Glioblastoma is an aggressive brain cancer whose cells can switch between different modes of invasion in response to their surroundings, making the disease difficult to predict and treat. How physical forces influence this adaptability remains poorly understood. Here, we used simulated microgravity together with engineered hydrogels that independently control adhesion, degradability, and mechanical properties to test how gravity affects glioblastoma invasion. Microgravity strongly reduced invasion and shifted cells from elongated, protrusive behavior to a more cohesive state. Proteomic analysis showed reduced invasive signaling together with increased cell-matrix and cell-cell adhesion, consistent with a redistribution of contractile forces toward the cell edge. Under normal gravity, blocking CD44, integrin β1, or N-cadherin reduced matrix-dependent invasion. In contrast, under microgravity, inhibiting these same adhesion pathways restored invasion, indicating that microgravity traps cells in an overly adhesive, cohesive state that limits movement rather than motility itself. These findings show that gravity is an important regulator of cancer cell plasticity and reveal a mechanically induced vulnerability in glioblastoma invasion. More broadly, combining defined biomaterials with gravitational modulation provides a new way to study how physical forces shape tumor behavior.

Metastasis is responsible for most cancer-related deaths. However, only a fraction of circulating cancer cells succeed in forming secondary tumours, indicating that adaptive mechanisms during circulation play a part in dissemination. Here, we report that constriction during microcapillary transit triggers reprogramming of melanoma cells to a tumorigenic cancer stem cell-like state. Using a microfluidic device mimicking physiological flow rates and gradual capillary narrowing, we show that compression through narrow channels causes cell and nuclear deformation, rapid chromatin remodelling and increased calcium signalling via mechanosensor PIEZO1. Within minutes, cells upregulate transcripts associated with metabolic reprogramming and metastatic processes. Over time, this results in the stable adoption of a cancer stem cell-like state. Squeezed cells express elevated melanoma stem cell markers, exhibit increased trans-endothelium invasion and display enhanced tumorigenicity in vitro and in vivo. Pharmacological inhibition of PIEZO1 blocks this transition, while activation with Yoda1 induces the stem cell-like state irrespective of constriction. Deletion of PIEZO1 completely abolishes the constriction-induced phenotype. Together, these findings demonstrate that compressive forces during circulation reprogram circulating cancer cells into tumorigenic, stem cell-like states, primed for extravasation and metastatic colonization.

In conditions of microgravity the human body undergoes extensive alterations in physiological function. However, it has proven challenging to determine how these changes are mediated at the molecular and cellular level. Here, we investigated whether ELKIN1, a mechanically activated ion channel, regulates changes in cellular and molecular structures in conditions of simulated microgravity. Deletion of ELKIN1 inhibited the simulated microgravity-induced alterations of cellular structure and attachment. In addition, cells lacking ELKIN1 did not exhibit changes in focal adhesion structures and redistribution of the YAP1 transcription factor in response to simulated microgravity, consistent with wild type cells. Finally, melanoma cell invasion of a collagen gel, from organotypic spheroids, was reduced in simulated microgravity, in an ELKIN1 dependent manner. Thus, the force sensing molecule, ELKIN1, modulates the impact of microgravity at both the molecular and cellular levels, revealing one of the molecular mechanisms that underpins cellular adaptations to conditions of microgravity.

Dendritic cells (DCs) are key players in melanoma immune surveillance, presenting tumor antigens to CD8+ T cells that target tumor cells. However, the interaction between DCs and tumor cells in the melanoma microenvironment is complex, shaped by tissue structures like endothelial vessels and dynamic biological, chemical, and physical factors. The immunosuppressive tumor microenvironment (TME) often hinders DC migration and their ability to initiate an effective anti-tumor response. Combining romidepsin, an epigenetic agent, with type I interferon (IFN), known to enhance DC function and counteract immune suppression, could enhance DC recruitment to the TME, improve tumor-DC interactions, and boost the anti-tumor immune response. Given the complex interactions between melanoma cells and DCs in the TME, organ-on-chip platforms provide a powerful, human-relevant tool for modeling these dynamics and enabling real-time studies of immune responses to drug treatments.In this study, we developed an innovative tumor-on-a-chip device, designed to closely mimic the human TME. The device features a microvascular structure with a fully integrated lumen, lined by endothelial cells that form tight intercellular junctions, ensuring proper barrier functionality. This engineered setup creates a highly physiologically relevant model of tumor growth at the interface of a flow-perfused blood vessel, offering a 3D environment that mimics the spatial, biochemical, and physical conditions found in vivo. Using this model, we concentrated on examining the migration of DCs through the endothelial barrier, a key process in immune response to tumorigenesis, allowing them to reach and interact with melanoma cells. By applying therapeutic treatments to the system, we demonstrated that DCs exhibit enhanced directional migration toward drug-treated versus untreated metastatic melanoma, likely driven by changes in the TME that promote immune cell trafficking. Furthermore, we report the occurrence of persistent interactions between DCs and drug-treated melanoma cells. These sustained interactions drive continuous DC activation, enhancing their ability to present tumor antigens to T cells and fostering a more robust and lasting anti-tumor immune response. Our findings demonstrate that our novel vascularized tumor-on-a-chip is a powerful tool for studying DC transmigration and tumor cell interactions. This model reveals how therapies influence immune cell dynamics in the TME and offers valuable insights into immune cell trafficking, aiding cancer immunotherapy strategies. Lucia Gabriele, Stefania Parlato, Giulia Silvani, Giovanna Peruzzi, Davide Caprini, Giorgia Sinibaldi, Stefania Rossi, Alesandra Fragale, Giulia Romagnoli, Irene Canini, Arianna Mencattini, Eugenio martinelli, Carlo Massimo Casciola. A novel vascularized tumor-on-a-chip model for tracking melanoma-dendritic cell interactions following combined romidepsin and type I IFN therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1209.

With the advancement in reusable rocket propulsion technology, space tourist trips into outer space are now becoming a possibility at a cost-effective rate. As such, astronauts will face a host of health-related challenges, particularly on long-duration space missions where maintaining a balanced healthy microbiome is going to be vital for human survival in space exploration as well as mission success. The human microbiome involves a whole list of micro-organisms that reside in and on the human host, and plays an integral role in keeping the human host healthy. However, imbalances in the microbiome have been directly linked to many human diseases. Research findings have clearly shown that the outer space environment can directly affect the normal microbiome of astronauts when the astronaut is exposed to the microgravity environment. In this study, we show that the simulation of microgravity on earth can mimic the outer space microgravity environment. Staphylococus aureus (S. aureus) was chosen for this study as it is an opportunistic pathogen, which is part of the normal human skin microflora and the nasal passages. This study's results show that S. aureus proliferation was significantly increased under a microgravity environment compared to Earth's gravity conditions, which complements previous work performed on bacteria in the outer space environment in the International Space Station (ISS). This demonstrates that this technology can be utilised here on Earth to mimic the outer space environment and to study challenging health-related questions. This in return saves us the cost on conducting experiments in the ISS and can help advance knowledge at a faster rate and produce countermeasures to mitigate the negative side effects of the hostile outer space environment on humans.

The acoustic force spectroscopy (AFS) tool was recently introduced as a novel tool for probing mechanical properties of biomolecules, expanding the application of sound waves to high-throughput quantification of the mechanical properties of single cells. By using controlled acoustic forces in the piconewton to nanonewton range, tens to hundreds of cells functionalized by attached microspheres can simultaneously be stretched and tracked in real-time with sub millisecond time response. Since its first application, several studies have demonstrated the potential and versatility of the AFS for high-throughput measurements of force-induced molecular mechanisms, revealing insight into cellular biomechanics and mechanobiology at the molecular level. In this chapter, we describe the operation of the AFS starting with the underlying physical principles, followed by a run-down of experimental considerations, and finally leading to applications in molecular and cellular biology.

The advancement of microgravity simulators is helping many researchers better understanding the impact of the mechanically unloaded space environment on cellular function and disfunction. However, performing microgravity experiments on Earth, using simulators such as the Random Positioning Machine, introduces some unique practical challenges, including air bubble formation and leakage of growth medium from tissue culture flask and plates, all of which limit research progress. Here, we developed an easy-to-use hybrid biological platform designed with the precision of 3D printing technologies combined with PDMS microfluidic fabrication processes to facilitate reliable and reproducible microgravity cellular experiments. The system has been characterized for applications in the contest of brain cancer research by exposing glioblastoma and endothelial cells to 24 h of simulated microgravity condition to investigate the triggered mechanosensing pathways involved in cellular adaptation to the new environment. The platform demonstrated compatibility with different biological assays, i.e., proliferation, viability, morphology, protein expression and imaging of molecular structures, showing advantages over the conventional usage of culture flask. Our results indicated that both cell types are susceptible when the gravitational vector is disrupted, confirming the impact that microgravity has on both cancer and healthy cells functionality. In particular, we observed deactivation of Yap-1 molecule in glioblastoma cells and the remodeling of VE-Cadherin junctional protein in endothelial cells. The study provides support for the application of the proposed biological platform for advancing space mechanobiology research, also highlighting perspectives and strategies for developing next generation of brain cancer molecular therapies, including targeted drug delivery strategies.

Glioblastoma multiforme (GBM) is one of the most aggressive malignant brain tumors and urgently requires the development of new therapeutic strategies. In this study, an innovative hybrid in vitro vascularized GBM‐on‐a‐chip model is presented as a strategic integration of microfluidics and 3D bioprinting technologies. The system can recreate the compartmentalized brain tumor microenvironment, comprising the functional blood brain barrier (BBB) and the adjacent 3D perivascular tumor niche, by selectively mimicking physiological shear stress and cell–cell, cell–matrix mechanical interaction. The GBM‐on‐a‐chip model was evaluated under simulated microgravity (µG) condition as a form of mechanical unloading showing a significant cell morphological and mechanotransduction response thereby indicating that gravitational forces play an important role in glioblastoma mechanical regulation. The proposed GBM‐on‐a‐chip represents a meaningful biological tool for further research in cancer mechanobiology and pre‐clinical approach in brain tumor therapy.

Advanced TherapeuticsVolume 4, Issue 11 2170033 Cover PictureFree Access A 3D-Bioprinted Vascularized Glioblastoma-on-a-Chip for Studying the Impact of Simulated Microgravity as a Novel Pre-Clinical Approach in Brain Tumor Therapy (Adv. Therap. 11/2021) Giulia Silvani, Giulia SilvaniSearch for more papers by this authorCarin Basirun, Carin BasirunSearch for more papers by this authorHanjie Wu, Hanjie WuSearch for more papers by this authorChristine Mehner, Christine MehnerSearch for more papers by this authorKate Poole, Kate PooleSearch for more papers by this authorPeta Bradbury, Peta BradburySearch for more papers by this authorJoshua Chou, Joshua ChouSearch for more papers by this author Giulia Silvani, Giulia SilvaniSearch for more papers by this authorCarin Basirun, Carin BasirunSearch for more papers by this authorHanjie Wu, Hanjie WuSearch for more papers by this authorChristine Mehner, Christine MehnerSearch for more papers by this authorKate Poole, Kate PooleSearch for more papers by this authorPeta Bradbury, Peta BradburySearch for more papers by this authorJoshua Chou, Joshua ChouSearch for more papers by this author First published: 18 November 2021 https://doi.org/10.1002/adtp.202170033AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract This cover image represents the bioconvergence between microfluidics, 3D bioprinting, lab-on-chip and microgravity technologies to develop a new approach towards the development and understanding of complex biological microenvironments including brain cancer which is described in this study. The emergence of space mechanobiology has demonstrated potential to unveiling biological mechanisms and disease function. This is reported by Joshua Chou and co-workers in article number 2100106. Volume4, Issue11November 20212170033 RelatedInformation

An endothelial-lined blood vessel model is obtained in a PDMS (Polydimethylsiloxane) microfluidic system, where vascular endothelial cells are grown under physiological shear stress, allowing -like maturation. This experimental model is employed for enhanced drug delivery studies, aimed at characterising the increase in endothelial permeability upon microbubble-enhanced ultrasound-induced (USMB) cavitation. We developed a multi-step protocol to couple the optical and the acoustic set-ups, thanks to a 3D-printed insonation chamber, provided with direct optical access and a support for the US transducer. Cavitation-induced interendothelial gap opening is then analysed using a customised code that quantifies gap area and the relative statistics. We show that exposure to US in presence of microbubbles significantly increases endothelial permeability and that tissue integrity completely recovers within 45 min upon insonation. This protocol, along with the versatility of the microfluidic platform, allows to quantitatively characterise cavitation-induced events for its potential employment in clinics.

Characterizing mechanical properties of cells is important for understanding many cellular processes, such as cell movement, shape, and growth, as well as adaptation to changing environments. In this study, we explore the mechanical properties of endothelial cells that form the biological barrier lining blood vessels, whose dysfunction leads to development of many cardiovascular disorders. Stiffness of living endothelial cells was determined by Acoustic Force Spectroscopy (AFS), by pull parallel multiple functionalized microspheres located at the cell-cell periphery. The unique configuration of the acoustic microfluidic channel allowed us to develop a long-term dynamic culture protocol exposing cells to laminar flow for up to 48 h, with shear stresses in the physiological range (i.e., 6 dyn/cm(2)). Two different Endothelial cells lines, Human Aortic Endothelial Cells (HAECs) and Human Umbilical Vein Endothelial Cells (HUVECs), were investigated to show the potential of this tool to capture the change in cellular mechanical properties during maturation of a confluent endothelial monolayer. Immunofluorescence microscopy was exploited to follow actin filament rearrangement and junction formation over time. For both cell types we found that the application of shear-stress promotes the typical phenotype of a mature endothelium expressing a linear pattern of VE-cadherin at the cell-cell border and actin filament rearrangement along the perimeter of Endothelial cells. A staircase-like sequence of increasing force steps, ranging from 186 pN to 3.5 nN, was then applied in a single measurement revealing the force-dependent apparent stiffness of the membrane cortex in the kPa range. We also found that beads attached to cells cultured under dynamic conditions were harder to displace than cells cultured under static conditions, showing a stiffer membrane cortex at cell periphery. All together these results demonstrate that the AFS can identify changes in cell mechanics based on force measurements of adherent cells under conditions mimicking their native microenvironment, thus revealing the shear stress dependence of the mechanical properties of neighboring endothelial cells.

The rapid progress in mechanobiology has brought together many scientific and engineering disciplines to work hand in hand toward better understanding of the role that mechanical force plays in functioning and evolution of different forms of life. New tools designed by engineers helped to develop new methods and techniques for investigation of mechanical properties of biological cells and tissues. This multidisciplinary approach made it clear that cell mechanics is tightly linked to intracellular signaling pathways, which directly regulate gene expression in response to mechanical stimuli originating outside or inside the cells. Mechanical stimuli act on mechanoreceptors which convert these stimuli into intracellular signals. In this chapter, we review the current knowledge about cell mechanics and the role cell mechanics plays for the function of mechanosensitive ion channels as a special class of mechanoreceptors functioning as molecular transducers of mechanical stimuli on a millisecond timescale.

ABSTRACTCellular processes including adhesion, migration and differentiation are governed by the distinct mechanical properties of each cell. Importantly, the mechanical properties of individual cells can vary depending on local physical and biochemical cues in a time-dependent manner resulting in significant inter-cell heterogeneity. While several different methods have been developed to interrogate the mechanical properties of single cells, throughput to capture this heterogeneity remains an issue. While new high-throughput techniques are slowly emerging, they are primarily aimed at characterizing cells in suspension, whereas high-throughput measurements of adherent cells have proven to be more challenging. Here, we demonstrate single-cell, high-throughput characterization of adherent cells using acoustic force spectroscopy. We demonstrate that cells undergo marked changes in viscoelasticity as a function of temperature, the measurements of which are facilitated by a closed microfluidic culturing environment that can rapidly change temperature between 21 °C and 37 °C. In addition, we show quantitative differences in cells exposed to different pharmacological treatments specifically targeting the membrane-cytoskeleton interface. Further, we utilize the high-throughput format of the AFS to rapidly probe, in excess of 1000 cells, three different cell-lines expressing different levels of a mechanosensitive protein, Piezo1, demonstrating the ability to differentiate between cells based on protein expression levels.

Targeting pharmaceuticals through the endothelial barrier is crucial for drug delivery. In this context, cavitation-assisted permeation shows promise for effective and reversible opening of intercellular junctions. A vessel-on-a-chip is exploited to investigate and quantify the effect of ultrasound-excited microbubbles-stable cavitation-on endothelial integrity. In the vessel-on-a-chip, the endothelial cells form a complete lumen under physiological shear stress, resulting in intercellular junctions that exhibit barrier functionality. Immunofluorescence microscopy is exploited to monitor vascular integrity following vascular endothelial cadherin staining. It is shown that microbubbles amplify the ultrasound effect, leading to the formation of interendothelial gaps that cause barrier permeabilization. The total gap area significantly increases with pressure amplitude compared to the control. Gap opening is fully reversible with gap area distribution returning to the control levels 45 min after insonication. The proposed integrated platform allows for precise and repeatable in vitro measurements of cavitation-enhanced endothelium permeability and shows potential for validating irradiation protocols for in vivo applications.

Traditional drug delivery systems, where pharmaceutical agents are conveyed to the target tissue through the blood circulation, suffer of poor therapeutic efficiency and limited selectivity largely due to the low permeability of the highly specialised biological interface represented by the endothelial layer. Examples concern cancer therapeutics or degenerative disorders where drug delivery is inhibited by the blood-brain barrier (BBB). Microbubbles injected into the bloodstream undergo volume oscillations under localised ultrasound irradiation and possibly collapse near the site of interest, with no effect on the rest of the endothelium. The resulting mechanical action induces a transient increase of the inter-cellular spaces and facilitates drug extravasation. This approach, already pursed in in vivo animal models, is extremely expensive and time-consuming. On the other hand in vitro studies using different kinds of microfluidic networks are firmly established in the pharmaceutical industry for drug delivery testing. The combination of the in vitro approach with ultrasound used to control microbubbles oscillations is expected to provide crucial information for developing cavitation enhanced drug delivery protocols and for screening the properties of the biological interface in presence of healthy or diseased tissues. Purpose of the present review is providing the state of the art in this rapidly growing field where cavitation is exploited as a viable technology to transiently modify the permeability of the biological interface. After describing current in vivo studies, particular emphasis will be placed on illustrating characteristics of micro-devices, biological functionalisation, properties of the artificial endothelium and ultrasound irradiation techniques.

The development of targeted delivery systems releasing pharmaceutical agents directly at the desired site of action may improve their therapeutic efficiency while minimizing damage to healthy tissues, toxicity to the patient and drug waste. In this context, we have developed a bio-inspired microdevice mimicking the tumour microvasculature which represents a valuable tool for assessing the enhancement of blood vessel permeability due to cavitation. This novel system allows us to investigate the effects of ultrasound-driven microbubbles that temporarily open the endothelial intercellular junctions allowing drug to extravasate blood vessels into tumour tissues. The blood vessel on a chip consists of a tissue chamber and two independent vascular channels (width 200 mu m, height 100 mu m, length 2762 mu m) cultured with endothelial cells placed side-by-side and separated by a series of 3 mu m pores. Its geometry and dimensions mimic the three-dimensional morphology, size and flow characteristics of microvessels in vivo. The early stage of this project had a twofold objective: 1. To define the protocol for culturing of Human Umbilical Vein Endothelial Cells (HUVECs) within the vascular channel; 2. To develop a fluorescence based microscopy technique for measuring permeability. We have developed a reliable and reproducible protocol to culture endothelial cells within the artificial vessels in a realistic manner: HUVECs show the typical elongated shape in the direction of flow, exhibit tight junction formation and form a continuous layer with a central lumen that completely covers the channels wall. As expected, the permeability of cell-free device is higher than the one cultured with HUVECs in the vascular channels. The proposed blood vessel on a chip and the permeability measurement protocol have a significant potential to allow for the study of cavitation-enhanced permeability of the endothelium and improve efficiency in screening drug delivery systems.