Integrative Biology

The tumor microenvironment plays a pivotal role in tumor development, harboring elements such as endothelial cells, immune cells, fibroblasts, and soluble factors such as transforming growth factor-{beta} (TGF-{beta}) family. TGF-{beta} family regulates cell development and promotes tumor invasion, metastasis, angiogenesis, and endothelial-to-mesenchymal transition (EndoMT). Here, we investigate the effects of TGF-{beta} signaling on vascular remodeling using a three-dimensional (3D) vascular network in a microfluidic device. Using both a co-culture (3D-Co) and simplified endothelial monoculture (3D-CM), we demonstrate that TGF-{beta} signaling reduces the quality and functionality of the vasculature by regressing them. In addition, we observed the upregulation of EndoMT-related markers in mRNA and protein expressions, suggesting the induction of EndoMT in 3D vascular networks. The increased vascular permeability stimulated by TGF-{beta}2 also supports the loss of endothelial identity in the 3D-Co. Transcriptomic analysis revealed the coordinated activation of pathways associated with cell migration and EndoMT, along with the suppression of cell cycle progression. A comparative analysis of two-dimensional (2D) and 3D cultures revealed a fundamentally distinct endothelial response to TGF-{beta}2 in the 3D context, including metabolic reprogramming. These findings demonstrate that the 3D microenvironment critically modulates endothelial responses to TGF-{beta} and enables the emergence of vascular phenotypes not captured in 2D systems. This study provides a more physiologically relevant platform to investigate endothelial dysfunction and vascular remodeling.

Anti-angiogenic cancer therapies attempt to withhold necessary nutrients and oxygen from growing tumors by targeting the major promoters of endothelial cell (EC) angiogenesis: vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2). Unfortunately, these treatments are often insufficient, even when coupled with chemotherapies, and fail to significantly increase survival rates. The tumor microenvironment (TME) is mechanically distinct compared to normal tissue, including increased matrix deformations or strains caused by cancer-associated fibroblasts (CAFs). In this report, we detail the specific and independent roles of two tyrosine residues, Y1054 and Y1214, on mechanical activation of VEGFR-2. Furthermore, we characterize CAF biochemical and mechanical signaling and demonstrate how ECs exhibit decreased vessel growth when co-cultured with CAFs and treated with a contractility inhibitor. Using non-phosphorylatable VEGFR-2 mutants, we reveal Y1054 and Y1214 are each necessary for EC angiogenesis, particularly in response to strain. Overall, this research highlights the need to study how mechanics in the TME promote vessel growth and thus tumor progression, which is important to consider when developing future anti-angiogenic therapies.

Glioblastoma (GBM), the most common primary brain tumor, is characterized by extensive infiltration into surrounding brain tissue. GBM tumors exhibit substantial intratumoral heterogeneity making it difficult to identify and target invasive cell subpopulations. Here, we use an in vitro model of the mechanical transitions at the tumor-brain interface to isolate highly invasive GBM cells from populations derived from unique patient tumors for downstream transcriptomic analysis or further culture. Using single-cell RNA sequencing combined with cell barcodes we were able to trace distinct cell lineages during migration and identify an intrinsically invasive subpopulation. This invasive subpopulation exhibits a distinct pre-invasive transcriptomic profile characterized by overexpression of galectin-1, a {beta}-galactoside binding protein. Our findings reveal galectin-1 overexpression is an innate characteristic of invasive GBM subpopulations, where expression level positively correlates with invasion rate and inhibition of galectin-1 binding to cell surface glycoproteins effectively prevented migration. While some studies have reported that galetcin-1 aids in cell migration, this study identifies and confirms that galectin-1 expression is a pre-existing characteristic of invading GBM cells and a target to prevent tumor recurrence.

The cortical neuron is a fundamental building block of the mammalian brain, and the morphology of its axonal projections is central to how functional circuits assemble. The trajectory along which an axon grows is a key determinant of connectivity, yet the kinematics of cortical axon outgrowth remain poorly quantified. Characterizing these dynamics is most tractable in vitro, where axonal growth can be measured directly and under controlled, reproducible conditions. Even in culture, however, this remains challenging because cortical neurons require dense plating for viability, and their soma is motile, so growth behavior is highly sensitive to local density and population context, complicating reproducible measurement of intrinsic dynamics. To overcome these limitations, we used size-controlled cortical neurospheres, which provide a fixed spatial origin and a reproducible environment, together with a custom semi-automated tracking pipeline to quantify single-axon kinematics across two functionalized substrates and two developmental phases. This approach revealed a substrate-dependent divergence in outgrowth: during the later developmental phase, axons on poly-D-lysine with laminin (PDL-LA) substrate grew faster than those on PDL, with a mean step size of 0.436 versus 0.339 {micro}m /min. Decomposing trajectories into Katz dynamic states, we built a generative biased random walk model that reproduces axonal behavior at both microscopic (single-axon) and macroscopic (network topology) scales. This open, reproducible framework links single-axon kinematics to network architecture, enabling the structural connectivity of neurospherebased circuits in vitro to be predicted from measurable growth dynamics, a necessary foundation for future studies linking circuit structure to emergent function.

Metastatic dissemination is initiated by tumor cells interpreting spatially organized biochemical and biophysical cues that remain difficult to reproduce using conventional migration assays. Here, we developed a computationally guided metastasis-on-a-chip (MET-on-a-chip) platform based on the concept of the Minimally Functional Unit (MFU), in which only the biological components required to answer a defined experimental question are incorporated. The platform consists of two independent culture chambers connected through an array of confined microchannels that permits diffusion of soluble factors while constraining tumor cell migration. Rather than relying on empirical optimization, finite-element COMSOL simulations were first used to predict molecular transport, define growth factor loading conditions, identify biologically relevant exposure regions, and guide the rational design of the microfluidic assay. Computational predictions were experimentally validated using 70-kDa FITC-dextran diffusion and VEGF release studies, confirming the formation of stable spatial concentration gradients across the microfluidic platform. The simulations further demonstrated that both growth factor loading and cell positioning relative to the predicted gradients critically influenced assay performance, leading to the optimization of the platform through spatial reconfiguration of the tumor compartment. Using the optimized configuration, we compared the migratory responses of neuroblastoma, Ewing sarcoma, and osteosarcoma cells to vascular (VEGF-A165) and lymphatic (VEGF-C) chemotactic cues. VEGF-C significantly increased migration through the microchannel array in Ewing sarcoma and osteosarcoma cells, whereas VEGF-A165 produced no significant effect. In contrast, neuroblastoma cells exhibited minimal migration under either condition, revealing tumor-specific differences in responsiveness to VEGF signaling. Together, these findings establish a computationally guided workflow for the rational design of metastasis-on-a-chip assays, in which predictive modeling informs experimental design before biological validation. By substantially reducing empirical trial-and-error while enabling quantitative control over growth factor exposure, this strategy provides a robust framework for developing minimally functional microphysiological systems capable of dissecting individual steps of the metastatic cascade under experimentally defined conditions. Translational Impact StatementMetastatic dissemination remains one of the greatest clinical challenges in pediatric oncology, yet experimental models capable of quantitatively evaluating early migratory events remain limited. The computationally guided MET-on-a-Chip workflow presented here provides a human-relevant platform in which soluble microenvironmental cues can be systematically investigated under controlled and predictive conditions. Although demonstrated here using VEGF-A165 and VEGF-C, the platform can be readily adapted to study virtually any chemotactic factor, cytokine, extracellular vesicle population, or therapeutic candidate involved in metastatic dissemination. The modular MFU design allows biological complexity to be incorporated progressively as dictated by the scientific question, providing a flexible framework for future applications. In the longer term, this workflow could be combined with patient-derived tumor cells, organoids, or biopsy material to investigate patient-specific metastatic behavior and evaluate anti-metastatic therapeutic strategies in a personalized setting. Beyond identifying pro-migratory signaling pathways, the platform may serve as a preclinical tool to prioritize compounds capable of preventing tumor cell dissemination before evaluation in more complex animal models or clinical studies.

Mesenchymal Stromal Cells (MSC) face several heterogeneity challenges hindering clinical and commercial success. Employing a multiple response model, interplay between donor heterogeneity, and critical processing parameters (CPPs), effects on MSC potency and cell expansion attributes were investigated through computed composite attribute scores. Twelve unique CPP combinations were tested in thirteen marrow-derived MSC(M) and five adipose-tissue MSC(AT) training and test datasets, respectively. Donor heterogeneity and select CPP conditions affected a curated gene panel (surrogate for MSC potency); while MSC expansion was primarily influenced by CPPs. Model performances were evaluated against clinical effectiveness data from a previously deployed clinical trial; top-performing model predicted donor rankings coincided with clinical effectiveness data, validating the modeling approach used. Our model predicted that only 8% of tested donors were agnostic to CPPs; a majority (62%) of donors showed CPP-dependent optimal composite quality attributes, with MSC seeding density as a key driver; medium supplementation and oxygen preferences were highly donor dependent. Approximately 30% of donors performed poorly at all conditions tested and may be prospectively identified using a subset of genes (TGFB, VEGF, PDCD1LG1, PDCD1LG2, IDO). Model predicted optimal parameters worked for 69% of tested donors, while sub-optimal parameters worked for only 23% of donors and were confirmed in an independent CD14+ macrophage assay. Our integrated computational and experimental framework predictably identified interactive effects of donor heterogeneity and CPP conditions to optimize MSC potency attributes.

IntroductionImmune checkpoint inhibitors (ICIs) have only shown limited efficacy for patients with ovarian cancer (OC), partly due to the immune suppressive tumour microenvironment (TME) and platelet cloaking of the cancer cells. We tested a nanomedicine strategy to enhance {gamma}{delta} T cell immunotherapy by conjugating the PD-L1 inhibitor (BMS202) to nanodiamonds (NDs). MethodsPatient-derived ascites cells were exposed to activated platelets to model platelet cloaking and the immunosuppressive phenotype in metastatic OC. ND/BMS202 nanocomplexes were then applied to platelet-conditioned OC cells and co-cultured with expanded {gamma}{delta} T cells. Cytotoxicity and immune activation were assessed by quantifying Granzyme B, CD107a, {gamma}-H2AX, cleaved caspase-3, and cleaved caspase-8. ResultsND-mediated delivery of BMS202 significantly enhanced {gamma}{delta} T cell-mediated killing of platelet cloaked OC cells in a dose-dependent manner, with greater efficacy than free BMS202 This enhanced cytotoxicity was supported by increased degranulation (CD107a), Granzyme B release, tumour cell apoptosis (caspase cleavage) and DNA damage ({gamma}-H2AX staining). ConclusionsND-based delivery of the PD-L1 inhibitor (BMS202) enhances {gamma}{delta} T cell-mediated killing of platelet-cloaked metastatic OC cells. While our data support enhanced {gamma}{delta} T cell cytotoxicity following BMS202 delivery, direct evidence of PD-L1 target engagement or PD-1/PD-L1 binding inhibition was not demonstrated in this study. These findings nonetheless justify further validation in patient-derived organoid models to optimize this {gamma}{delta} T cell-based combination immunotherapy and advance its development as a precision therapeutic strategy for metastatic OC.

ObjectiveStructural adaptations of capillary networks, through angiogenesis, arterialization, and regression, are implicated in many diseases, and gaining a better understanding of the cell-cell interactions that underpin these adaptations may lead to novel therapeutic discoveries for disease management. Endothelial cells and pericytes are the two cell types that comprise capillary networks. Experimental model systems have been developed to study the dynamic interactions between endothelial cells and pericytes, providing valuable insights into capillary development, cell-to-cell communication, and responses to growth factors and therapeutic agents. MethodsIn this study, we present a novel and simple co-culture system that uses commercially available primary human endothelial cells and pericytes, does not require microfluidic perfusion, and allows simultaneous observation of cell morphologies and interactions over time in 60 samples, enabling high-throughput analysis of multiple culture conditions with replicates. ResultsImage analysis pipelines were created to quantify microvascular adaptations, including one to measure colocalization between endothelial cells and pericytes, capturing dynamic coupling and uncoupling associated with capillary stability, angiogenesis, and regression. We validated the ability of our co-culture system to reproducibly represent the effects of fibrotic and angiogenic activation signals, including an FDA-approved drug, on endothelial cells, pericytes, and their coupling. ConclusionThis novel, high-throughput microvascular screening assay enables quantification of microvascular dynamics in response to disease-relevant stimuli and therapeutics in a repeatable, real-time manner.

Alterations in epidermal growth factor receptor (EGFR) dynamics can influence tumor initiation by changing receptor abundance, ligand-dependent activation, and downstream proliferative signaling. Mathematically linking these receptor-scale processes to population-level tumor growth remains challenging because they couple molecular, cellular, and tissue-scale dynamics. Here, we develop multiscale models that explicitly captures receptor-ligand dynamics. We analyze the dynamics of a refined version of a 3D stochastic multicellular model with explicit EGFR-EGF interactions to derive a receptor-structured continuum model in which cells are organized by active receptor clusters. This model is further reduced into a population dynamics model that tracks the mean number of active receptors. It captures the main qualitative behaviours of the higher-dimensional models while enabling analytical and numerical characterization of model-derived thresholds for sustained growth. After calibration and comparison with available in vivo tumor-growth data under EGFR overexpression, we use the model hierarchy to quantify how initiation thresholds depend on EGF availability, EGFR abundance, receptor-ligand unbinding, and genetic potential. The models predict that EGFR overexpression, stronger receptor-ligand binding, and more aggressive cell phenotypes each lower the EGF molecular counts required for sustained tumor growth. Overall, the proposed framework provides a flexible mathematical approach for connecting receptor-ligand kinetics with population-level tumor-initiation dynamics.

Tumors consist of heterogeneous phenotypic cells, such as normoxic cells, which are highly proliferative, and hypoxic cells, which are less proliferative. Their phenotypic switching depends on tumor microenvironmental factors, such as oxygen and nutrient concentrations supplied by local blood vessels. However, during ongoing angiogenesis, the process of sprouting new blood vessels at the tumor site from pre-existing blood vessels, and how this phenotypic switching affects and impacts tumor growth, remains poorly understood. In this article, we formulate a mathematical model to elucidate the crosstalk between vasculature and tumor cellular heterogeneity during tumor progression. The model results show a strong agreement with the experimental data. Our simulation results demonstrate that ongoing angiogenesis increases tumor growth rate. In addition, we observe that the influence of hypoxic cells on phenotypic switching from normoxic to hypoxic is more pronounced than their influence on the transition from hypoxic to normoxic. Furthermore, we perform a global sensitivity analysis using the Sobol's method to assess the importance of the model's parameters. It highlights that the volume at which blood vessels attain half-maximal rate has the maximum effect on the model.

Integrin v{beta}8 is a major activator of latent transforming growth factor-{beta} (TGF-{beta}) and an emerging therapeutic target in cancer and immune regulation. However, functional assays that directly measure v{beta}8-mediated activation of latent TGF-{beta} in a physiologically relevant context remain limited. Here, we report a co-culture cell-based reporter assay for quantitative measurement of v{beta}8-mediated activation of latent TGF-{beta}1. NIH/3T3 reporter cells were engineered to express a SMAD-responsive NanoLuc reporter, constitutive firefly luciferase for internal normalization, and cell-surface GARP-latent TGF-{beta}1. When co-cultured with v{beta}8-expressing LN-229 cells, reporter cells produced a robust signal that directly reflected localized latent TGF-{beta}1 activation. The assay demonstrated stable expression of the required biological components, reproducible signal-to-background performance, and sensitivity to benchmark v{beta}8-blocking antibodies. Inhibition studies showed potent dose-dependent blockade by an anti-v{beta}8 antibody. In contrast, pan-TGF-{beta} neutralizing antibody displayed markedly weaker apparent potency, suggesting that targeting localized v{beta}8-mediated activation is more effective than neutralizing released TGF-{beta} in this assay context. The assay also enabled screening and ranking of anti-v{beta}8 antibodies, identifying several high-potency clones, and detected v{beta}8-mediated activation of a non-cleavable latent TGF-{beta}1 mutant. This platform provides a sensitive, internally normalized, and scalable approach for mechanistic studies and therapeutic discovery targeting the v{beta}8-TGF-{beta} axis.

During tumor growth and progression, cancer cells are exposed to sustained physical confinement and volumetric compression that can alter cell volume, cytoskeletal organization, mechanotransduction, and invasive behavior. However, whether breast cancer cells retain a compression-induced mechanical memory after release from sustained volumetric compression, and how this memory influences subsequent migration and invasion, remains poorly understood. Here, by controlling cell volume using PEG - mediated volumetric compression, we investigated the compression and post-compression recovery responses of MCF-7 breast cancer cells. Cells were compressed for four days, followed by four days of recovery after PEG removal, and analyzed using daily morphological tracking, single-cell time-lapse imaging, F-actin and YAP staining, wound healing assays, and 3D spheroid invasion assays. We show that sustained volumetric compression shifts MCF-7 cells into a compact, jammed-like, low-motility state characterized by reduced morphodynamic remodeling, suppressed collective migration, and limited spheroid invasion. In contrast, post-compression recovery induces a distinct mechanobiological state marked by increased cell area and perimeter, altered single-cell trajectories, heterogeneous F-actin remodeling, enhanced YAP nuclear localization in enlarged recovered cells, accelerated wound closure, and increased spheroid invasion and cell dissemination. These findings suggest that prior volumetric compression can prime breast cancer cells for enhanced migration and invasion after stress release, supporting post-compression recovery as a form of mechanical memory that may contribute to tumor dissemination.

Current {gamma}{delta} T-cell expansion protocols often sacrifice functionality for yield and largely ignore the context of activation. Here we utilize a tunable alginate microgel system functionalized with anti-CD3 and co-stimulatory antibodies (CD28 or CD2) to investigate the impact of biochemical signaling and substrate mechanics on {gamma}{delta} T-cell activation. Microgel-mediated expansion was compared to conventional soluble antibodies and TransAct beads. The microgels enhanced {gamma}{delta} T-cell expansion compared to soluble antibodies, allowed for controlled tuning of differentiation state, and promoted higher NKG2D, IFN-{gamma} and TNF- expression levels. Functionally, microgel-expanded {gamma}{delta} T-cells exhibited superior cytotoxicity against both solid and liquid tumor targets. This system also allowed elucidation of the differences in stimulation requirements for various donors, based on the starting phenotype. These findings establish a tunable platform for engineering {gamma}{delta} T-cells with improved therapeutic potential. Significance Statement{gamma}{delta} T-cells have shown promising therapeutic effects when used for T cell-based immunotherapy to treat solid tumor. However, achieving rapid expansion of {gamma}{delta} T-cells while maintaining their functionality remains a major challenge, especially given the heterogeneous responses from donors. We demonstrate that a tunable microgel system with flexible presentation of stimulatory cues improves {gamma}{delta} T-cell expansion while preserving cytotoxic function and reveal how starting phenotypes influence responses to activation. These understandings will provide design rationale to enable patient-specific treatment for optimal therapeutic outcomes.

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.

Glioblastoma (GBM) is a highly aggressive brain tumor with a five-year survival rate of less than 5%. The current standard of care established 20 years ago includes maximal surgical resection and administration of alkylating agent temozolomide (TMZ). GBM is highly invasive, and GBM cells that evade surgical resection can become resistant to TMZ and develop new aggressive secondary tumors. Post-relapse there are few treatment options available to patients. Tissue engineering approaches suggest the opportunity to develop in vitro models of the GBM tumor microenvironment that may accelerate the discovery of novel therapies for GBM. Here, we report the adaptation of hydrogel microdroplets (microgels) to encapsulate GBM cells in a tailorable 3D matrix to assess patterns of growth and to screen TMZ drug response using patient-derived xenograft (PDX) specimens. We exploit a unique aspect of the microgel system to account for the cellular heterogeneity within the tumor microenvironment (TME). We combine cell-laden microgels generated from TMZ-resistant and TMZ responsive variants of the same PDX specimens to create heterogeneous populations with varying levels of drug sensitivity. We demonstrate a range of drug resistance phenotypes as a function of the ratio of TMZ-responsive to resistance cells and identify the population required for TMZ-resistance to overtake take the response. We then investigate the influence of tumor mimetic shifts in hyaluronic acid bioavailability and hypoxia on patterns of TMZ resistance. We show exposure to matrix-bound hyaluronan increases TMZ resistance and the glioma stem cell population in both cell variants. Lastly, we report an increase in TMZ sensitivity but divergent changes in the GSC subfraction for TMZ resistant vs responsive GBM in the presence of hypoxia. Together, we demonstrate the versatility of cell-laden microgel approach to replicate heterogenous tumor populations, model shifts in the tumor microenvironment, and rapidly screen therapeutic response.

BackgroundThe development of clinically viable tissue-engineered heart valves (TEHVs) remains limited by inconsistent host cell infiltration. The dynamic hemodynamic environment may play a central role in driving or inhibiting cell invasion, yet the effects of cyclic stretch on cell migration and proliferation remain largely unexplored in 3D tissues and scaffolds. Given evidence that uniaxial constraint promotes directional invasion in 3D matrices, we hypothesized that uniaxial cyclic stretch would enhance cell invasion, particularly along the stretch direction. MethodsWe embedded multicellular spheroids into collagen hydrogels and subjected them to uniaxial cyclic stretch (3-10%, 1 Hz) for two days and quantified invasion into the surrounding extracellular matrix using a custom image-processing program. Smooth muscle cells, valvular interstitial cells, and dermal fibroblasts were examined to represent cell populations relevant to TEHVs and for comparison across cell types with different contractility. To determine the mechanisms underlying changes in invasion with stretch, effects of cell tension were evaluated using gel compaction assays and inhibition of myosin IIA, and proliferation was assessed by Ki67 immunostaining. ResultsContrary to our hypothesis, cyclic stretch profoundly inhibited cell invasion into the matrix across all cell types and magnitudes of stretch. Invasion decreased by >50% in smooth muscle cells and fibroblasts and by up to 99% in valvular interstitial cells. Invasion suppression was inversely correlated with cell contractility, implicating a role for cell-generated tension. Inhibition of myosin IIA partially rescued invasion with stretch, though not to static levels. Stretched spheroids also exhibited reduced cell proliferation relative to static controls. ConclusionsThese findings implicate actomyosin-mediated mechanotransduction in stretch-induced suppression of cell invasion and suggest that the dynamic valve environment may limit host-cell repopulation of TEHVs. More broadly, this work provides insight into how cyclic stretch regulates 3D cell invasion in mechanically active tissues with implications for wound healing and cancer metastasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/732094v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@2a21b1org.highwire.dtl.DTLVardef@9fbf6org.highwire.dtl.DTLVardef@17ceb17org.highwire.dtl.DTLVardef@2e3bf9_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Despite major improvements in molecular characterization of breast cancer, current biomarkers still fall short in accurate treatment prediction. Interrogating tumor tissue ex-vivo in its native conformation is a direct strategy for guiding treatment of individual patients but presents a challenge. In this study, we developed a microfluidic tissue array (FTA) using small biopsy samples (< 1mm3) mimicking physiological flow for consistent exchange of nutrients and waste, retaining the tumor native stroma. Cell/patient-derived breast cancer xenograft tissues were maintained over 2 weeks in the array and their response to therapeutic agents, doxorubicin or neratinib, were interrogated. Drug response in the uFTA showed >2-fold reduction in tumor cell viability which corroborated tumor size shrinkage in mice bearing the same tumor load. EdU/Ki67 assays indicated selective retention of cells with higher proliferative capacity after drug treatment, underscoring in vivo clinical relevance . We have also developed a valved-FTA to increase throughput and variety of treatment conditions on the same chip. Together, this FTA can be staged as a powerful, low-cost benchtop theranostic tool for personalized cancer therapeutics compatible with FDA New Approach Methods.

Solid tumors are often surrounded by abnormal vasculature and a dense collagen-rich extracellular matrix that severely restrict the infiltration of T cells, including tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR)-T cells. These physical barriers represent a major obstacle to the efficacy of adoptive T cell therapies in solid tumors. We previously identified Formin-like 1 (FMNL1) as a cytoskeletal regulator critical for T cell extravasation and migration through restrictive environments, making it a promising target to improve T cell infiltration into tumors. Here, we developed a bioengineering platform to enhance T cell cytoskeletal dynamics by overexpressing FMNL1 in TILs and CAR-T cells. FMNL1 overexpression significantly increased T cell migration through restrictive pores in transwell assays, supporting enhanced migratory capacity of T cells under mechanically constraining conditions. Importantly, FMNL1 overexpression did not impair T cell reactivation or cytotoxic function in vitro. In murine models of melanoma and lung carcinoma characterized by limited effector T cell infiltration, FMNL1-overexpressing TILs and CAR-T cells had significantly increased accumulation at tumor sites compared to controls. Importantly, enhanced tumor accumulation resulted in improved therapeutic activity, as adoptive transfer of FMNL1-overexpressing CAR-T cells limited tumor growth and prolonged the survival of tumor-bearing mice in multiple melanoma models. Together, our findings identify FMNL1 as a broadly applicable cytoskeletal engineering target to enhance T cell accumulation and persistence in restrictive tumor microenvironments, thereby overcoming a fundamental limitation of adoptive cellular immunotherapy in solid tumors.

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.