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.

Chimeric antigen receptor (CAR) T-cells engineered from {gamma}{delta} T-cells show limited clinical benefit in solid tumors such as ovarian cancer (OVCA). Although {gamma}{delta} T-cells can detect malignant transformation and exert cytotoxicity, these properties confer potent preclinical efficacy but limited therapeutic activity and the basis for this discrepancy remains unclear. Here, we established an ex-vivo culture consisting of highly inflammatory patient-derived malignant ascites and hypoxia conditions, to simulate key obstacles encountered by CAR T-cells in the immunosuppressive OVCA tumor microenvironment (TME). Using this condition, we first demonstrated the presence of CAR-expressing {gamma}{delta} T-cells within conventional (conv)CAR T-cells, which displayed greater proliferative capacity than {gamma}{delta}neg T-cells. We therefore produced {gamma}{delta}CAR T-cells for parallel comparison with conventional (conv)CAR T-cells, evaluating differences in gene expression and functionality. Under TME-like conditions, {gamma}{delta}CAR T-cells demonstrated lower viability but consistently outperformed convCAR T-cells, demonstrating favorable gene expression profiles, enhanced cytotoxicity, improved retention of cytokine secretion and degranulation and increased proliferative capacity. Importantly, we characterized an ascites protein profile that impacted the viability of both cell types comparably. Together, these findings reveal specific functional advantages of {gamma}{delta}CAR T-cells over convCAR T-cells in the immunosuppressive OVCA TME-like condition while facing different cellular fitness constraints, providing insights into the use and limitations of {gamma}{delta}CAR T-cells.

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.

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.

Radiotherapy (RT) significantly improves outcomes and reduces the risk of recurrence in breast cancer. However, in patients that experience recurrence despite treatment, the irradiated breast tissue may create a pre-metastatic niche that promotes tumor cell infiltration. Because current models of metastasis lack key physiological and microenvironmental cues, we developed a mammary vasculature-on-chip (MVoC) to model how the vasculature responds to RT and impacts immune and tumor cell behavior in mammary tissue. Murine and human MVoCs incorporated endothelial cells (ECs) cultured under physiologic shear stress and mammary-specific fibroblasts to mimic the vascular-fibrous stroma interface. MVoCs exhibited a transient, acute response to RT that led to a persistent phenotypic shift. Neutrophils, which play key roles in pre-metastatic niche formation, adhered more to irradiated ECs and induced persistent damage to the endothelial barrier, preventing recovery. Neutrophils also significantly increased tumor cell adhesion to the endothelium in MVoCs. These findings demonstrate that MVoCs can be used to study RT-induced pre-metastatic niche formation and suggest a positive feedback loop between damaged ECs and immune cell activation that may facilitate tumor cell colonization. MVoCs provide a biologically relevant approach to probe molecular and cellular interactions, improving translation toward developing novel therapies that improve patient outcomes.

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.

Oncofetal fibronectin (oFN) is a known biomarker of cancer progression and is involved in events such as epithelial mesenchymal transition and multidrug resistance. It is also understood that fibronectin (FN) and its splice variants are differentially arranged by cancer associated fibroblasts (CAFs) to support cancer cell proliferation and migration. Contrastingly, little is understood about the mechanobiology of oFN, a post-translationally modified FN isoform defined by O-glycosylation of a specific threonine residue. To investigate the architectural features of the oFN matrix, CAFs were simulated by culturing human mammary fibroblasts (HMFs) on stiff polyacrylamide gels (20 kPa) in media collected from HCC-1937 (primary site breast adenocarcinoma) and MDA-MB-231 (metastatic breast adenocarcinoma) cells. As a control, HMFs were also cultured on softer gels representing healthy adipose tissue (2 kPa) in media not exposed to cancer cells. After immunocytochemical analysis, tumor conditioned HMFs displayed an expected CAF-like phenotype that was most prominent with MDA-MB-231 conditioned media. Matrices produced by tumor-conditioned HMFs contained more oFN with structural features mostly following that of the overall dense, aligned FN matrix. Cancer cells reseeded on their corresponding decellularized tumor-associated matrices had greater F-actin/oFN colocalization than control matrix. The lack of distinct oFN topographical cues may indicate that oFN O-glycosylation is more directly involved in biochemical signaling than mechanosignaling through matrix architecture. Together, this study established a platform for investigating stromal oFN which revealed that tumor-conditioned media sourced from cancer cells with different metastatic potential influences HMF activation, leading to distinct total FN and thus oFN matrix microarchitecture features.

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.

The lung is a common site of metastasis and is continuously deformed by breathing. Whether physiological breathing mechanics influence tumor cell extravasation is unknown. To address this, we developed a mechanically actuated microphysiological system combining perfusable lung-specific microvascular networks with breathing-like cyclic deformation. Under static conditions, lung-specific networks exhibited more lung-like vascular features and higher tumor cell extravasation rates than generic networks. The mechanically actuated device reproduced physiological tissue and vascular deformation, while 24 h of cyclic actuation did not alter vessel viability, permeability, or morphology compared with unactuated controls. Pre-actuation for 24 h, followed by tumor cell perfusion under static conditions, did not alter subsequent extravasation, whereas actuation initiated immediately after perfusion significantly increased extravasation compared with unactuated controls. This biomimetic platform enables direct investigation of how breathing-like vascular deformation influences metastasis and provides a versatile tool for studying vascular mechanobiology in the lung and other mechanically active organs.

Endothelial cells integrate biomechanical and biochemical cues to regulate vascular function, which can be modeled using cell signaling networks. Yet, the extent to which their signaling behavior is determined by network topology versus reaction-specific parameterization remains unclear. We developed a continuous logic-based ordinary differential equation model of vascular endothelial signaling containing 70 species and 123 regulatory reactions assembled from the literature. Model predictions were evaluated against 78 literature-defined directional responses to increased wall stress, wall shear stress, hypoxia, and TNF. We first tested whether a single global parameter triplet describing reaction weight (w), Hill coefficient (n), and half-maximal activation (EC50) could reproduce these observations. Investigator-selected global parameters reproduced 62 of 78 responses (79.49%). Monte Carlo sampling of 100,000 globally constant parameter sets improved agreement to 69 of 78 responses (88.46%), indicating that network topology alone captured most qualitative signaling behavior but remained sensitive to parameter choice. Allowing reaction-specific parameters increased the best agreement to 75 of 78 responses (96.15%). We then used a genetic algorithm to identify the smallest subset of reactions requiring reaction-specific specification while retaining a common constant-parameter spine for the remainder of the network. Complete agreement (100%) with all 78 literature-defined responses was achieved with reaction-specific parameterization of only 10 of 123 reactions. The optimized model was subsequently used to simulate continuous changes in wall shear stress, revealing pathway-dependent nonlinear responses, including monotonic increases in nitric oxide and pSmad1/5, nonmonotonic responses in endothelin-1 and NF-{kappa}B, and a biphasic pSmad2/3 response. These findings show that most observed endothelial signaling behavior can be reproduced by a fixed network topology with broadly shared reaction parameters with only a limited subset of interactions requiring quantitative specialization. This framework provides a compact endothelial signaling model for investigating mechanobiological regulation and for future integration with multiscale models of vascular adaptation and disease.

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.

Cancer survival rates increase with earlier diagnosis. However, many cancers are diagnosed only after symptoms develop, in the later stages of cancer progression. We need a diagnostic tool that can detect cancer earlier. Circulating tumour cells (CTCs) are cells that detach from the main tumour and can enter the bloodstream. The capture and analysis of CTCs can provide an early indicator of cancer. However, rarity of CTCs in blood makes their efficient capture challenging. Inertial microfluidics can be utilised for a label-free separation of CTCs from white blood cells (WBCs) by manipulating cells in the microchannel based on cell properties, achieving good separation performance. Inertial microfluidic devices mainly rely on size-based separation. However, residual WBC carryover limits complete separation. Here, we show that cell mechanical heterogeneity plays a large role in the separation process and should be considered in the design of these devices. We simulate the migration dynamics of CTCs and WBCs in a straight microchannel using a 3D lattice-Boltzmann-immersed-boundary-finite-element solver. Our results demonstrate that WBC migration behaviour changes depending on the deformability of a CTC. The size and deformability of the cells have been shown to determine the cell-specific heteroclinic orbits leading to different interaction types, these interactions alter WBC migration resulting in more/less WBC carryover. This work highlights that both cell-specific heteroclinic orbits and single cell migration rates should be considered in the design of inertial microfluidics for separation.

Cell therapies involving human adipose-derived stromal cells (hASCs) have shown promise for a range of clinical applications due to their ability to stimulate angiogenesis and dampen inflammation via paracrine mechanisms. However, a major barrier to the successful clinical translation of hASC-based therapies is that standard culture methods for expansion on rigid 2D tissue-culture polystyrene under static conditions diminish the pro-regenerative functionality of the cells. To address these limitations, the current project focused on the development of an in vitro bioreactor system for preconditioning hASCs to augment their capacity to stimulate regeneration through paracrine mechanisms. Specifically, the combined effects of decellularized adipose tissue (DAT) coatings, shear-stress stimulation, and varying oxygen tensions on hASC expansion and paracrine factor secretion were assessed. Additional studies were performed to characterize the effects of stimulating hASCs within the rocking bioreactor system using the pro-inflammatory cytokines IFN-{gamma} and TNF-. Expansion in the bioreactor under all conditions supported hASC growth with no observable morphological differences. However, dynamic culture on DAT coatings enhanced intracellular indoleamine 2,3-dioxygenase (IDO) expression in hASCs cultured under 20% O2. Moreover, culturing under dynamic conditions and/or on DAT coatings significantly increased secretion of the pro-angiogenic factors VEGF, HGF, and angiogenin. When pro-inflammatory cytokine priming was introduced, the expression of all tested paracrine factors was enhanced, particularly the immunomodulatory factors IL-6, IL-8 and MCP-1. Overall, a novel bioreactor system was developed for hASC expansion and preconditioning, demonstrating that the cell microenvironment can be tuned to modulate hASC paracrine factor secretion.

Muscle regeneration relies on the coordinated activation of muscle stem cells, whose fate decisions are regulated by intracellular gene expression dynamics and intercellular coupling via the Notch-Dll1 signaling pathway. Central components of this regulatory network include the transcriptional repressor Hes1, its target gene Dll1, and the myogenic regulator MyoD. Experimental and theoretical studies have shown that proliferating muscle stem cells exhibit oscillatory dynamics of these molecules, whereas sustained expression is associated with differentiation. Here, we investigate the dynamics of a previously established delay differential equation model of two coupled muscle stem cells. Using linear stability analysis, we systematically characterize how model parameters affect the transition between stable and unstable steady states. In addition, numerical bifurcation analysis is employed to study the influence of intercellular coupling strength and delay on the system dynamics. Our analysis shows that continuous variation of the coupling delay induces repetitive changes in the stability of the steady state. However, this sensitivity towards the coupling delay is confined to a narrow region of parameter space and therefore requires a fine tuning of all other parameters. Beside the identification of parameter sets for in-phase and out-of-phase oscillations, we demonstrate the possibility of coexisting stable in-phase and out-of-phase oscillations, a dynamical feature that has not been reported previously. While oscillation periods are largely determined by intracellular regulatory mechanisms, oscillation amplitudes can be strongly modulated by intercellular coupling. These results provide new insight into how the intracellular network and intercellular communication interact to generate different collective dynamics.

Background: Chimeric Antigen Receptor (CAR) T-cell therapy has transformed the treatment of hematologic malignancies, yet, its broader clinical application often faces major challenges, including slow expansion rates, variable transduction efficiency, exhaustion, and functional heterogeneity. Recent studies have demonstrated that a Synthetic Immune Niche (SIN) composed of immobilized CCL21 and ICAM1 promotes the proliferation of murine and human T-cells while preserving their cytotoxic potency. In this study, we explored the capacity of immobilized CCL21 and ICAM1 to enhance the production of highly potent CAR T-cells by facilitating both their expansion and cytotoxic capacity. Methods: CD19-directed CAR T-cells were generated from PBMCs of healthy donors using a clinical-grade protocol. Following retroviral transduction, the cells were expanded on CCL21 and ICAM1 coated plates, or on uncoated control plates. The effects of the SIN treatment on CAR T-cell expansion, morphology, physical properties, phenotypic markers, and potency were systematically evaluated. Results: SIN exposure significantly enhanced CAR T-cell expansion, achieving a 9.3-fold higher total cell yield by day 13, compared with control cultures. This proliferative advantage persisted even after withdrawal of the cells from the synthetic niche on day 10. SIN stimulation preferentially expanded the CAR-transduced population, increasing CAR T-cell frequencies from 45.4% to 77.6%, resulting in a 14.6-fold increase in the absolute number of CAR T-cells compared with untreated cultures. Morphological and phenotypic analyses revealed distinct activated cell morphology, manifested by increased cell size, polarity and granularity, and elevated expression of the activation markers CD137 and CD69. Importantly, CAR T-cells transiently exposed to the SIN retained cytokine secretion and cytotoxic activity comparable to that of continuously SIN-treated cells, indicating that the SIN effect is persistent. Overall, SIN-conditioned CAR T-cells displayed robust antigen-dependent IFN-{gamma} secretion and cytotoxicity against CD19-expressing target cells. Conclusion: Stimulation of CAR T-cells with a synthetic immune niche consisting of immobilized CCL21 and ICAM1 enhances overall expansion while selectively enriching the CAR-transduced population, thereby substantially increasing both the number and prominence of therapeutically relevant CAR T-cells. This effect is accompanied by a persistent activation phenotype and high functional potency. We propose that incorporating SIN stimulation into CAR T-cell manufacturing represents a simple and scalable strategy for improving CAR T-cell yield while maintaining high cytotoxic efficacy.

Lenalidomide (Revlimid), bortezomib (Velcade), and dexamethasone, are used alone or in combination (RVd therapy) as first-line therapies for hematologic malignancies including the plasma cell dyscrasia, Multiple Myeloma (MM). The effects of RVd treatment on tumor cells have been documented, but little is known about their impacts on "healthy" immune cells. New therapeutics, including chimeric antigen receptor T cell (CAR-T) and bispecific T-cell engagers (BiTEs) depend on robust T cell function but these are often harvested for cellular therapies after or in tandem with RVd therapy. Understanding the molecular effects of these drugs on healthy T cells is therefore of particular importance. Since the molecular effects of each drug includes regulation of key transcription factors, we used a tri-modal, single-cell assay (TEA-seq), simultaneously profiling mRNA transcripts, cell surface proteins, and chromatin accessibility in primary human peripheral blood T cells treated for 4, 24, or 72 hours in vitro. Synergies and conflicts potentially arising from combinatorial therapy were identified and validated by flow cytometry. Doublet and triplet drug combinations were used to assess effects on cell-surface marker expression and on T cell activation. Our results suggest that T cell function can be optimized by the administration of these drugs individually and that sequential administration may offer an opportunity to enhance T cell function by affecting localization and altering T cell states. This work provides those interested in immunology, oncology, and cell therapies an opportunity to explore molecular pathways that could be manipulated by these commonly used drugs to modify T cell function.

Biochemical gradients and mechanical microenvironments synergistically regulate tumor cell proliferation, migration, and phenotypic evolution, which are critical for recapitulating in vitro tumor physiological microenvironments. In this work, a multilayer-assembled microfluidic chip integrated with Tesla-valve flow channels was developed for 3D SKOV3 cell culture under combined biochemical gradients and mechanical stiffness interfaces. The reconfigurable multilayer PMMA structure enables flexible construction of diverse concentration gradient fields, while the embedded Tesla-valve geometry effectively stabilizes fluid flow, reduces flow velocity fluctuation, and generates smoother, more stable biochemical gradients compared with conventional curved channel designs. Multiphysics simulations were performed to systematically verify the flow field distribution and gradient formation performance of the optimized channel structure. Furthermore, by constructing heterogeneous hydrogel microenvironments consisting of rigid Pluronic F127 and soft dextran hydrogel inside microfluidic chambers, stable mechanical stiffness interfaces were successfully fabricated. Combined with TGF {beta}1 biochemical stimulation, the platform was applied to explore the durotactic migration and morphological changes of 3D-cultured SKOV3 cells at stiffness interfaces. Fluorescence staining results demonstrated that mechanical boundary conditions significantly affected cell migration behavior and cell aggregation phenotypes under consistent biochemical induction. With the advantages of low cost, simple assembly, good biocompatibility, and controllable dual physicochemical microenvironments, this multilayer microfluidic platform provides a reliable and efficient strategy for in vitro tumor microenvironment simulation and tumor cell mechanobiology research.