite a lack of understanding regarding the mechanisms which promote its multiple effects. Here, we show that specific activation of GPER inhibits EMT, mechanotransduction and cell contractility in cancer cells via the GTPase Ras homolog family member A (RhoA). We further show that GPER activation inhibits invasion through an in vitro basement membrane mimic, similar in structure to the pancreatic basement membrane that we reveal as an asymmetric bilayer, which differs in composition between healthy and cancer patients.

Where No Hand Has Gone Before: Probing Mechanobiology at the Cellular Level.

Physical forces and other mechanical stimuli are fundamental regulators of cell behavior and function. Cells are also biomechanically competent: they generate forces to migrate, contract, remodel, and sense their environment. As the knowledge of the mechanisms of mechanobiology increases, the need to resolve and probe increasingly small scales calls for novel technologies to mechanically manipulate cells, examine forces exerted by cells, and characterize cellular biomechanics. Here, we review novel methods to quantify cellular force generation, measure cell mechanical properties, and exert localized piconewton and nanonewton forces on cells, receptors, and proteins. The combination of these technologies will provide further insight on the effect of mechanical stimuli on cells and the mechanisms that convert these stimuli into biochemical and biomechanical activity.

Tamoxifen mechanically deactivates hepatic stellate cells via the G protein-coupled estrogen receptor.

Tamoxifen has been used for many years to target estrogen receptor signalling in breast cancer cells. Tamoxifen is also an agonist of the G protein-coupled estrogen receptor (GPER), a GPCR ubiquitously expressed in tissues that mediates the acute response to estrogens. Here we report that tamoxifen promotes mechanical quiescence in hepatic stellate cells (HSCs), stromal fibroblast-like cells whose activation triggers and perpetuates liver fibrosis in hepatocellular carcinomas. This mechanical deactivation is mediated by the GPER/RhoA/myosin axis and induces YAP deactivation. We report that tamoxifen decreases the levels of hypoxia-inducible factor-1 alpha (HIF-1α) and the synthesis of extracellular matrix proteins through a mechanical mechanism that involves actomyosin-dependent contractility and mechanosensing of tissue stiffness. Our results implicate GPER-mediated estrogen signalling in the mechanosensory-driven activation of HSCs and put forward estrogenic signalling as an option for mechanical reprogramming of myofibroblast-like cells in the tumour microenvironment. Tamoxifen, with half a century of safe clinical use, might lead this strategy of drug repositioning.

Chemically Functionalised Graphene FET Biosensor for the Label-free Sensing of Exosomes.

A graphene field-effect transistor (gFET) was non-covalently functionalised with 1-pyrenebutyric acid N-hydroxysuccinimide ester and conjugated with anti-CD63 antibodies for the label-free detection of exosomes. Using a microfluidic channel, part of a graphene film was exposed to solution. The change in electrical properties of the exposed graphene created an additional minimum alongside the original Dirac point in the drain-source current (Ids) - back-gate voltage (Vg) curve. When phosphate buffered saline (PBS) was present in the channel, the additional minimum was present at a Vg lower than the original Dirac point and shifted with time when exosomes were introduced into the channel. This shift of the minimum from the PBS reference point reached saturation after 30 minutes and was observed for multiple exosome concentrations. Upon conjugation with an isotype control, sensor response to the highest concentration of exosomes was negligible in comparison to that with anti-CD63 antibody, indicating that the functionalised gFET can specifically detect exosomes at least down to 0.1 µg/mL and is sensitive to concentration. Such a gFET biosensor has not been used before for exosome sensing and could be an effective tool for the liquid-biopsy detection of exosomes as biomarkers for early-stage identification of diseases such as cancer.

Cost-effective rapid prototyping and assembly of poly(methyl methacrylate) microfluidic devices.

The difficulty in translating conventional microfluidics from laboratory prototypes to commercial products has shifted research efforts towards thermoplastic materials for their higher translational potential and amenability to industrial manufacturing. Here, we present an accessible method to fabricate and assemble polymethyl methacrylate (PMMA) microfluidic devices in a "mask-less" and cost-effective manner that can be applied to manufacture a wide range of designs due to its versatility. Laser micromachining offers high flexibility in channel dimensions and morphology by controlling the laser properties, while our two-step surface treatment based on exposure to acetone vapour and low-temperature annealing enables improvement of the surface quality without deformation of the device. Finally, we demonstrate a capillarity-driven adhesive delivery bonding method that can produce an effective seal between PMMA devices and a variety of substrates, including glass, silicon and LiNbO3. We illustrate the potential of this technique with two microfluidic devices, an H-filter and a droplet generator. The technique proposed here offers a low entry barrier for the rapid prototyping of thermoplastic microfluidics, enabling iterative design for laboratories without access to conventional microfabrication equipment.

Liquid biopsies for management of pancreatic cancer.

Pancreatic cancer is one of the main causes of cancer-related deaths worldwide. It is asymptomatic at an early stage, and most diagnosis occurs when the disease is already at a late stage, by which time the tumor is nonresectable. In order to increase the overall survival of patients with pancreatic cancer, as well as to decrease the cancer burden, it is necessary to perform early diagnosis, prognosis stratifications and cancer monitoring using accurate, minimally invasive, and cost-effective methods. Liquid biopsies seek to detect tumor-associated biomarkers in a variety of extractable body fluids and can help to monitor treatment response and disease progression, and even predict patient outcome. In patients with pancreatic cancer, tumor-derived materials, primarily circulating tumor DNA, circulating tumor cells and exosomes, are being studied for inclusion in the management of the disease. This review focuses on describing the biology of these biomarkers, methods for their enrichment and detection, as well as their potential for clinical application. Moreover, we discuss the future direction of liquid biopsies and introduce how they can be exploited toward point of care personalized medicine for the management of pancreatic cancer.