Biology subjects

Vassalli, M.

Publications and source records attributed to Vassalli, M..

13 recordsLinked to original sources

bioRxiv2026

Culture enhanced MSCs to boost haematopoietic engraftment in a model of the post-leukaemic bone marrow niche.

Relapse remains a leading cause of treatment failure in acute myeloid leukaemia (AML), making haematopoietic stem cell transplantation (HSCT) the only curative option for many patients. Yet HSCT efficacy is often limited by impaired engraftment, driven by AML-induced remodelling of the bone marrow stem cell niche. Mesenchymal stromal cells (MSCs) are key mediators of niche formation and could, in principle, restore a supportive microenvironment when introduced alongside HSC therapy; but this strategy remains largely untested. A key obstacle to MSC-based therapy is that MSCs acquire a senescent, pro-inflammatory phenotype during standard in vitro expansion. We addressed this by engineering a polymer-laminin presentation system that suppresses senescence and preserves a proliferative, regenerative MSC phenotype during expansion. Then, to investigate potential cell therapy use, we developed a bioengineered in vitro model as a new approach methodology (NAM) for studying disease-driven niche modification. The system consists of MSC spheroids embedded in a synthetic hydrogel within a transwell platform, allowing controlled co-culture of healthy or AML-derived haematopoietic cells, therapeutic MSCs, and chemotherapeutic agents. Using this platform, we modelled an AML-like niche and showed that MSCs expanded via the polymer-laminin system, when introduced alongside HSCs, significantly improved HSCT engraftment relative to both standard-expanded MSCs and HSCT performed without MSC support. These results establish MSC phenotype maintenance as a critical determinant of therapeutic efficacy, and position this NAM as a platform for pre-clinical screening of niche-targeted therapies in AML.

bioengineering↗

bioRxiv2026

Nucleoli as drivers of nuclear remodelling in cardiomyocytes during Heart Failure

Cardiomyocyte mechanotransduction has traditionally focused on the sarcomere and cytoskeleton, yet emerging evidence highlights the nucleus as an active mechanical responder. To adapt to the dynamic mechanical environment, the nucleus forms nuclear invaginations (NIs), double-membrane folds that provide structural support to chromatin and incorporate nuclear pore complexes to facilitate nucleo-cytoplasmic transport, including Ca2+ transport. However, how these structures are formed is not yet understood. We leveraged advances in high-resolution microscopy, mechanical stimulation, rat models and human samples, to study the formation, function and remodelling of cardiac NIs in Heart Failure (HF). Here, we demonstrate that the formation of NI in cardiomyocytes is regulated by both, cytoskeleton such as actin and detyrosinated microtubules as well as intranuclear nucleolar interactions with NI disruption resulting in elevated baseline nuclear Ca2+. In a 16-week post-Myocardial Infarction (MI) end-stage HF rat model, as well as in human Dilated Cardiomyopathy samples, a marked reduction in NIs was observed. Importantly, NI loss was already evident at 8 weeks post-MI, preceding detectable cytoskeletal stiffening. At this stage, we observed increased DNA damage in the peri-nucleolar region, accompanied by nucleolar remodelling and a shift in nucleolar biomechanical properties. In conclusion, preserving nucleolar integrity emerges as a potential target for intervention.

cell biology↗

bioRxiv2026

Viscoelasticity drives EMT in pancreatic intraepithelial neoplasia

Pancreatic intraepithelial neoplasia (PanIN) is a precursor of pancreatic adenocarcinoma (PDAC) and therefore critical to understand for identifying early-stage diagnostic and therapeutic targets. During PanIN, epithelial-to-mesenchymal transition (EMT) of pancreatic epithelial cancer cells is a crucial event which promotes invasion and early dissemination of cells into circulation before the full development of PDAC tumours. Changes in tissue mechanics are apparent during progression from PanIN to PDAC and increased local and global elasticity has been mathematically modelled in PanIN tissue as a predictive tool for diagnostics and development of personalized therapies. Aside from elasticity, viscoelasticity is emerging as a key feature of cancer which affects tissue mechanics through a combination of elastic and viscous components. Viscoelasticity has recently been shown to drive mechanosensitive cell behaviour and is known to change dramatically in PDAC progression. Hydrogels, as water-swollen polymer networks, are effective extracellular matrix (ECM) models that can recapitulate the viscoelastic properties of natural tissue. Despite this, hydrogels developed for studying cell behaviour in PanIN use purely elastic materials or have neglected the viscous component. Here, using PDAC mouse models, we show that viscoelasticity dynamically alters between healthy and PanIN-bearing tissue and have decoupled the role of elasticity and viscosity during EMT of pancreatic epithelial cancer cells using two-dimensional (2D) polyacrylamide (PAAm) hydrogels. Our work shows viscosity is critical in driving phenotypic changes associated with EMT in a pancreatic epithelial cancer cell line. These findings identify viscosity as an integral component of cell mechanosensing as PanIN develops, which may contribute to initial metastatic events via dissemination from the developing primary tumour. This should be explored further to potentially reveal novel diagnostic and therapeutic targets.

cancer biology↗

bioRxiv2026

Stiffness Drives Endothelial Senescence and Inflammation in Aging and Doxorubicin-Induced Vascular Dysfunction

Age-related arterial stiffening affects over [~] 60% of elderly individuals and is a major independent risk factor for cardiovascular mortality. Similarly, doxorubicin-induced cardiotoxicity affects up to [~] 48% of cancer patients, limiting therapeutic options. Here, we demonstrate that vascular stiffness mechanically amplifies endothelial senescence phenotypes, identifying the extracellular matrix as a potential therapeutic target for both cardiovascular aging and chemotherapy-induced vascular dysfunction. Using polyacrylamide (PAAm) hydrogels to mimick soft (3.3 kPa) and physiological (30 kPa) arterial stiffness, and glass to reproduce pathological stiffening, we show that substrate rigidity enhances senescence markers including {beta}-galactosidase activity, DNA damage, and inflammatory cytokine secretion in both therapy-induced and replicative senescence models. Critically, we identify a protective effect at physiological stiffness, where IL-6 and IL-8 secretion is minimized compared to both softer and stiffer conditions, suggesting an optimal mechanical therapeutic window. RNA sequencing reveals stiffness-dependent activation inflammatory pathways including chemotaxis and leukocyte migration. Our findings position vascular stiffening not just as a consequence but as driver of endothelial dysfunction, creating a positive feedback loop amenable to therapeutic intervention. These mechanobiological insights provide rationale for developing mechanical-based therapies in cardiovascular medicine and cardio-oncology, where targeting tissue mechanics alongside conventional approaches could improve clinical outcomes.

bioengineering↗

bioRxiv2025

Bioengineered bone marrows that can support stem cells and remodel to mimic human disease metabolism and chemotherapeutic effects

Blood cancer drug discovery is reliant on poorly predictive and costly animal models. Therefore, bioengineered, human cell containing, models are attractive to Pharma. Designing effective bone marrow (BM) models is complicated as they need to both regulate haematopoietic stem cell (HSC) phenotype and be able to undergo remodelling to mimic the blood cancer microenvironment. Here, we develop synthetic hybrid niches using poly(ethylacrylate) to organise laminin on hard, bone mimicking, surfaces and interface with soft, marrow mimicking, polyethylene glycol-fibronectin hydrogels. Optimisation of mesenchymal stromal cell (MSC) mechanobiology within the model offers both support for HSCs and remodelling in response to acute myeloid leukaemia derived cells. The remodelling has many parallels to in vivo and patient data including increased dependency on nestin+ve MSCs, enhanced cytoprotection and increased taurine metabolism. We also use the model to demonstrate, as has been seen in vivo, that targeting taurine enhances effects of chemotherapy.

bioengineering↗

bioRxiv2025

Age-associated Tissue Organisation Shifts Retinal Pigment Epithelium Actomyosin Plasticity in Phagocytosis

Tissue homeostasis relies on mechanical feedback loops balanced by cell loss and proliferation. However, maintaining this balance becomes particularly challenging in postmitotic tissues, where alternative mechanisms replace compensatory proliferation. In the postmitotic retinal pigment epithelium (RPE), these mechanisms include significant structural adaptations over time. Yet, how these adaptations relate to epithelial mechanical homeostasis and age-associated functional decline remains poorly understood. To establish the relationship between structural changes, mechanical homeostasis and function, we developed an in vitro reductionistic model mimicking age-related reduction in RPE cell density. Inducing large-scale apoptosis in postmitotic stem cell-derived RPE monolayers recapitulates structural hallmarks of aged tissue, such as reduced cell height, shortened microvilli and cytoskeletal reorganisation. This new structure acquires a new mechanical equilibrium, evidenced by tissue stiffening and enhanced junctional contractility. Functionally, the monolayers display impaired vision-supporting phagocytosis of photoreceptor outer segments. Mechanistically, modulation of actin nucleators, Arp2/3 and formins, demonstrates that apicolateral monolayer deformation is critical for phagocytosis and may be compromised in aged RPE. Our findings suggest that a shift in mechanical homeostasis due to cell loss is a major driver of age-related RPE functional decline. Importantly, we show that structural remodelling in ageing alone can compromise tissue function, independent of other stressors.

cell biology↗

bioRxiv2025

Intestinal Tissue Mechanics Regulate Angiogenesis and Stem Cell Proliferation via Vascular Piezo

The vasculature is a prominent component of developmental and adult tissue microenvironments. How tissue specific characteristics and environmental states influence vascular biology and function, remains largely understudied. Using the fruit fly Drosophila melanogaster midgut and its associated vascular-like tracheal system, we discover reciprocal, mechanochemical interorgan communication between the adult intestine and its vascular microenvironment, which influences vascular and stem cell adaptations during intestinal regeneration. Mechanistically, apoptotic epithelial cells contribute to biomechanical remodelling of the regenerating midgut, which is associated with activation of Piezo in a subset of gut tracheal cells. Piezo drives activation of the mechanosensitive transcription factor Yorkie/YAP, leading to tracheal remodelling and intestinal stem cell proliferation. Furthermore, we identify a conserved role of mammalian vascular Piezo1 influencing remodelling of the intestinal crypt vasculature, and intestinal regeneration upon DNA damage and inflammation in mice. Our cross-species in vivo study reveals the presence of biomechanically distinct compartments in the regenerative intestine and identifies mechanosensory interorgan signalling, which contributes to intestinal regeneration through the vascular-stem cell niche. Furthermore, our work highlights the importance of studying tissue- and context-specific vascular biology to better understand intestinal compartmentalization, plasticity, and the complexity of tissue/vasculature interactions within a living organ.

developmental biology↗

bioRxiv2024

Cryptosporidium Oocyst Wall Proteins are true oocyst wall proteins, with COWP8 functioning to hold the inner and outer layers of the oocyst wall together

Cryptosporidiosis is a significant cause of diarrhoeal disease contributing to substantial morbidity and mortality for the immunocompromised and for young children, especially those who are malnourished. There are no vaccines available and no effective treatments for these patients. Another challenge is that Cryptosporidia are waterborne and resistant to common water treatments including chlorination. Cryptosporidia are transmitted as an oocyst that is made up of a hardy oocyst wall that protects four parasites. Little is understood about how the oocyst is constructed, its composition, and the how it resists chlorination. A family of predicted Cryptosporidium Oocyst Wall Proteins (COWPs) was identified from the genome. Using a genetic approach, we confirm that all members of the COWP family localise to the oocyst wall. Our studies indicate that COWP2, 3 and 4 localise specifically to the oocyst "suture", a zipper-like structure on the oocyst wall from which parasites emerge during infection. In parasites lacking COWP8, we observe that the inner and outer layers of the oocyst wall are no longer associated suggesting a role for COWP8 in oocyst wall morphology. Despite loss of COWP8, these transgenic parasites are viable, unchanged in mechanical strength, and retain resistance to chlorination. This work sets the foundation for future exploration of Cryptosporidium transmission.

microbiology↗

bioRxiv2024

Piezo1 is a mechanosensor of soft matrix viscoelasticity

Mechanosensitive ion channels have emerged as fundamental proteins in sensing extracellular matrix (ECM) mechanics. Among those, Piezo1 has been proposed as a key mechanosensor in cells. However, whether and how Piezo1 senses time-dependent ECM mechanical properties (i.e., viscoelasticity) remains unknown. To address this question, we combined an immortalised mesenchymal stem cell (MSC) line with adjustable Piezo1 expression with soft (400 Pa) and stiff (25 kPa) viscoelastic hydrogels with independently tuneable Youngs modulus and stress relaxation. We demonstrate that Piezo1 is a mechanosensor of viscoelasticity in soft ECMs, consistent with the molecular clutch model. By performing RNA sequencing (RNA-seq), we identified the transcriptomic phenotype of MSCs response to matrix viscoelasticity and Piezo1 activity, highlighting gene signatures that drive MSCs mechanobiology in soft and stiff viscoelastic hydrogels.

biophysics↗

bioRxiv2024

Matrix viscoelasticity controls epithelial cell mechanobiology through dimensionality

In recent years, matrix viscoelasticity has emerged as a potent regulator of fundamental cellular processes and has been implicated in promoting cancer progression. Alongside viscoelasticity, additional ECM cues have been shown to influence migration decision-making of cancer cells, and spatial confinement is now considered as a potential regulator of metastasis. However, our understanding of these complex processes predominantly relies on purely elastic hydrogels, and the exact relationship between matrix viscoelasticity and spatial confinement in driving epithelial cell mechanotransduction and migration during cancer progression remains unclear. Here, we systematically investigated the interplay between matrix stiffness, viscoelasticity and spatial confinement by engineering soft ([~]0.3 kPa) and stiff ([~]3 kPa) polyacrylamide hydrogels with varying degrees of viscous dissipation, mirroring the mechanical properties of healthy and tumoral conditions in breast tissue. We observed that viscoelasticity modulates cell spreading, focal adhesions and YAP nuclear import in opposite directions on soft and stiff substrates. Strikingly, viscoelasticity enhances migration speed and persistence on soft substrates, while impeding them on stiff substrates via actin retrograde flow regulation. Combining soft micropatterning with viscoelastic hydrogels, we also show that spatial confinement restricts cell migration on soft matrices regardless of matrix viscoelasticity and promotes migration on stiff matrices in a viscoelasticity-dependent fashion. Our findings establish substrate viscoelasticity as a key regulator of epithelial cell functions and unravel the role of the matrix dimensionality in this process. SignificanceWhile matrix elasticity has received significant attention, recent findings underscore the importance of its natural dissipative properties and spatial confinement in regulating cellular processes and tumour invasiveness. However, the intricate interplay between viscoelasticity and spatial confinement in orchestrating epithelial cell behaviour during cancer progression remains elusive. Using micropatterned viscoelastic hydrogels to replicate the mechanical properties encountered during breast tumour progression, we unveil that viscoelasticity modulates cell behaviour and mechanotransduction signals differently on soft and stiff substrates. Increased viscoelasticity enhances migration speed and persistence on soft substrates while impeding them on stiff substrates via actin retrograde flow regulation. Furthermore, spatial confinement restricts cell migration on soft matrices regardless of viscoelasticity, while promoting migration on stiff matrices in a viscoelasticity-dependent manner.

biophysics↗

bioRxiv2020

Nanovibrational stimulation of mesenchymal stem cells induces therapeutic reactive oxygen species and inflammation for 3D bone tissue engineering

There is a pressing clinical need to develop cell-based bone therapies due to a lack of viable, autologous bone grafts and a growing demand for bone grafts in musculoskeletal surgery. Such therapies can be tissue engineered and cellular, such as osteoblasts combined with a material scaffold. Because mesenchymal stem cells (MSCs) are both available and fast growing compared to mature osteoblasts, therapies that utilise these progenitor cells are particularly promising. We have developed a nanovibrational bioreactor that can convert MSCs into bone-forming osteoblasts in 2D and 3D but the mechanisms involved in this osteoinduction process remain unclear. Here, to elucidate this mechanism, we use increasing vibrational amplitude, from 30 nm (N30) to 90 nm (N90) amplitudes at 1000 Hz, and assess MSC metabolite, gene and protein changes. These approaches reveal that dose-dependent changes occur in MSCs responses to increased vibrational amplitude, particularly in adhesion and mechanosensitive ion channel expression, and that energetic metabolic pathways are activated, leading to low-level reactive oxygen species (ROS) production and to low-level inflammation, as well as to ROS- and inflammation-balancing pathways. These events are analogous to those that occur in the natural bone-healing processes. We have also developed a tissue engineered MSC-laden scaffold designed using cells mechanical memory, driven by the stronger N90 stimulation. These new mechanistic insights and cell-scaffold design are underpinned by a process that is free of inductive chemicals.

bioengineering↗

bioRxiv2020

The use of nanovibration to discover specific and potent bioactive metabolites that stimulate osteogenic differentiation in mesenchymal stem cells

Bioactive metabolites have wide-ranging biological activities and are a potential source of future research and therapeutic tools. Here, we use nanovibrational stimulation to induce the osteogenic differentiation of MSCs, in the absence of off-target differentiation. We show that this differentiation method, which does not rely on the addition of exogenous growth factors to the culture media, provides an artefact-free approach to identifying bioactive metabolites that specifically and potently induce osteogenesis. We first identify a highly specific metabolite as the endogenous steroid, cholesterol sulphate. Next, a screen of other small molecules with a similar steroid scaffold identified fludrocortisone acetate as being both specific and having highly potent osteogenic-inducing activity. These findings demonstrate that physical priciples can be used to identify bioactive metabolites and then metabolite potency can be optimised by examining structure-function relationship.

bioengineering↗

bioRxiv2019

Disruption of membrane cholesterol organization impairs the concerted activity of PIEZO1 channel clusters

The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment or depletion by methyl-{beta}-Cyclodextrin (MBCD) and disruption of membrane cholesterol organization by dynasore affects PIEZO1-GFPs response to mechanical force. Electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1-GFP pressure-response curve, increased channel latency in response to mechanical stimuli and markedly slowed channel inactivation. The same effects were seen in native PIEZO1 in N2A cells. STORM super-resolution imaging revealed that, at the nano-scale, PIEZO1-GFP channels in the membrane associate as clusters sensitive to membrane manipulation. Both cluster distribution and diffusion rates were affected by treatment with MBCD (5 mM). Supplementation of poly-unsaturated fatty acids appeared to sensitize the PIEZO1-GFP response to applied pressure. Together, our results indicate that PIEZO1 function is directly dependent on the membrane mechanical properties and lateral organization of membrane cholesterol domains which coordinates the concerted activity of PIEZO1 channels.\n\nSUMMARYThe essential mammalian mechanosensitive channel PIEZO1 organizes in the plasma membrane into nanometric clusters which depend on the integrity of cholesterol domains to rapidly detect applied force and especially inactivate syncronously, the most commonly altered feature of PIEZO1 in pathology.

biophysics↗