Cellular and Molecular Bioengineering

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.

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

Synthetic blood contacting devices frequently fail due to the lack of requisite biochemical and biomechanical cues needed to support transanastamotic endothelialization. During transanastomotic endothelialization, endothelial cells experience dynamic changes in extracellular mechanical cues as they migrate from compliant native vessels onto stiffer blood contacting device surfaces. However, how substrate stiffness and mechanical memory from prior mechanical environments influence temporal integrin remodeling and downstream endothelialization processes necessary to establish a stable endothelial layer remains poorly understood. In this study, human coronary artery endothelial cells (HCECs) were cultured on substrates spanning physiologically relevant stiffnesses to determine how substrate mechanics regulate collagen binding integrins and endothelialization. Increasing substrate stiffness promoted time dependent upregulation of 2{beta}1 integrin expression, whereas 1{beta}1 expression remained unchanged. Enhanced 2{beta}1 expression on stiff substrates was accompanied by increased vinculin associated focal adhesion maturation and accelerated endothelialization, characterized by increased proliferation, migration, and progression to confluence prior to reaching quiescence after 1 week. To better model transanastomotic migration and investigate mechanical history effects, cells initially expanded on compliant hydrogels were transferred to stiff substrates. Although these cells exhibited transient reductions in 2{beta}1 expression at early timepoints compared with tissue culture polystyrene expanded controls, no persistent differences in focal adhesion maturation, proliferation, migration, confluence, or quiescence were observed. Collectively, these findings demonstrate that substrate stiffness is a primary regulator of the early endothelialization processes required to establish a stable endothelial monolayer, whereas the influence of mechanical history is transient and ultimately superseded by the current mechanical environment. These findings also identify 2{beta}1 mediated mechanotransduction as a potential design target for blood contacting biomaterials that promote rapid endothelialization while supporting long-term endothelial cell quiescence.

Treatments for advanced melanoma have markedly improved, but a significant proportion of patients still receive little to no survival benefit with standard-of-care therapies due to resistance and relapse1-5. The discovery and development of novel targets and therapies are needed to continue to improve patient outcomes in advanced melanoma. The identification of ligand-dependent BMP signaling that inhibits differentiation and promotes survival of melanoma cells suggests it is a potential therapeutic target that could complement current therapies6. Expression of the BMP ligand GDF6 (a.k.a BMP13) is responsible for this activity, and its expression is correlated with poor outcomes for melanoma patients. Here, we describe a novel monoclonal antibody targeting GDF6 that causes melanoma cell differentiation and death and blunts tumor growth in vivo. Together, these results indicate BMP-directed therapy has significant potential as a novel therapy for patients with advanced melanoma.

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.

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.

Leptomeningeal disease (LD), involving the metastasis of cells to the leptomeningeal membranes in the central nervous system (CNS), can be a deadly complication of several different types of cancer originating in the periphery or CNS, including breast cancer (BC) and pediatric medulloblastoma (MB). Targeted therapy represents a promising new approach to improve overall survival for LD patients. To this date, angiopep-2 (Ang2) and transactivating transcriptional activator (TAT), two well-known peptides for their brain delivery capability, have been reported to transport therapeutic cargos into the CNS for treatment of disease. Current administration strategies, however, still rely on oral delivery or intravenous injection (IV), where the substances need to travel through complex biological barriers to reach the subarachnoid space (SAS), which is the primary location of LD. Our research group has focused on the intrathecal (IT) route of administration as an alternative approach that can potentially enable high exposure of drug to CSF exposed tissues. However, there is a major field gap in understanding how targeting peptides can access (or not access) LD as a function of their route of administration. Therefore, our work was focused on comparing the targeting capability of Ang2 vs TAT by IT vs IV routes of administration. We first generated two xenograft models of LD by directly infusing breast cancer cells (MDA-MB231) or medulloblastoma cells (HDMB03) into the SAS via intracisternal magna injection (ICM) to form BC-LD and MB-LD models, respectively. These tumor models were characterized for overall survival, tumor growth patterns, and presence of hydrocephalus. Second, we further administered fluorescently labeled Ang2 or TAT peptides either IV or ICM into tumor bearing mice. Neuraxial fluorescence images were examined to evaluate the targeting ability of these two peptides based on colocalization between peptide signal and tumor tissues ex vivo. We discovered that the median survival of both models was negatively related to the number of the cells infused. While HDMB03 cells tended to metastasize preferentially to the brain region, MDA-MB231 cells tended to metastasize preferentially to the spinal cord. Both models present hydrocephalus as one of the common clinical symptoms in LD patients. Compared to the healthy control, MB-LD yielded a 7.3-fold increase and BC-LD a 26.5-fold increase in ventricular volume. Furthermore, targeting achieved by TAT was significantly higher than targeting achieved by Ang2 in thoracic spine for the MB-LD model. For BC-LD model, TAT signal was found to be significantly higher than Ang2 signal in the olfactory bulbs, brain stem, thoracic spine, and lumbar spine regions. While both peptides showed a strong signal at 2 hours post ICM injection, signal was not detectable 24 hours after administration, reflecting washout or degradation. Significantly, these data provide evidence that ICM will be a preferable route of administration over IV for the purpose of maximally targeting LD.

Tumor models that recapitulate 3D architecture are essential for understanding how cellular organization and microenvironmental interactions govern therapeutic response in human cancers. Here, we developed a microfluidic microphysiological system that enables controlled and scalable culture and drug testing of non-small cell lung cancer spheroids and patient-derived organoids. The platform integrated U-shaped microwells with dual-channel loading to support de novo spheroid formation, efficient trapping of pre-formed spheroids, and loading of intact organoids with reduced size heterogeneity. Tumor spheroids and organoids maintained high viability and structural integrity during long-term on-chip culture, and constrained microscale confinement produced ellipsoidal geometries that deviate from idealized spherical assumptions. Baseline genotype-dependent responses to KRAS G12C and EGFR inhibitors were preserved across agarose and microfluidic formats, establishing a validated reference state. Building on this baseline, fibroblast- and endothelial-derived cues consistently attenuated responses to targeted therapies across conditioned media, mixed co-culture, and spatially organized configurations. Resistance phenotypes converged on a dominant role for paracrine signaling, while increasing architectural complexity primarily enhanced morphological fidelity rather than altering therapeutic response. These findings establish a microphysiological framework that decouples tumor-intrinsic drug sensitivity from microenvironment-mediated modulation, enabling the systematic evaluation of paracrine resistance mechanisms in NSCLC.

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.

PurposeOculocutaneous albinism type 1 (OCA1) is an inherited disorder caused by tyrosinase (TYR) gene mutations. Affected individuals experience visual impairment and severe photosensitivity from ocular hypomelanosis, with no current treatments. We evaluated the safety and efficacy of a TYR-encoding adeno-associated virus (AAV) vector in healthy rhesus macaques as a potential OCA1 treatment. MethodsA novel AAV2-based capsid (ATX002) was packaged with the human VMD2 promoter and TYR (hTYR) fused with mGreenLantern (mGL). Two adult rhesus macaques were injected with ATX002-hVMD2-hTYR-mGL subretinally (OD) and intravitreally (OS). Safety and efficacy were assessed via comprehensive ophthalmic examination, fundus photography, spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinography at baseline and defined timepoints up to 12 weeks post-injection, followed by post-mortem immunohistochemistry (IHC). ResultsBoth subretinal doses induced localized hypermelanosis by 3 weeks post-injection, which persisted through the study endpoint and was accompanied by measurable thickening of the retinal pigment epithelium (RPE) on SD-OCT. Histological IHC confirmed successful RPE transduction via robust mGL fluorescence, corroborating in vivo findings by revealing localized RPE hyperplasia and transgene-expressing cells adjacent to regions of de novo hypermelanosis. Intravitreal delivery did not induce any changes to the RPE. Transient uveitis was observed but successfully managed with anti-inflammatory treatment. ConclusionsSubretinal AAV-TYR delivery is a safe and effective approach with the potential to induce RPE pigmentation. These findings support the use of AAV-TYR gene therapy for OCA1, demonstrating efficacy and a manageable safety profile in a large-animal model, and provide a critical bridge toward human clinical translation.

The development of cancer immunotherapies requires preclinical models that capture intravenous delivery, immune cell recruitment and intratumoral T cell activation. Conventional 3D in vitro systems lack perfused vascular networks, whereas mouse models are limited by throughput. The chick chorioallantoic membrane (CAM) assay enables rapid growth of vascularized tumors derived from human cancer cells in ovo, but its application to human T cell-based immunotherapy testing is constrained by CAM-derived vascularization and species-specific barriers between human immune cells and avian endothelium. Here, we establish an endothelial graft-enhanced CAM tumor model that incorporates an immortalized murine endothelial cell line capable of anastomosing with the chick vasculature. This generates a perfused and branched mammalian vascular interface within human tumor xenografts growing on the CAM. Human ICAM-1 expression on the grafted endothelial cells further enhances recruitment of intravenously delivered human T cells into CAM tumors. Using this platform, we demonstrate target-dependent intratumoral T cell activation by bispecific T cell engagers (TCEs) across tumor models, including evaluation of the clinically approved DLL3-targeting TCE tarlatamab in small cell lung cancer models.

Idiopathic pulmonary fibrosis is a devastating chronic lung disease characterized by progressive scarring of the lung, which leads to impaired gas exchange and ultimately death. While research has provided us with extensive understanding on end-stage disease, the factors that lead to forward-feedback loops of fibrotic progression are still not fully known. Cell intrinsic activation, pathological extracellular matrix (ECM) composition, and increased tissue stiffness are all hallmarks of advanced fibrosis, but the relative contribution of these factors to disease has been difficult to disentangle using classic in vivo models. In this study we created biomaterials-based 3D lung models that incorporate geometrically relevant co-culture of lung epithelial cells and fibroblasts with tunable stiffness, ECM-containing hybrid-hydrogels. Using this model system, we demonstrated that environmental stiffness has the strongest effect on overall fibroblast activation. RNAseq analysis revealed unique gene-level changes in both fibroblasts and epithelial cells due to both composition and stiffness, highlighting the importance of incorporating both factors into any in vivo disease models. Overall, these results reinforce the value of biomaterials-based models in understanding disease pathogenesis, and their potential for screening of treatment responses.

Low response rates to immune checkpoint inhibitors (ICIs) in solid tumors are often driven by insufficient tumor-infiltrating CD8 T cells and immunosuppressive tumor microenvironment (TME). Although interleukin-2 (IL-2) potently expands and activates CD8 T cells, its clinical use is limited by rapid clearance, dose-limiting toxicity, and regulatory T cell (Treg) stimulation. Engineered IL-2 variants have not yet achieved meaningful clinical efficacy. Here, polymer-modified mesoporous silica nanoparticles displaying dense, unmodified wild-type IL-2 on their surface (IL2-NP) are developed, conferring proteolytic stability and tumor retention. IL2-NP enables avidity-mediated CD8 T cell binding and enhances proliferation and effector function without increased Treg binding or proliferation. Intratumoral IL2-NP expands CD8 T cells, increases CD8/Treg ratios, and reprograms TME through dendritic cell activation and M1-like macrophage polarization. IL2-NP induces regression of both treated and untreated distant colorectal tumors in a CD8 T cell-dependent manner. IL2-NP synergizes with ICIs and leads to complete tumor regression and immunological memory that protect against rechallenge. Treatment is well tolerated, with strong efficacy also observed in triple-negative breast and metastatic ovarian cancer models. Overall, intratumoral IL2-NP elicits robust systemic antitumor immunity, offering a promising strategy to enhance ICIs, cancer vaccines, and adoptive T-cell therapies. Graphical abstractThis work introduces a nanoparticle platform that overcomes major shortcomings of IL-2 immunotherapy by presenting wild-type IL-2 at high density on the nanoparticle surface, thereby increasing binding avidity to effector T cells. The resulting IL-2 nanoparticles enhance cytotoxic T cell expansion, reprogram the tumor microenvironment, and augment responses to immune checkpoint blockade to achieve robust ant-tumor immune response in mouse tumor models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/738558v1_ufig1.gif" ALT="Figure 1"> View larger version (82K): org.highwire.dtl.DTLVardef@12f8c8corg.highwire.dtl.DTLVardef@b46b1forg.highwire.dtl.DTLVardef@e4efc5org.highwire.dtl.DTLVardef@3993e6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cancer of the uterine corpus is the fourth leading cancer and the fifth leading cause of cancer-related death in women in the United States. Chemotherapeutic resistance, specifically platinum-resistance, contributes to this problem. Therefore, an alternative treatment regimen is required. Using inorganic copper oxide nanoparticles (CuO NPs), we evaluated cancer cell responses indicative of anti-neoplastic activity. CuO NPs were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), dynamic light scattering (DLS) and laser Doppler velocimetry (LDV). The nanoparticles were rod-like and had a diameter of 70 {+/-} 30 nm and a copper content ranging from 77% - 82.6%. The hydrodynamic diameter and the zeta potential significantly decreased with more particles in solution. These materials were also used in four endometrial cancer cell lines and one cervical cancer cell line to evaluate cell viability, apoptosis, migration, and reactive oxygen species. In endometrial cancer cell lines, the IC50 values ranged from 1.028 ug/mL in HEC-1A cells to 73.62 ug/mL in Ishikawa cells, indicating that different cells have vastly different responses to CuO NPs. The results also indicated cell line-dependent differences in apoptosis, oxidation potential, and migration. Further, the cervical cancer cell line was modified using CRISPR technology to highlight a common germline mutation that causes earlier onset and more aggressive cancer progression. These genetic mutations resulted in differences in a loss of redox potential without observable changes in apoptosis or migration. The results of these studies indicate that CuO NPs elicit effects dependent upon the stage of cancer. Anticipated long-term applications of these studies includes the potential as a target-specific anti-cancer agent, designed using knowledge at the interface of colloids and the tumor environment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/729888v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1e0fe68org.highwire.dtl.DTLVardef@5e776dorg.highwire.dtl.DTLVardef@1f2aad6org.highwire.dtl.DTLVardef@add7fc_HPS_FORMAT_FIGEXP M_FIG C_FIG Copper oxide nanoparticles (CuO NPs) were characterized upon receipt using electron microscopy, elemental analysis, dynamic light scattering, laser Doppler velocimetry, and Fourier-transform infrared spectroscopy. These CuO NPs were exposed to HeLa cells with and without DNA mismatch repair deficiencies to assess the impacts on cancer cell migration, apoptosis, and redox potential.

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.

Silicones are elastomers that have a wide variety of uses, including biomedical applications such as the coating of biomedical devices and as implants. Soft silicones with mechanical properties similar to those of biological tissues have particularly gained use as substrates for cell culture in mechanobiology studies. In this context, it would be desirable to be able to alter their surface mechanical properties with a relatively simple physical treatment. While deep ultraviolet (deep UV) or ultraviolet C (UV-C) treatment has been previously used as a surface treatment method for stiffer silicones formulations, the effect of this treatment on soft silicones relevant for mechanobiology applications is still uncharacterized. We first used nanoindentation to determine the Youngs modulus of two types of soft silicones, Qgel and GEL-8100/Syl (GEL-8100 with Sylgard-184 crosslinker), both with initial moduli in the kilopascal range. We show that nanoindentation in the presence of 1% sodium dodecyl sulfate avoids adhesion between the nanoindentation glass probe and the soft silicones. After deep UV exposure in the presence of air, nanoindentation revealed that the apparent Youngs moduli of the soft silicones Qgel and GEL-8100/Syl increased by 70% and 33%, respectively. The bulk rheology of the soft silicones were not affected, suggesting that this corresponds to a surface stiffening effect with a topical stiffening of at least several hundred kilopascals. Energy-dispersive X-ray spectroscopy results show an increase in the mole fraction of oxygen, consistent with oxidation of the surface. Attenuated Total Reflectance Fourier-Transform Infrared spectra show evidence of Si-OH group formation in GEL-8100/Syl and silicon sub-oxide formation in Qgel. Consistent with this, water contact angle measurements show enhanced hydrophilicity after deep UV treatment. Our results have implications for using soft silicones as substrates in mechanobiology studies and in processes where deep UV light is used in the surface treatment of soft silicones.

BackgroundRecombinant expression of the SARS-CoV-2 receptor-binding domain (RBD) is essential for vaccine development, serological diagnostics, and mechanistic studies. Primary human fibroblasts offer physiologically relevant protein folding and post-translational modification, yet their short lifespan limits scalable production. We used an immortalized human splenic fibroblast cell line to stably express RBD-sfGFP for longitudinal characterization and downstream studies. MethodsImmortalized human primary splenic fibroblasts were transfected by electroporation with a plasmid encoding SARS-CoV-2 RBD fused to superfolder GFP (sfGFP), with a neomycin resistance cassette (neoR) for G418 selection. Four independent G418-resistant cultures (n=4), designated HPSF-IM-RBD-BHSKPU T1-T4, were established from distinct selection flasks. Based on previous screenings, two cultures (T1, T3) were monitored for 98 days (14 passages, P1-P14); two cultures (T2, T4) were monitored for 42 days (6 passages, P1-P6). RBD-sfGFP expression was assessed by fluorescence microscopy at 7-day intervals. For each timepoint, 2 fields were imaged and analyzed for relative fluorescence intensity (normalized to global maximum = 100%) and mean fluorescence intensity (MFI, normalized to global maximum = 100%). Coefficient of variation (CV), linear regression, and Pearson correlation were calculated. ResultsAll four cultures exhibited robust GFP fluorescence, confirming stable transgene retention. Expression ranking: T1 (93.1% +/- 3.6%) > T3 (89.2% +/- 3.4%) > T2 (84.2% +/- 3.2%) > T4 (79.7% +/- 3.9%). Long-term cultures T1 and T3 retained [~]100% of Day 7 signal at Day 98 (T1: 100.7%; T3: 100.0%). Expression exhibited passage-dependent oscillation rather than progressive silencing. CV increased over time in T1 (1.5% -> 8.5%), indicating growing inter-cellular heterogeneity. A strong positive correlation between fluorescence and MFI (Pearson r = 0.823, p = 7.44 x 10-11) suggested coherent population-level regulation. ConclusionsHPSF-IM-RBD-BHSKPU cells stably retain RBD-sfGFP expression for over 3 months, validating their utility as a recombinant protein production platform. However, oscillatory dynamics and increasing heterogeneity are consistent with position-effect variegation at distinct integration loci. Consequently, early passages (P1-P4) are optimal for applications requiring maximal uniformity. Ultimately, these cells provide a practical tool for RBD production and a valuable model for studying epigenetic regulation of transgene expression in human primary fibroblast backgrounds.

Cancer phenotypic plasticity drives invasion, treatment resistance, and relapse. Quantifying how cells dynamically couple morphology and migration in real time, without molecular labels, remains unsolved. Static molecular markers report on protein expression state rather than functional migratory behavior. Existing image-based metrics treat shape and migration as independent features, missing the coordinated coupling that defines plastic migratory states. We introduce Directional Shape Coupling (DSC), a quantitative metric purpose-built for live label-free imaging. DSC integrates movement direction consistency, shape deformation, and directional-shape alignment into a single interpretable score. Component weights are derived from PCA, adapting automatically to any dataset without manual tuning. Applied to differential interference contrast imaging of pancreatic cancer cells on a tissue-mimicking substrate recapitulating desmoplastic tumor stroma, DSC exhibited a large phenotype-associated effect size,{varepsilon} 2 = 0.65, across five distinct migratory phenotypes within a genetically homogeneous population, demonstrating that behavioral heterogeneity is structured and non-genetic. DSC encodes information orthogonal to classical shape and motion descriptors. Critically, DSC reveals that dynamic shape adaptation to mechanical cues rather than directional commitment drives phenotypic identity in this system. DSC provides the label-free imaging community a transparent, generalizable framework for quantifying dynamic non-genetic plasticity directly from live imaging data.

Pancreatic ductal adenocarcinoma (PDAC) develops within a desmoplastic and stiffened microenvironment that critically shapes tumor progression and therapeutic resistance, yet these features are not reproduced by conventional rigid plastic culture systems. Here, we leverage a tuneable bioengineered platform that mimics stromal stiffening to investigate how mechanical cues regulate PDAC cell behaviour and to identify pharmacological strategies that counteract stiffness-driven malignancy. We show that increasing matrix stiffness promotes key hallmarks of PDAC aggressiveness, including enhanced cell spreading, focal adhesions maturation, and cytoskeletal tension. Notably, we identify the glucocorticoid budesonide as a selective suppressor of stiffness-induced malignant phenotypes. Transcriptomic profiling reveals that budesonide counteracts stiffness-associated gene programs, prominently affecting pathways governing cytoskeletal dynamics, nuclear envelope organization, and YAP nucleocytoplasmic transport. Consistently, budesonide reduced force transmission to the nucleus, restoring nuclear wrinkling and constraining nuclear size and shape. These effects are mediated through both glucocorticoid receptor-dependent and -independent mechanisms, revealing a previously unrecognized mode of action. Together, our findings establish mechanical context as a critical determinant of PDAC vulnerability and identify budesonide as a candidate for therapeutic repurposing to target stiffness-driven cancer progression.

Biomaterial scaffolds for repairing traumatic muscle injuries require restoration of both the anisotropic architecture and basement membrane extracellular matrix cues critical to normal muscle function. To address this need, we establish a collagen-glycosaminoglycan (CG) scaffold platform pairing an aligned pore microstructure, produced via directional freeze-drying, with basement membrane protein functionalization via carbodiimide crosslinking. Laminin and/or collagen IV are successfully tethered and retained within CG scaffolds over 7 days without significantly altering pore size or alignment, confirming stable protein functionalization and preservation of scaffold architecture. Human muscle progenitor cells show excellent viability and metabolic activity in all scaffold groups, with collagen IV functionalization significantly enhancing myotube number and fusion index. Toward establishing scaffold compatibility with non-myogenic support cells, we show that neural stem cells remain viable and metabolically active across all scaffold conditions. Overall, these findings highlight the combination of aligned scaffold architecture and collagen IV functionalization as potentially impactful for skeletal muscle tissue engineering.