Advanced Biology

Circadian rhythms are critical for maintaining homeostasis and regulating physiological functions, and consequentially impact disease progression; yet, they remain largely overlooked in in vitro models used in preclinical research. One major barrier to rigorously testing the role of circadian rhythms in these models is the lack of accessible tools that seamlessly integrate into standard culture setups and are capable of sustainably delivering time cues to cells and tissues in long term experiments. Here, we present the ThermoClock, a low-cost, Arduino-based automated temperature control system capable of delivering independent temperature programs to multiple cultures simultaneously. Using circadian reporter U2OS cell lines (Bmal1:Luc and Per2:Luc), we demonstrated that ThermoClock-driven temperature cycles (36{degrees}C/38.5{degrees}C, 12h:12h) produced significantly higher amplitude entrainment than a programmable incubator delivering identical temperature trajectories, suggesting that the ramp time to setpoint is a critical determinant of entrainment strength. We further applied ThermoClock to skin explants from keratinocyte-specific Dbp:Luc reporter mice, showing that circadian temperature cycles (T24: 12h:12h and T25: 12.5h:12.5h) extended synchronized circadian rhythms ex vivo, while a shortened T-cycle (T20: 10h:10h) induced rhythm disruptions. We also observed reduced cell migration in T20 temperature-entrained explants wounded ex vivo, closely recapitulating attenuated wound healing observed in T20 light-cycle-disrupted mice in vivo. Finally, we show that wounding can act as a phase-resetting cue but its efficacy depends on pre-injury entrainment state, with circadian entrained tissues (T25) resisting reset, while disrupted (T20) and unentrained tissues showed resetting sensitivity. These findings establish ThermoClock as a versatile platform for incorporating circadian regulation and, for the first time, disruption into 2D and 3D in vitro systems and demonstrate that peripheral clock disruption and its functional consequences can be modeled ex vivo.

High content imaging is being applied to achieve quantitative fluorescence readouts in increasingly complex 3-dimensional (3D) cell culture models such as spheroids and organoids. Compared to conventional 2D assays, 3D assays better represent biological heterogeneity but require more complex sample preparation, 3D imaging and 3D image analysis that can affect the accuracy and precision of such assays. We used spheroids formed from the NRAS-activated melanoma cell line 19161 modified to express an ERK kinase translocation reporter (KTR) as an exemplar 3D phenotypic assay carried out in 96-well plates. The spheroids were treated with the ERK activator TPA and a range of concentrations of the MEK inhibitor Binimetinib. 3D live-cell imaging with sub-cellular spatial resolution was performed using a dual-view oblique plane microscope (dOPM) - a form of single-objective light-sheet microscope - and the experiment was performed separately at 4 different institutes. The results were analysed using an identical 3D analysis pipeline and parameters. We assessed the variation in assay readout using a linear mixed effects model. Random variance at the well level was negligible (SD = 0.0048 relative to range of KTR biosensor readout at reference site of 0.17), indicating low technical noise. Treatment effects were dose-dependent and highly statistically significant compared to DMSO control across all sites (Dunnett-corrected p < 0.001). The range in KTR readout between the minimum (3.5 M Binimetinib) and maximum (100 nM TPA) treatments varied between 59 to 96% relative to the reference site. Measured bias in KTR readout between sites was between 6 and 12% of the range of the reference site. This study quantifies the reproducibility of a 3D live spheroid-based assay employing a fluorescence biosensor requiring readout out at the per-cell level using the dOPM platform and discusses areas where experimental protocol could be improved in the future to further improve reproducibility.

Muscle satellite cells (SCs) regenerate skeletal muscle, but their regenerative capacity declines with age, in part due to extracellular matrix (ECM) remodeling and aberrant fibroblast activation within the SC niche. In regenerating young mouse muscle, fibronectin remodeling is transient, whereas in aged mouse muscle, fibronectin remodeling is prolonged and disorganized. Fibroblasts in aged mice are activated, increasing fibronectin deposition and expressing elevated -smooth muscle actin (SMA), which negatively influence SC fate. We develop a viscoelastic hydrogel co-encapsulation system, enabling three-dimensional co-culture of intact myofibers with primary fibroblasts. Using this 3D co-culture system, we show that fibroblasts from young mice support SC quiescence and self-renewal, whereas fibroblasts from aged mice aberrantly activate SCs and promote their differentiation on myofibers isolated from either young or aged mice. Knocking down fibronectin (Fn1) in fibroblasts from aged mice partially restores SC function, promoting quiescence and limiting differentiation. Using a novel 3D hydrogel co-culture system, we demonstrate that fibroblast-deposited fibronectin is a key age-associated regulator negatively affecting SC fate within the SC niche of aged mice.

Generation of otic progenitors from pluripotent stem cells requires precise timed regulation of signalling pathways, including bone morphogenetic protein 4 (BMP4). Because endogenous levels of BMP4 varie between cell lines, the optimal concentration of exogenous BMP4 must be determined individually to achieve efficient otic differentiation. Three different human induced pluripotent stem cell lines (hiPSCs) underwent ectodermal differentiation to early otic induction stages in the presence of various concentrations of BMP4 (0-5 ng/ml). Differentiation outcomes were assessed by immunofluorescence staining, and quantitative gene expression analysis. Raman microscopy was used to characterize biochemical differences between hiPSC differentiated cultures exposed to different BMP4 concentration. We observed distinct ectodermal fate were after 8 days of in vitro differentiation depending on BMP4 concentration, including neural, non-neural/otic ectoderm and surface epidermal fates. The proportion of PAX2-otic progenitors varied substantially between cell lines and culture conditions, ranging from approximately 9% to 77%. Raman spectroscopy revealed concentration dependent spectral differences and enabled discrimination between differentiating condition within individual hiPSC lines. Analysis of Raman spectral features indicated differences in nucleic acid, lipid, protein, and collagen associated signatures across culture conditions and cell lines. These findings demonstrate that Raman microscopy provides a non-destructive, label-free method for monitoring molecular changes associated with early otic differentiation. By complementing conventional molecular and immunocytochemical analyses, Raman spectroscopy offers a valuable tool for optimizing BMP4-mediated otic induction protocols and improving the reproducibility of stem cell-based strategies for inner ear research and regenerative medicine.

Cell death is a critical process involved in physiological and pathological conditions, including neurodegenerative diseases and cancer. This study explores the use of optogenetic techniques to induce cell death by employing light sensitive proteins. By manipulating mitochondrial function with light-sensitive proteins, we investigated three distinct strategies: 1) inhibiting oxidative phosphorylation through Gloeobacter rhodopsin-mediated alkalization, 2) inducing mitochondrial depolarization with reverse proton-pumping rhodopsins (RPPR) and anion-conducting channelrhodopsins, and 3) generating reactive oxygen species (ROS) using mitochondria-targeted miniSOG. Our findings highlight the potential of optogenetic approaches to induce cell death, offering promising avenues for therapeutic interventions in diseases characterized by aberrant cell survival.

Regulated keratinocyte differentiation is required for formation of the stratified epidermis and a functional barrier. Understanding genetic drivers of keratinocyte differentiation is crucial for understanding several skin diseases. Perturb-seq is a single-cell CRISPR screen that measures transcriptomic responses to perturbations. To date, Perturb-seq experiments have principally focused on 2-dimensional cell culture models lacking hallmarks of skin development - physiological desmosome formation and barrier function. Here, we leverage Perturb-seq in an epidermal organoid model that recapitulates physiologically relevant differentiation programs. We demonstrate that our perturbations significantly impact diverse differentiation programs and reveal bidirectional function of non-canonical NF-{kappa}B signaling in late keratinocyte differentiation.

Different in vitro models are widely used as experimental platforms to assess neuronal responses to metabolic stress and test potential treatments for patients with ischemic stroke. Results of those studies depend on the stress models used, and the link between cell viability-based readouts and electrophysiological activity remains poorly explored. We investigated the neuronal network activity of human-derived neuronal networks generated from human induced pluripotent stem cells (hiPSCs) under three commonly used metabolic stress models: hypoxia alone, oxygen and glucose deprivation (OGD), and hypoxia combined with different concentrations of glutamate. We aim to clarify the differences between three commonly used in vitro models, including the relation between microscopic and electrophysiological readouts. These conditions produced distinct effects on neuronal network activity. Hypoxia alone induced a progressive decline in activity over time. In contrast, OGD triggered a biphasic response, characterized by an early increase in activity followed by a decline. High concentration glutamate exposure under hypoxia also altered network dynamics, inducing a triphasic pattern consisting of a rapid activity decrease, a transient increase, and a subsequent decline. Across all these pathological conditions, neuronal activity progressively declined and converged toward network failure after prolonged hypoxia. Following reoxygenation, recovery was limited and condition-dependent: hypoxia alone, OGD, and high glutamate conditions showed limited recovery. On the other hand, low glutamate concentration was associated with good recovery. Microscopic assessment revealed that cellular viability was differentially affected across conditions. OGD was associated with the highest levels of cell death, whereas glutamate exposure, particularly at high concentrations, led to a marked reduction in synaptic puncta despite partial preservation of cell viability. These findings highlight that commonly used in vitro ischemia models induce distinct neuronal responses and highlight the importance of integrating electrophysiological and structural analyses to better characterize metabolic stress in human neuronal networks better.

A cnidarian photosymbiont alga, Breviolum minutum, is an emerging model to study symbiosis due its ability to colonise host in absence of light, and amenability to genetic and physiological manipulations. This alga undergoes subcellular reorganisation in response to stress conditions such as elevated temperature and nutrient deprivation. However, subcellular visualisation of this alga is challenging because of its broad spectrum autofluorescence (400-700 nm) and relatively small size (6-8 m). We developed a super resolution imaging, Expansion Microscopy (ExM) workflow - a hydrogel-based technique for mechanical enlargement of cells, that reveals previously inaccessible subcellular features in B. minutum. This ExM workflow presents a set of thermic and enzymatic conditions which enables 4-fold expansion of B. minutum, optical clearing of autofluorescence as well as the removal of its thick cellulose rich cell wall. We implemented a recently described platinum (II)-based tri-functional linker 1, to retain in situ hybridised oligonucleotides targeted to 18S rRNA within ExM hydrogel and exploited its azide reactive group for post-ExM fluorophore labelling (DBCO modification). We observed actin patches (a cytoskeletal feature) and calmodulin (a calcium binding signalling protein) that are not previously visualised in B. minutum. This approach overcomes some of the long-standing problems in visualisation of B. minutum using commonly available reagents and commercially available low-cost ExM compatible chemistries. The broader uptake of this tool for the visualisation of diverse species of photosymbionts will pave the way for fundamental discoveries underpinning cellular reorganisation in formation and breakdown of symbiosis.

Monitoring pH and extracellular acidification rate (ECA) in biological samples containing live mammalian cells can provide valuable information on the glycolytic activity and bioenergetic status of cells. Compared to pH electrodes, optochemical pH sensors look more advantageous, since they allow rapid, non-invasive parallel analysis of multiple samples with stable readout of pH. We have developed new fluorescent pH sensors based on hydrophobic protonable metal-free porphyrins,OEP and OEPK, embedded in a plasticized PVC matrix containing a proton transfer agent. These pH sensors provide internally-referenced calibration-free operation, both in ratiometric intensity and lifetime-based detection modes. Sensor development included optimization of the indicator dye and its photophysical characteristics, screening of different proton transfer agents to minimize sensor toxicity, tuning of the protonation range and pKa, long-term storage stability and response time studies. Optimised pH sensor coatings were then deposited on plastic substrates (96-well microplates) and used for real-time monitoring of Extracellular Acidification Rate (ECAR) for cultured cancer cells and 3D spheroid structures on standard laboratory equipment (multi-label plate reader and confocal FLIM microscope). The advanced pH sensors tailored for use with biological samples have high potential for cell analysis and related applications.

Introduction No widely adopted guidelines exist for collecting and reporting donor-level metadata in tissue-based research, limiting interpretability, reproducibility, and potentially introducing bias. This study aimed to inform ethical and appropriate metadata practices. Methods Semi-structured interviews were conducted with 16 investigators from the Human BioMolecular Atlas Program. Thematic analysis using inductively derived codes identified metadata elements and perspectives on their collection and reporting. Results Participants identified 80 metadata variables across six domains: demographic, sociodemographic, medical history, personally identifying information, cause of death, and tissue/organ data. Most supported routine collection and reporting of demographics and medical history, whereas views on cause of death and sociodemographic data were mixed. Conclusion We recommend routinely collecting and reporting demographics and medical history, while restricting cause of death and sociodemographic variables to situations with explicit consent or justification. These findings provide initial evidence to inform ethical donor metadata guidelines, with further stakeholder engagement and consensus-building needed.

Accurate evaluation of extracellular matrix (ECM) mineralization at the nano-scale is essential for establishing relevant in vitro bone models. This is particularly important with the development and increased application of three-dimensional (3D) cell models for biological research. Transmission electron microscopy (TEM) allows to perform ultra-structural analysis of cells and ECM organization, but its application in in vitro bone models remains limited, due to the potential alteration or loss of the mineral phase during sample preparation. In this study, we compared two TEM sample preparation methods - the conventional chemical fixation and the anhydrous methods - to evaluate their ability to preserve the mineralized ECM in MC3T3-E1 cells cultured as monolayers and as alginate-encapsulated bone spheroids. Chemical fixation preserved cellular ultra-structure and collagen organization, allowing for detailed assessment of cells and ECM organization. Although mineral deposits were detected and their needle-like morphology assessed, characterization of more immature deposits was partially limited by the effects of uranyl acetate and the overall sample preparation process, which could lead to alteration or loss of less stable mineral phases. The anhydrous preparation method resulted in limited preservation of cellular and ECM morphology and did not allow reliable identification of mineral deposits. When applied to spheroids, the chemical fixation method preserved the 3D architecture, collagen-rich ECM and inner mineral deposits, confirming spheroids as a relevant model for bone studies. Overall, these results highlight the need for optimized sample preparation strategies that preserve both ultra-structure and mineral components for accurate nano-scale characterization of bone mineralization.

Human skeletal muscle is the principal site of insulin-stimulated glucose disposal and a major mediator of exercise-induced metabolic benefits, yet human models that preserve metabolic and exercise responsiveness remain limited. We generated primary human skeletal muscle organoids from donor-derived CD56+ myoblasts using a collagen-based extracellular matrix and serum-free IGF1-guided differentiation. The organoids formed aligned contractile tissues containing oxidative and glycolytic fiber type-like myotubes, displayed enhanced mitochondrial respiration, insulin-stimulated glucose uptake, and reproducible force generation. Electrical pulse stimulation induced AMPK activation, increased glucose utilization and lactate production, and upregulated canonical exercise-responsive genes including NR4A3 and PPARGC1A. Notably, transcriptional responses to in vitro exercise overlapped with acute exercise responses observed in skeletal muscle biopsies from the same donors. The organoids further detected functional impairments of skeletal muscle performance induced by TGF-{beta}1 and metformin and increased speed generation by testosterone treatment. These findings establish a donor-specific human skeletal muscle platform that recapitulates key features of insulin action and exercise adaptation and may enable mechanistic studies of skeletal muscle metabolism, exercise responsiveness, and therapeutic interventions relevant to diabetes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/735246v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@2ea1c9org.highwire.dtl.DTLVardef@17fa8c1org.highwire.dtl.DTLVardef@2045d5org.highwire.dtl.DTLVardef@c8b059_HPS_FORMAT_FIGEXP M_FIG C_FIG Article highlightsWe generated primary human skeletal muscle organoids under serum-free IGF1-guided conditions to reproduce key metabolic and exercise-responsive features of skeletal muscle. The organoids were insulin-responsive, displayed enhanced mitochondrial function and force-generating contractility, reproduced hallmark molecular and metabolic responses to exercise, overlapping with exercise responses observed in the same donors in vivo. The organoids were suitable to detect functional alterations after treatment with endogenous hormones and cytokines and diabetes medication This platform provides a human donor-specific system for studying skeletal muscle mechanisms underlying insulin sensitivity, exercise benefits, and therapeutic responses relevant to diabetes and metabolic disease.

Photobiomodulation (PBM) is a therapeutic approach based on illumination with red or near-infrared (NIR) light. Cytochrome c oxidase (COX), a terminal enzyme of the mitochondrial respiratory chain, contains copper centers (CuA and CuB) that absorb light within the red and NIR spectral range, making it a potential primary photoacceptor at wavelengths around 820 nm. PBM appears to be a promising strategy for the treatment and prevention of neurological disorders. Elucidating its precise molecular mechanisms may help optimize therapeutic outcomes. Using patch-clamp method, we showed that illumination with 820 nm light activates mitochondrial large-conductance calcium-activated potassium (mitoBKCa) channels in rat hippocampal mitochondria. Moreover, 820 nm light caused neuroprotective effect in NMDA-treated organotypic hippocampal cultures. Consistently, activation of mitoBKCa channel by 820 nm light illumination was observed in mitochondria isolated from glioma U-87 MG cells. To further investigate the role of mitoBKCa channel, we used CRISPR/Cas9- developed U-87 MG cells lacking the -subunit of the BKCa channel (dBK cells). Comparative transcriptomic analysis of illuminated wild-type and dBK cells revealed significant differences in gene expression profiles. In summary, our results show two types of cellular responses to the PBM. An acute effect involving activation of the mitoBKCa channel and a long-term effect associated with extensive transcriptome remodeling. Both mechanisms may contribute to the cytoprotective effect of 820 nm near-infrared light. HighlightsO_LI820 nm light activates hippocampal mitochondrial BKCa channels C_LIO_LI820 nm light induces hippocampal neuroprotection under excitotoxic conditions C_LIO_LI820 nm light causes intensive transcriptome remodeling in glioma cells C_LIO_LIBKCa channels modulate a subset of transcriptomic responses to 820 nm light C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/731043v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@5a5595org.highwire.dtl.DTLVardef@a8ddb2org.highwire.dtl.DTLVardef@72ec20org.highwire.dtl.DTLVardef@ec46da_HPS_FORMAT_FIGEXP M_FIG C_FIG

Epithelial cells in Hydra perform an unusual combination of functions: they divide continuously like adult stem cells while simultaneously executing the complex physiological tasks of differentiated epithelia. This challenges the traditional distinction between proliferative stem cells and terminally differentiated tissue, raising the question of how a single cell type integrates these opposing roles. Using electron microscopy, we examined morphological characteristics that define the stem-like and differentiated states of Hydras ectodermal and endodermal epithelial cells. Stemness is reflected by nuclear characteristics of active proliferation, including extensive euchromatin, large nucleoli, and the presence of nuage. However, differentiated epithelial cells exhibit strong apical-basal polarity, various endomembrane compartments for endocytosis and transport, specialized secretion mechanisms, and basal muscle processes with dense-core vesicles implicated in hormonal communication. Cryofixation improved ultrastructure preservation, elucidating the pleiomorphic configurations of complex intracellular channel systems traditionally presenting as singular vacuoles. This may shed new light on possible functions of this compartment. Taken together, Hydra epithelial cells combine ancient stem cell traits with highly specialized differentiated functions. This multifunctionality provides insight into the cellular organization of early-branching animals and suggests that multifunctional epithelia may represent an ancestral condition preceding the strict segregation of stem and differentiated cell lineages in bilaterians.

Mechanotransduction via the actin cytoskeleton is linked to fundamental cellular processes such as morphogenesis, cell division, and motility, requiring the control of tensile forces mediated by the motor protein non-muscle myosin 2 (NM2). Formins such as mDia1 have been shown to elongate actin structures that are under mechanical tension; conversely, mDia1s elongation rates are modulated by the applied force. Despite their relevance at the membrane/cortex interface, reported values for tension in formin-elongated actin filaments stem from theoretical estimates and simulations, but have not been amenable experimentally so far. Thus, we developed a Forster resonance energy transfer (FRET)-based, tension-sensitive probe (mDia1TS) and quantified the measured tension in live U2OS cells using fluorescence lifetime imaging microscopy (FLIM). Through whole-cell ROI analysis we show a short and long lifetime component, reporting an intensity-weighted, averaged lifetime corresponding to [~]3.5 pN. Upon mitogen stimulation of cells using EGF, we show that the tension homeostasis changed significantly, with a measurable increase in tension in the cells periphery and relaxation in its center. Furthermore, the reported average tension relaxed by 2 pN after adding the NM2 inhibitor para-nitroblebbistatin. We utilized siRNA knockdowns of individual NM2 paralogs (NM2-A, NM2-B, or NM2-C) to measure their individual contribution, revealing NM2-A as the main paralog to produce tensile force in this system. Taken together, we demonstrate that mDia1TS is able to directly determine that active mDia1 in cells is under tension, and that subcellular quantification with pN precision is possible. SignificanceDespite the fundamental importance of formins in regulating actin-based processes, reported values for tension in formin-mediated actin structures stem from simulations and theoretical estimates. In this study we developed a FRET-based, tension-sensitive reporter probe for formin mDia1, which we termed mDia1TS. Given the expanding clinical spectrum of DIAPH1/mDia1 mutations, our tool mDia1TS provides a quantitative tool for elucidation of changes in cytoskeletal assemblies.

Senescence human bone marrow mesenchymal stem cells (BMSCs), vulnerable to age-related defects, is poor in tissue regeneration. Cells in bone marrow accumulated senescent contributing to the development of metabolic energy regulation hold prospects for therapeutic advances. This study aimed to evaluate energy metabolic changes in male bone marrow mesenchymal stem cells senescence process. Our research established cell specific surface marker and enzymes expression level changes, as well as ECAR and OCR resonance. Notably, CD14, HLA-DRB1 and CD90 upregulated, glycolysis-related genes are increased, tricarboxylic acid cycle-related genes are decreased. We firstly identified links between time-dependent cell aging process and energy metabolism in BMSCs.

Fluorescent voltage-sensitive dyes (VSDs) enable non-invasive, high-throughput optical measurement of membrane potential in living cells, but the analytical reliability of such measurements depends critically on whether the dye and associated imaging conditions perturb the system under study. Here, we systematically characterise the photophysical performance and cell-perturbing effects of FluoVolt, a widely adopted VSD, across cancer cell lines (GIN31 glioblastoma and SK-MEL-30 melanoma) and primary human macrophages. Photobleaching kinetics were strongly cell-type-dependent, with SK-MEL-30 cells exhibiting complete fluorescence loss within 400 seconds under standard widefield conditions. FluoVolt staining combined with laser excitation caused an approximately 2.5-fold increase in cell detachment relative to unstained controls, and dual-wavelength excitation (488 + 405 nm) reduced GIN31 cell viability by approximately 17.5%. Critically, morphological changes, a transition from elongated to amoeboid-like phenotypes, were detected under staining conditions alone, prior to any laser exposure, indicating baseline dye-induced perturbation independent of phototoxicity. Halving dye concentration and loading time significantly attenuated these effects while preserving measurable fluorescence signal. These findings identify FluoVolt staining and excitation as previously uncharacterised sources of systematic measurement artefact and provide practical, actionable guidance for protocol design, control selection, and data interpretation in optical membrane potential imaging.

Understanding how aging reshapes retinal metabolism requires methods that can resolve molecular and structural changes across the retinas highly organized cellular layers. Here, we applied a nonlinear multimodal imaging platform that integrates fluorescence lifetime imaging microscopy (FLIM), second-harmonic generation (SHG), hyperspectral stimulated Raman scattering (HS-SRS), and deuterium oxide-based stimulated Raman scattering (DO-SRS) to map age-associated metabolic and compositional alterations in young and aged mouse retinas. FLIM analysis of the outer nuclear layer (ONL) revealed increased free NADH and NADPH fractions in aged retinas, consistent with reduced oxidative phosphorylation and enhanced lipid anabolic activity. SHG imaging of the sclera showed pronounced age-related remodeling of collagen organization, including increased fiber density, elevated anisotropy, and the emergence of densely crosslinked bundles in the central sclera. DO-SRS further demonstrated elevated lipid turnover in rod photoreceptor outer segments and the retinal pigment epithelium (RPE) with aging which was confirmed by lipidomic analysis. Complementary HS-SRS analysis revealed reduced triacylglycerol and cholesterol content together with localized sphingosine accumulation in the RPE. Together, these findings provide a spatially resolved view of metabolic remodeling in the aging retina and establish multimodal optical imaging as a powerful framework for studying alterations associated with age-related retinal disease.

High-grade serous carcinoma (HGSOC) is the deadliest subtype of ovarian cancer, characterized by high metastatic rates. HGSOC is typically diagnosed at late stages, and treatment options are limited, resulting in a 60% recurrence rate. HGSOC cells exhibit metabolic plasticity, dynamically shifting between glycolysis and oxidative phosphorylation (OXPHOS) to meet energy demands for tumor progression. To evaluate therapeutic strategies that target metabolic vulnerabilities, we developed a microphysiological system (MPS) that recapitulates the heterogenous cell states and bioenergetic distribution of HGSOC solid tumors. Our platform utilized HGSOC spheroids embedded in a collagen hydrogel that mimics the extracellular matrix to capture tumor progression in the ovary. We used atovaquone (ATO), an FDA-approved OXPHOS inhibitor, to prototype the capabilities of our platform to investigate metabolic plasticity in HGSOC. Treatment with ATO decreased viability and invasion of HGSOC spheroids. Crucially, ATO exhibited no cytotoxicity toward biomimetic blood vessels, preserving their integrity and permeability. Metabolic imaging revealed that ATO induces an oxidative state in the outer region of the spheroids. At the invasive front, ATO disrupted mitochondrial organization, forcing collective cell migration and eventually inducing breakdown of mitochondrial networks. Furthermore, ATO decreased YAP/TAZ pathway activity in the outer region of the spheroid, providing a potential mechanism for hindered cell invasion. Collectively, our data demonstrates that a low-potency OXPHOS inhibitor like ATO can effectively target metabolic plasticity to suppress HGSOC spheroid progression. Overall, this platform successfully recapitulated metabolic heterogeneity and provided a workflow for safely testing other drugs that target cancer metabolism.

Multicellular spheroids better recapitulate native cardiac tissue than two-dimensional systems, preserving cell-cell and cell-matrix interactions and relevant signalling. However, analytical tools for extracting quantitative data from these complex models remain limited. Here, we present an optimised holotomography (HT) workflow for fixed spheroids, enabling label-free quantification of protein concentration and dry mass across conditions. Using a hypoxia-reperfusion injury model to mimic myocardial infarction, HT measurements reveal a statistically significant reduction in the protein concentration of cardioids, reflecting impaired structural integrity and declining viability, subtle changes often missed by conventional approaches. These findings establish HT as a robust, scalable method for quantitative analysis of 3D cardiac models, with direct relevance for disease modelling and preclinical research.