Integrative Biology

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.

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.

Tunneling nanotubes (TNTs) mediate intercellular communication and contribute to cancer progression. Tumour cells produce excess lactate through increased glycolysis, but whether lactate regulates TNT formation has not been studied. Here, we show that lactate promotes TNT formation in U-87 MG glioblastoma (GBM) cells under both glucose-restricted and glucose-replete conditions, with a similar effect in MDA-MB-231 breast cancer cells. This effect depends on lactate uptake and metabolism rather than receptor-mediated signalling: inhibition of monocarboxylate transporter 1 (MCT1) or lactate dehydrogenase (LDH) reduced TNT formation, while exogenous lactate restored TNT formation during LDH inhibition. 3-chloro-5-hydroxybenzoic acid, an agonist of the plasma membrane-localized lactate receptor (GPR81/HCAR1), did not reproduce the effect of lactate. Lactate also increased intercellular mitochondrial transfer, attenuated temozolomide (TMZ)-induced growth suppression of GBM spheroids, and increased expression of the TNT-associated genes CDC42, MYO10 and FSCN1. Inhibition of p300/CBP-mediated lactylation with A-485 reduced both protein lactylation and TNT formation, implicating lactylation in TNT biogenesis. Lactate increased basal and ATP-linked respiration that persisted during MCT1 inhibition and reduced on LDH inhibition, indicating that the effects of lactate on respiration and on TNT formation are at least partly separable. Together, these findings identify lactate as a regulator of TNT formation and link tumour metabolism to intercellular connectivity and TMZ response, highlighting lactate metabolism and lactylation as potential therapeutic targets.

In breast cancer, collagen fiber alignment and the second harmonic generation forward-to-backward ratio (F/B), an optical measurement sensitive to fibril-scale organization within collagen fibers, have each been associated with metastatic outcome. Although both are measured at the tumor-stromal interface, in vitro studies have largely examined them separately, because experimental systems have not allowed the two to be varied independently. To address this technical gap, we used a microfluidic biofabrication approach to generate paired aligned and unaligned regions within 3D collagen hydrogels via extensional strain and varied measured F/B across gels by changing the pre-gel pH. The resulting library covered alignment values reported in breast tumors and F/B values associated with poorer metastatic outcome in invasive ductal carcinoma at matched matrix stiffness, pore diameter, and fiber fraction. In MDA-MB-231 cells, alignment biased migration along the fiber axis without changing migration speed, as expected, but the magnitude of the contact guidance response varied with measured F/B. In contrast, MCF-7 cells showed little overall contact guidance, yet migrated ~30% faster in high F/B than low F/B gels in both aligned and unaligned regions. Together, these results show that cells migrated differently in matrices with similar fiber alignment. Thus, including measured F/B may provide additional information to help anticipate how cells migrate in collagen environments that appear similar based on alignment alone.

Nanoparticle albumin-bound paclitaxel (Abraxane) was among the first clinically approved nanomedicines and is a first-line chemotherapeutic. It enhances therapeutic efficacy and reduces side effects of paclitaxel. Despite its extensive clinical use, mechanisms of Abraxane's therapeutic effects are the subject of ongoing study. This work posits a new mechanism for Abraxane's effects in lung cancers and metastasis. Tracing the albumin component of Abraxane with radioisotopes or fluorophores, we find that Abraxane homes to the lungs. Abraxane's lung tropism is elevated by inflammation and is eliminated in complement protein C3 knockout mice, showing that complement pathway drives the uptake. Flow cytometry and histology show that neutrophils are the dominant cell type taking up Abraxane in the lungs. We demonstrate Abraxane's tropism to pulmonary neutrophils in mice and in human donor lungs. We note previously undocumented side effects associated with Abraxane's lung tropism, including pro-thrombotic and pro-inflammatory responses in acute lung inflammation. We show that Abraxane's efficacy against lung metastases is affected when lung uptake is eliminated by C3 knockout. Our results provide insight into why Abraxane is effective in lung cancer and metastases. These findings may be used to improve outcomes in patients receiving Abraxane and help design next-generation cancer nanomedicines.

Solid tumors are known to be refractory to treatment due to a poorly perfused microenvironment that blocks immune cell infiltration and effective drug delivery. Histotripsy is a non-invasive focused ultrasound modality that generates cavitation microbubbles to mechanically disrupt solid tumors, resulting in decreased hypoxia and stimulation of tumor-targeted adaptive immune responses. In this study, we aim to investigate whether post-histotripsy hypoxia reduction and cytotoxic immune cell infiltration are associated with normalization of tumor vasculature and enhanced perfusion. Structural analysis of both treated and contralateral off-target tumors following partial histotripsy revealed significant vascular remodeling, enhanced vascular integrity, and decreased vascular leakage indicative of vascular normalization in multiple in vivo models of melanoma, hepatocellular carcinoma, and pancreatic adenocarcinoma. These effects appeared antigen-specific, as no remodeling was observed in contralateral off-target tumors of discordant pathology. Mechanistically, CXCR3+CD8+ T cells played a role in mediating these vascular changes, as genetic knockout of CD8 and pharmacological antagonism of CXCR3 impaired both remodeling and normalization. Additionally, significant changes in endothelial CXCR4 and Angiopoietin-1 and -2 expressions were observed following histotripsy, indicating their potential involvement in promoting vascular normalization and remodeling. Significant vascular remodeling was accompanied by enhanced tumor perfusion and intratumoral delivery of chemotherapeutic agents and therapeutic monoclonal antibodies. These findings suggest that histotripsy not only creates a more favorable microenvironment for immunotherapy, but also optimizes drug delivery into solid tumors. Combinatorial approaches using histotripsy with chemotherapy or immunotherapy may be a novel therapeutic strategy to overcome current limitations of conventional solid tumor therapies.

Lack of robustness and high variability in cardiomyocyte (CM) differentiation from human induced pluripotent stem cells (hiPSCs) remain critical challenges for cardiac regenerative medicine and biological research. Guided by developmental principles of lineage specification, we investigated whether early WNT inhibition could expand the process window of CM differentiation and thereby improve robustness. To test this, we compared early WNT inhibition from days 1--5 with standard inhibition from days 3--5 following a 1-day CHIR99021 (CHIR) treatment for WNT activation. Focusing on CHIR concentration as a critical parameter, we found that early WNT inhibition substantially broadened the concentration window yielding [≥]80% differentiation efficiency across distinct hiPSC lines. Extending the analysis to a two-dimensional parameter space incorporating cell density revealed a markedly expanded [≥]80% process window. Temporal analysis of lineage-specific transcription factor expression showed that early WNT inhibition suppressed paraxial mesoderm (PM) specification, likely through MSGN1 suppression, in the range of high initial WNT activation that otherwise favored PM fate, thereby extending the process window. It also suppressed definitive endoderm (DE) specification within the expanded window, promoting a more favorable lateral plate mesoderm/cardiac fate. We further demonstrated substantially reduced variability under parameter perturbations and across wells and batches, accompanied by higher differentiation efficiency, without compromising CM phenotypes. These findings demonstrate early WNT inhibition as an effective strategy for expanding the process window of CM differentiation and improving robustness, while exemplifying the process engineering principle that a broader window can support more robust differentiation, with potential relevance to stem cell--based bioprocesses.

Lymphatic endothelial cell (LEC) function is strongly tied to transitions between button- and zipper-like junctions that control fluid flow into vessels. Published methods quantifying junction morphology are limited to single cell analysis or use few quantitative metrics to describe junctions across LEC monolayers. Therefore, a novel 3-step image analysis pipeline was developed to identify new metrics that quantify differences in button- and zipper-like junctions in LECs. The pipeline used Cellpose, CellProfiler, and Fiji to analyze VE-cadherin expression across lymphatic vessels within a 3D microfluidic chip and quantify VE-cadherin radial intensity, number of objects, object size, branchpoints, end-points, and junction thickness. Tumor necrosis factor (TNF)- exposure produced but-ton-like junctions, and unexposed cells were zippered. Radial intensity was heightened at the cell membrane in zipper-like junctions while perinuclear intensity increased for but-ton-like junctions. Increased branchpoints, endpoints, objects per cell, and junction thick-ness were associated with junction buttoning, while increased object size was associated with junction zippering. Principle component analysis results reinforced these trends and identified metric combinations that represented each junctional state. Overall, this semi-automated pipeline quantified differences in VE-cadherin junction morphology across LEC monolayers and generated new quantitative metrics to expand the tools available for studying junction morphology in lymphatic vasculature.

Purpose: Lung metastases are the primary cause of death in osteosarcoma patients and treatment response often vary due to convergence of genetic and phenotypic influences in the context of a dynamic tumor-host tissue environment. The purpose of this study was to determine how dynamic tumor- and host-driven receptor tyrosine kinase (RTK)-ERK signaling interactions contribute to phenotypic drug response heterogeneity. Experimental Design: Live-cell time-lapse microscopy and data-driven dynamical modeling was used to resolve ERK activity, FOSL1/Fra-1 reporter expression, and spatiotemporal cell fate in living, biosensor-expressing osteosarcoma lung metastases ex vivo following MCL1 inhibitor exposure. RTK inhibitors were then evaluated for their ability to suppress adaptive signaling and enhance MCL1 inhibitor efficacy ex vivo and in vivo. Results: MCL1 inhibition induced extensive tumor-cell death; however, persistent ERKhigh/Fra-1high reporter cells emerged and were enriched at the tumor-lung interface, consistent with adaptive resistance. Experimental and computational approaches revealed that tumor-cell lysis amplified fibroblast growth factor receptor (FGFR)-mediated ERK signaling in neighboring cells, promoting a targetable drug-resistant state marked by adaptive ERK induction, FOSL1/Fra-1 expression, and correlated with upregulation of known drug resistance pathways Nrf2 and HMOX1. Combinatorial inhibition of FGFR with fexagratinib, or broad-spectrum RTK inhibitor, pazopanib, suppressed adaptive ERK activation and enhanced MCL1 inhibitor response resulting in greater efficacy ex vivo and in vivo. Conclusions: Tumor-host and tumor-cell-lysis-induced FGFR-ERK signaling contribute to spatiotemporally heterogeneous adaptive resistance in metastatic osteosarcoma. These data support combinatorial targeting of RTK signaling with cytotoxic or targeted agents as a strategy to block adaptive resistance and improve treatment efficacy.

We propose a minimal mathematical framework to describe the go-or-grow dynamics of tumor cells comprising two phenotypically distinct populations. One population is migratory and undergoes constant diffusion, while the other proliferates in an oxygen-dependent manner. The local oxygen concentration governs transitions between these phenotypes. We then ask whether the time scale of cyclic hypoxia, where oxygen is spatially homogeneous but fluctuates between normoxic and hypoxic levels over time, alters the dynamics of the reduced generalist model in the fast phenotype-switching regime compared with the full go-or-grow model. We observe that the reduced generalist phenotype model fails to capture the go-or-grow dynamics under rapid oxygen fluctuations. Our results predict that the tumor growth rate increases in the generalist-phenotype model under rapid oxygen fluctuations compared with tumors of the proposed go-or-grow model. We then explore whether oxygen consumption, which leads to spatial heterogeneity in oxygen concentration, plays a role in distinguishing the dynamics of the reduced model from the proposed go-or-grow model. We demonstrate that the tumor in the proposed go-or-grow model exhibits a higher growth rate than the tumor in the reduced generalist-phenotype model under a slow oxygen consumption rate.

The human pancreas secretes digestive fluid that flows through an extensive ductal network before emptying into the duodenum. However, little is known about how hydrodynamic forces influence pancreatic function and development. We hypothesized that pancreatic cells respond to physiological levels of fluid shear stress. In this study, we developed a microfluidic system featuring reversible sealing between the device layer and the cell culture substrate, enabling (1) conventional open-well cell culture prior to fluid shear stress application and (2) selective patterning of proteins and cells for migration studies. Channel dimensions were chosen to support 24-hour perfusion using standard syringe pumps. Using this platform, we investigated effects of physiological shear stress levels representing basal and activated pancreatic function (2, 8 and 33 mPa) on human iPSC-derived pancreatic progenitor cells. Pancreatic progenitor cells exhibit graded sensitivity to shear stress at the transcriptomic level. Specifically, shear stress upregulated expression of cell cycle-related gene sets; endocrine-associated genes including NEUROG3; and Notch receptor ligands (DLL1, DLL3) in a magnitude-dependent manner. The exposure to unidirectional fluid flow also resulted in a reduction in YAP1 protein content, decreased WWTR1 gene expression, and downregulation of YAP/TAZ target genes. Furthermore, barrier-based migration assays under shear stress revealed that cell patches expanded their boundaries both along and against the flow, with greater distance in the direction of flow. Our system and findings may help lay the foundation for understanding biophysical regulation of developmental processes in a physiologically important, yet hard-to-access, epithelial model system.

Oxygen (O2) transport is a major limitation in high-density cell culture, particularly as three-dimensional (3D) spheroids are increasingly used to better mimic in vivo tissue environments. Here, we developed a poly(4-methyl-1-pentene) (PMP)-based microwell platform, leveraging its lower molecular absorption and greater mechanical stability than the commonly used O2-permeable polydimethylsiloxane (PDMS), while retaining high O2 permeability. We measured O2 permeability in 50-m-thick PMP membranes and found approximately 200-fold greater O2 permeability than the conventional 1-mm polystyrene culture-plate material. PMP membrane-based microwell plates with 350-m-diameter wells maintained spatial separation of pre-formed hepatocyte spheroids while supplying O2 from the plate bottom, synergistically enhancing spheroid oxygenation. Compared with non-O2-permeable plates, the platform reduced localized hypoxia and hypoxia-associated cell injury while improving spheroid viability and growth. Collectively, PMP-based O2-permeable microwell plates provide a promising, scalable, high-throughput platform for spheroid culture with multiple applications in tissue engineering, drug screening and beyond.

Diffuse midline gliomas are a group of tumours for which no effective therapies exist. These tumours have substantial intratumoural heterogeneity and harbour beneficial subclonal interactions. Therapeutic failure due to resistance is a major concern in these tumours. We demonstrate how distinct subpopulations derived from the same tumour can interact with one another to alleviate the impacts of therapeutic intervention. This is achieved through the establishment of a mathematical extension to the Lotka-Volterra competition model, where interactions perturbing the Allee effect were demonstrated to exist. These interactions had substantial downstream consequences as they were determined to be perturbed by therapeutic interventions and strikingly, when simulating a two-drug paradigm, the interactions were demonstrated to substantially later the outcomes of treatment scheduling.

The immunosuppressive tumor microenvironment (TME) poses formidable challenges for cancer immunotherapies. Among the complex TME landscape, the cytokine TGF-{beta}1 is an attractive soluble target for reinvigoration of anti-tumor immunity due to its pivotal function in immune cell suppression, extracellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT) promotion, while immune checkpoint receptors exemplified by PD-1 and CD24 are promising surface targets. Though adaptive immune checkpoint inhibition is well established in clinical practice, TGF-{beta} blockade or innate immune checkpoint blockade is yet to be harnessed for tumor treatment. Here, leveraging CD24 overexpression on multiple tumor tissues and its unique recycling kinetics, we designed an innate Immune Checkpoint TArgeting Chimera (ICTAC) G01, a bifunctional CD24 antibody-TGF-{beta}1 receptor fusion protein that disrupts TGF-{beta}1 signaling by TGF-{beta}1 degradation instead of simple blockade and simultaneously inhibits the "don't eat me" CD24 signaling. We demonstrated that G01 effectively suppressed TGF-{beta}1-Smad signaling, reversed the EMT, and attenuated fibrotic collagen deposition. Additionally, it antagonized CD24 signaling to unleash robust antibody-dependent effector activity, further reprogramming the immunologically cold TME toward inflammatory phenotype. By selectively targeting malignant cells while sparing CD24-expressing B cells, G01 demonstrated a favorable safety profile. Notably, in patient-derived tumor fragments (PDTFs), G01 also inflamed the TME, underscoring its translational potential. Together, these results establish ICTAC as an effective strategy for tumor-specific receptor-mediated extracellular protein depletion, which supports its application for reversing TGF-{beta}1; and innate immune checkpoint-mediated tumor immunosuppression by concurrent targeting of both surface and soluble tumor-promoting factors.

Bone formation and remodeling depend on dynamic biochemical and biomechanical signaling from the collagen-rich osteoid that precedes mineralization, yet the role of osteoid nonlinear mechanics in regulating osteocyte-like network formation remains poorly understood. Here, we engineered a synthetic bottlebrush polymer hydrogel (BB) that mimics key mechanical features of osteoid and compared it to collagen type-I (Col1) matrices with matched shear modulus (~70 Pa) and strain-stiffening behavior. Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) were cultured for 28 days in growth (GM) or osteogenic media (OM) to examine network formation, and functional connectivity using live cell fluorescence recovery after photobleaching. hMSCs cultured in BB networks and OM showed upregulation of early osteocyte markers compared to Col1. We find that strain-stiffening materials with minimal stress relaxation promote osteocyte-like cell differentiation with functional connectivity, establishing osteoid-mimetic BB hydrogels as a promising matrix to study human osteocytogenesis and osteocyte mechanotransduction in vitro.

Matrix stiffness is a hallmark of aggressive solid tumors and strongly associated with therapeutic resistance and poor prognosis. This mechanical barrier represents a major unmet challenge in oncology, as current strategies rarely address how matrix mechanics themselves drive treatment resistance. Here, we identified ferroptosis, an iron-dependent form of cell death, as a potential long-sought vulnerability that can be exploited to eliminate mechanically resistant tumors. Using genetic and chemical approaches, we investigated how matrix stiffness sensitizes cancer cells to ferroptosis and whether this vulnerability can be therapeutically targeted. We found that cancer cells cultured on stiff matrices were more sensitive to ferroptosis than those on soft matrices, as evidenced by reduced cell viability and elevated lipid peroxidation. Furthermore, we found that matrix stiffness induced YAP activation, which upregulated its target gene SKP2, a key mediator required for the heightened ferroptosis sensitivity observed under stiff conditions. Finally, using an injectable hydrogel system, we demonstrated that stiffened tumors were resistant to chemotherapy but remained highly susceptible to ferroptosis induction, resulting in markedly reduced tumor volume and weight. Our findings established ferroptosis as a stiffness-dependent vulnerability mediated by a YAP-SKP2 signaling axis, revealing a mechanotransductive pathway that may be leveraged to target treatment-refractory tumors.

Oscillatory and saturating dynamics in regulatory gene networks govern processes such as cell cycle timing, circadian rhythms, and inflammatory signaling, and sparse regression methods such as SINDy offer an interpretable method for recovering their governing equations from time-series data. However, these networks are typically governed by Hill-type nonlinearities that become nearly collinear with polynomial candidates over most bounds of an oscillating curve, and placing them in a shared library produces ill-conditioned regressions and unreliable coefficient estimates. Standard approaches such as ridge regularization, library normalization, and orthogonalization can marginally improve conditioning but cannot remove the structural multicollinearity of these systems, in turn limiting our ability to produce accurate parameter estimates. To address this issue, we propose the Regulation-Informed Sparse Identification of Nonlinear Dynamics (RI-SINDy) method, which removes known regulatory terms from the regression and recovers their coefficients directly from an asymptotic drift-balance condition. Because the time averaged derivative of a bounded trajectory approaches zero, the coefficients of regulatory nonlinearities can be computed independently by constraining them to offset the mean contribution of the remaining terms. We demonstrate that this method effectively reduces library collinearity and improves conditioning numbers by 3-45x, substantially improving coefficient recovery and regression accuracy. We test the RI-SINDy method on experimental Hes1 data and on synthetic Goodwin and NF-{kappa}B models. On Hes1, it achieves the lowest trajectory error among the compared methods (Traditional SINDy, SR3, and Nullcline Reconstruction). On the synthetic systems, it recovers the correct sparse structure with lower trajectory error than SR3 and Nullcline Reconstruction, and in a Goodwin noise sweep it recovers all active terms at noise levels up to 15%, where Traditional SINDy and E-SINDy fail to. In conclusion, the RI-SINDy method provides a robust framework to overcome the issue of multicollinearity for system identification in biological systems.

Abstract Background: Experimental models that retain endogenous tumor immune complexity are needed to investigate heterogeneous responses to immune checkpoint inhibitors (ICIs) in melanoma. We established patient-derived melanoma organoids (PDMOs) to examine retention of parental tumor cellular components and investigate patient-specific and context-dependent responses to checkpoint blockade. Methods: Fresh melanoma specimens (n=50), including primary tumors, neoadjuvant-treated tumors and tumor-infiltrating lymphocyte (TIL) associated samples were processed for Matrigel-embedded culture of patient-derived melanoma organoids (PDMOs). Characterization and functional studies were performed in subsets of established cultures. Immunofluorescence assessed tumor, stromal, immune, and checkpoint-marker expression in PDMOs and matched parental tissues. NGFR expression was evaluated in relation to organoid establishment and growth. Responses to anti PD1 alone or combined with anti-LAG3 or anti-CTLA4 therapies were evaluated using viability and morphological analyses, with selected models undergoing immune phenotyping and multiplex cytokine profiling. Available clinical outcomes were used for exploratory comparison. PDMOs and matched two-dimensional cultures were evaluated under 21% and 5% oxygen conditions. Results: PDMOs were established from 30 of 50 specimens (60%) and retained melanoma, stromal, lymphoid, and myeloid components identified in matched parental tissues during early culture, together with immune checkpoint expression. Higher NGFR (CD271) expression was associated with greater organoid-forming capacity in an exploratory subset. Responses to checkpoint blockade varied across patient-derived models and regimens, with combination blockade not uniformly producing lower viability than PD1 monotherapy. Ex vivo responses showed parallels with available clinical outcomes, with discordant cases also observed. In three selected PDMOs, divergent PD-1 responses were accompanied by differences in T-cell and myeloid representation, MHCII and AXL expression, and inflammatory versus immunoregulatory cytokine profiles. Under normoxic conditions, PDMOs and matched two-dimensional cultures exhibited divergent treatment sensitivities, including clinically responding cases in which sensitivity was retained in PDMOs but attenuated in monolayers. Changing oxygen tension further altered checkpoint sensitivity within individual PDMOs, with the direction and magnitude of the observed shifts varying by model and regimen. Conclusions: Early passage PDMOs retain key endogenous tumor, immune, and stromal components and support functional investigation of patient-derived melanoma biology. Their heterogenous responses across checkpoint regimens and oxygen conditions provide a platform for investigating how tumor-immune composition and environmental context influence treatment sensitivity.

Moment-closure methods provide a widely used approach for approximating low-order statistics of stochastic biochemical reaction networks, but the resulting closed equations may exhibit physically meaningless dynamics. Here, we systematically investigate the stability of second-order moment-closure approximations for a bursty gene expression model with nonlinear protein degradation. We consider the Gaussian, log-normal, and negative-binomial closures. By analyzing the resulting dynamical systems, we show that the Gaussian closure fails the proposed stability criterion for all parameter values, whereas both the log-normal and negative-binomial closures possess a unique physically accessible fixed point and are stable if and only if kB(1 + 2B) [≥] 1, where B is the mean burst size and k is the dimensionless protein synthesis rate. Moreover, this condition is automatically satisfied when the steady-state mean protein copy number is at least one, suggesting that it is mild in biologically relevant regimes. Numerical comparisons over a broad parameter range further show that the negative-binomial closure is the most accurate, while the Gaussian closure is the least accurate. We further develop higher-order negative-binomial moment-closure schemes and demonstrate that increasing the closure order substantially improves predictive accuracy. These results highlight the importance of considering dynamical stability, in addition to numerical accuracy, when assessing moment-closure approximations.

Gelatin methacrylate (GelMA) hydrogels offer many advantageous properties such as excellent biocompatibility, tunable stiffness, and rapid fabrication, making them attractive materials for neural systems-on-a-chip devices used to study axonal outgrowth in three-dimensional (3D) environments. In this study, we evaluate properties of GelMA hydrogels crosslinked with visible blue light and application of these hydrogels for encapsulation of primary dorsal root ganglion (DRG) explants inside a custom microscale device. We selected low GelMA polymer concentrations of 3 % and 6 % w/v for hydrogel preparation to create soft materials and demonstrate the effect of degree of functionalization (DoF) on stiffness of GelMA in a blue light-induced crosslinking system. Additionally, a decrease in crosslinking efficiency and stiffness of GelMA was observed upon reconstitution of the polymer in DMEM cell culture media compared to PBS. To evaluate the axonal outgrowth, DRG explants were encapsulated in various GelMA formulations and cultured for 7 days. Following incubation, the explants were fixed and immunochemically labelled with anti-beta-III-tubulin antibody. We further describe an application of custom-developed semi-automated image analysis tool together with linear mixed effects modeling to evaluate axonal outgrowth across different GelMA formulations. Among the formulations tested in our study, 3 % GelMA 95 % DoF hydrogels supported the longest neurite outgrowth suggesting that matrix properties influenced neurite extension.