Experimental Model System Research Articles

Experimental models of antibiotic exposure and atopic disease.

In addition to numerous clinical studies, research using experimental models have contributed extensive evidence to the link between antibiotic exposure and atopic disease. A number of mouse models of allergy have been developed and used to uncover the specific effects of various microbiota members and perturbations on allergy development. Studies in mice that lack microbes entirely have also demonstrated the various components of the immune system that require microbial exposure. The importance of the early-life period and the mechanisms by which atopy "protective" species identified in human cohorts promote immune development have been elucidated in mice. Finally, non-animal models involving human-derived cells shed light on specific effects of bacteria on human epithelial and immune responses. When considered alongside clinical cohort studies, experimental model systems have provided crucial evidence for the link between the neonatal gut microbiota and allergic disease, immensely supporting the stewardship of antibiotic administration in infants. The following review aims to describe the range of experimental models used for studying factors that affect the relationship between the gut microbiota and allergic disease and summarize key findings that have come from research in animal and in vitro models.

TRESK channel activation ameliorates migraine-like pain via modulation of CGRP release from the trigeminovascular system and meningeal mast cells in experimental migraine models

AimsAccumulating evidence indicates the involvement of TRESK potassium channels in migraine, however, effects of TRESK activation on migraine-related mechanisms remain unclear. We explored effects of TRESK channel modulation on migraine-related behavioral and molecular markers in in-vivo and ex-vivo rat models of migraine. Main methodsThe selective TRESK activator cloxyquin at different doses, the TRESK inhibitor A2764, and the migraine drug sumatriptan were tested alone or in different combinations in nitroglycerin (NTG)-induced in-vivo model, and in ex-vivo meningeal, trigeminal ganglion and brainstem preparations in which CGRP release was induced by capsaicin. Mechanical allodynia, CGRP and c-fos levels in trigeminovascular structures and meningeal mast cells were evaluated. Key findingsCloxyquin attenuated NTG-induced mechanical allodynia, brainstem c-fos and CGRP levels, trigeminal ganglion CGRP levels and meningeal mast cell degranulation and number, in-vivo. It also diminished capsaicin-induced CGRP release from ex-vivo meningeal, trigeminal ganglion and brainstem preparations. Specific TRESK inhibitor A2764 abolished all effects of cloxyquin in in-vivo and ex-vivo. Combining cloxyquin and sumatriptan exerted a synergistic effect ex-vivo, but not in-vivo. SignificanceOur findings provide the experimental evidence for the anti-migraine effect of TRESK activation in migraine-like conditions. The modulation of TRESK channels may therefore be an attractive alternative strategy to relieve migraine pain.

Decorin, an exercise-induced secretory protein, is associated with improved prognosis in breast cancer patients but does not mediate anti-tumorigenic tissue crosstalk in mice

BackgroundRegular exercise can reduce incidence and progression of breast cancer, but the mechanisms for such effects are not fully understood. MethodsWe used a variety of rodent and human experimental model systems to determine whether exercise training can reduce tumor burden in breast cancer and to identify mechanism associated with any exercise training effects on tumor burden. ResultsWe show that voluntary wheel running slows tumor development in the mammary specific polyomavirus middle T antigen overexpression (MMTV-PyMT) mouse model of breast cancer but only when mice are not housed alone. We identify the proteoglycan decorin as a contraction-induced secretory factor that systemically increases in patients with breast cancer immediately following exercise. Moreover, high expression of decorin in tumors is associated with improved prognosis in patients, while treatment of breast cancer cells in vitro with decorin reduces cell proliferation. Notwithstanding, when we overexpressed decorin in murine muscle or injected recombinant decorin systemically into mouse models of breast cancer, elevated plasma decorin concentrations did not result in higher tumor decorin levels and tumor burden was not improved. ConclusionExercise training is anti-tumorigenic in a mouse model of luminal breast cancer, but the effect is abrogated by social isolation. The proteoglycan decorin is an exercise-induced secretory protein, and tumor decorin levels are positively associated with improved prognosis in patients. The hypothesis that elevated plasma decorin is a mechanism by which exercise training improves breast cancer progression in humans is not, however, supported by our pre-clinical data since elevated circulating decorin did not increase tumor decorin levels in these models.

Biogenic Sulfide-Mediated Iron Reduction at Low pH

Acid mine drainage (AMD) pollutes natural waters, but some impacted systems show natural attenuation. We sought to identify the biogeochemical mechanisms responsible for the natural attenuation of AMD. We hypothesized that biogenic sulfide-mediated iron reduction is one mechanism and tested this in an experimental model system. We found sulfate reduction occurred under acidic conditions and identified a suite of sulfate-reducing bacteria (SRB) belonging to the groups Desulfotomaculum, Desulfobacter, Desulfovibrio, and Desulfobulbus. Iron reduction was not detected in microcosms when iron-reducing bacteria or SRB were selectively inhibited. SRB also did not reduce iron enzymatically. Rather, the biogenic sulfide produced by SRB was found to be responsible for the reduction of iron at low pH. Addition of organic substrates and nutrients stimulated iron reduction and increased the pH. X-ray diffraction and an electron microprobe analysis revealed that the polycrystalline, black precipitate from SRB bioactive samples exhibited a greater diversity of iron chalcogenide minerals with reduced iron oxidation states, and minerals incorporating multiple metals compared to abiotic controls. The implication of this study is that iron reduction mediated by biogenic sulfide may be more significant than previously thought in acidic environments. This study not only describes an additional mechanism by which SRB attenuate AMD, which has practical implications for AMD-impacted sites, but also provides a link between the biogeochemical cycling of iron and sulfur.

Optimizing Numerical Weather Prediction Utility of the Maryland Mesonet with Observing System Simulation Experiments

Abstract The Maryland Mesonet Project will construct a network of 75 surface observing stations with aims that include mitigating the statewide impact of severe convective storms and improving analyses of record. The spatial configuration of mesonet stations is expected to affect the utility newly provided observations will have via data assimilation, making it desirable to study the effects of mesonet configuration. Furthermore, the impact associated with any observing system configuration is constrained by errors inherent to the prediction systems used to generate forecasts, which may change with future advances in data assimilation methodology, physical parameterization schemes, and resource availability. To address such possibilities, we perform sets of observing system simulation experiments using a high-resolution regional modeling system to assess the expected impact of four candidate mesonet configurations. Experiments cover seven 18-hour case-study events featuring moist convective regimes associated with severe weather over the state of Maryland and are performed using two versions of our experimental modeling system: a ’standard-uncertainty’ configuration tuned to be representative of existing convective-allowing prediction systems, and a ’constrained-uncertainty’ configuration with reduced boundary condition and model error that reflects a possible trajectory for future prediction systems. We find that the assimilation of mesonet data produces definitive improvements to analysis fields below 1000 m that are mediated by modeling system uncertainty. Conversely, mesonet impact on forecast verification is inconclusive and strongly variable across verification metrics. The impact of mesonet configuration appears limited by a saturation effect that caps local analysis improvements past a minimal density of observing stations.

Numerical integration of mechanical forces in center-based models for biological cell populations

Center-based models are used to simulate the mechanical behavior of biological cells during embryonic development or cancer growth. To allow for the simulation of biological populations potentially growing from a few individual cells to many thousands or more, these models have to be numerically efficient, while being reasonably accurate on the level of individual cell trajectories. In this work, we increase the robustness, accuracy, and efficiency of the simulation of center-based models by choosing the time steps adaptively in the numerical method and comparing five different integration methods. We investigate the gain in using single rate time stepping based on local estimates of the numerical errors for the forward and backward Euler methods of first order accuracy and a Runge-Kutta method and the trapezoidal method of second order accuracy. Properties of the analytical solution such as convergence to steady state and conservation of the center of gravity are inherited by the numerical solutions. Furthermore, we propose a multirate time stepping scheme that simulates regions with high local force gradients (e.g. as they happen after cell division) with multiple smaller time steps within a larger single time step for regions with smoother forces. These methods are compared for a model system in numerical experiments. We conclude, for example, that the multirate forward Euler method performs better than the Runge-Kutta method for low accuracy requirements but for higher accuracy the latter method is preferred. Only with frequent cell divisions the method with a fixed time step is the best choice.

Study on the Rules of Ground Settlement and Pipeline Deformation Considering the Combined Effects of Pipeline Damage Leakage and Shield Tunneling Construction

A pipeline with long-term “hidden leakage” will greatly reduce the stability of the ground between the pipeline and tunnel in the process of tunneling through existing pipelines in unsaturated soil. Excessive settlement of the surrounding strata and pipelines can occur when the shield excavation face approaches below a pipeline, which can lead to engineering accidents. This study is based on a self-developed model experimental system for tunneling through an existing pipeline with a double-line tunnel shield. The ground settlement and pipeline deformation caused by shield construction with small-scale and no leakages are investigated. An experimental study is conducted and the accuracy of the results is verified through a comparison with theoretical solutions. The results demonstrate that there is a significant increase in ground settlement and pipeline deformation under the influence of leakage water. It is also determined that the displacement field generated by the excavation of a double-line tunnel is not simply a superposition of the displacement field generated by the excavation of a single-line tunnel. The repeated disturbances caused by the excavation of a double-line tunnel significantly influences the redistribution of the displacement field. Additionally, a three-dimensional (3D) model of shield construction considering the influence of pipeline leakage is established. This study discusses the ground settlement and pipeline deformation patterns caused by changes in the vertical and horizontal leakage diffusion ranges. The computational results indicate that the diffusion depth of a leakage is the primary factor controlling the extent of settlement.

Constrained motion of self-propelling eccentric disks linked by a spring.

It has been supposed that the interplay of elasticity and activity plays a key role in triggering the non-equilibrium behaviors in biological systems. However, the experimental model system is missing to investigate the spatiotemporally dynamical phenomena. Here, a model system of an active chain, where active eccentric-disks are linked by a spring, is designed to study the interplay of activity, elasticity, and friction. Individual active chain exhibits longitudinal and transverse motions; however, it starts to self-rotate when pinning one end and self-beat when clamping one end. In addition, our eccentric-disk model can qualitatively reproduce such behaviors and explain the unusual self-rotation of the first disk around its geometric center. Furthermore, the structure and dynamics of long chains were studied via simulations without steric interactions. It was found that a hairpin conformation emerges in free motion, while in the constrained motions, the rotational and beating frequencies scale with the flexure number (the ratio of self-propelling force to bending rigidity), χ, as ∼(χ)4/3. Scaling analysis suggests that it results from the balance between activity and energy dissipation. Our findings show that topological constraints play a vital role in non-equilibrium synergy behaviors.

Functional loss of ERBB receptor feedback inhibitor 1 (MIG6) promotes glioblastoma tumorigenesis by aberrantactivation of epidermal growth factor receptor (EGFR).

Dysregulation of epidermal growth factor receptor (EGFR) is one of the most common mechanisms associated with the pathogenesis of various cancers. Mitogen-inducible gene 6 [MIG6; also known as ERBB receptor feedback inhibitor 1 (ERRFI1)], identified as a feedback inhibitor of EGFR, negatively regulates EGFR by directly inhibiting its kinase activity and facilitating its internalization, subsequently leading to degradation. Despite its proposed role as an EGFR-dependent tumor suppressor, the functional consequences and clinical relevance in cancer etiology remain incompletely understood. Here, we identify that the stoichiometric balance between MIG6 and EGFR is crucial in promoting EGFR-dependent oncogenic growth in various experimental model systems. In addition, a subset of ERRFI1 (the official gene symbol of MIG6) mutations exhibit impaired ability to suppress the enzymatic activation of EGFR at multiple levels. In summary, our data suggest that decreased or loss of MIG6 activity can lead to abnormal activation of EGFR, potentially contributing to cellular transformation. We propose that the mutation status of ERRFI1 and the expression levels of MIG6 can serve as additional biomarkers for guiding EGFR-targeted cancer therapies, including glioblastoma.

Abstract Mo071: Engineered Platform to Uncover Cellular Cross-Talk and Arrhythmogenesis in Idiopathic Ventricular Fibrillation (IVF)

Background: Idiopathic VF (IVF) represents an unmet clinical need with ~50% of patients having no known monogenic cause. This has resulted in the lack of an appropriate experimental model system, limiting effective treatment through an evidence-based approach. Within IVF, several subphenotypes have emerged through Deep Phenotyping. We recruited patients with Purkinje cell premature ventricular contraction-driven IVF (PVC-IVF) to generate induced pluripotent stem cells (iPSCs). Aim: To characterize PVC-IVF patient-specific iPSCs using a physiologically relevant engineered in vitro model. We hypothesize this model will allow for mechanistic characterization of PVC-IVF. Methods: Patient-specific iPSC-cardiomyocytes (CMs) and iPSC-cardiac fibroblasts (CFs) were plated on 10 kPa polydimethylsiloxane (PDMS) culture platform with Matrigel in either monolayers or interconnected 40 µm lanes ( Fig. 1A ). Optical mapping was used to characterize Ca 2+ handling and action potentials. Control cells were from non-diseased persons. Results: CM-CF coculture results in a monolayer yet maintains the underlying organization ( Fig. 1B-C ). Patterned constructs have increased conduction velocities and anisotropy. Custom stimulation protocols were developed to probe arrhythmia susceptibility and restitution kinetics ( Fig. 1D-E ). A genetic algorithm was used to optimize a biophysically detailed CM model which enabled identification of differential model parameters between IVF and control data ( Fig. 2A-B ). These data will be used to develop a Ca 2+ handling–conduction–contractility paradigm specific to PVC-IVF as well as a mechanistic-based understanding of the disorder. Conclusions: Topography informs CM structure, while CFs remodel the matrix to form a monolayer, enabling full field electrophysiological definition at the cell and tissue scale. The in vitro model provides a unique microenvironment to determine the arrhythmogenic mechanism of PVC-IVF.

Stable isotope-resolved metabolomics analyses of metabolic phenotypes reveal variable glutamine metabolism in different patient-derived models of non-small cell lung cancer from a single patient.

Stable isotope tracers have been increasingly used in preclinical cancer model systems, including cell culture and mouse xenografts, to probe the altered metabolism of a variety of cancers, such as accelerated glycolysis and glutaminolysis and generation of oncometabolites. Comparatively little has been reported on the fidelity of the different preclinical model systems in recapitulating the aberrant metabolism of tumors. We have been developing several different experimental model systems for systems biochemistry analyses of non-small cell lung cancer (NSCLC1) using patient-derived tissues to evaluate appropriate models for metabolic and phenotypic analyses. To address the issue of fidelity, we have carried out a detailed Stable Isotope-Resolved Metabolomics study of freshly resected tissue slices, mouse patient derived xenografts (PDXs), and cells derived from a single patient using both 13C6-glucose and 13C5,15N2-glutamine tracers. Although we found similar glucose metabolism in the three models, glutamine utilization was markedly higher in the isolated cell culture and in cell culture-derived xenografts compared with the primary cancer tissue or direct tissue xenografts (PDX). This suggests that caution is needed in interpreting cancer biochemistry using patient-derived cancer cells in vitro or in xenografts, even at very early passage, and that direct analysis of patient derived tissue slices provides the optimal model for ex vivo metabolomics. Further research is needed to determine the generality of these observations.

Neuropathy and pain in Fabry disease

Fabry disease (FD) is a multiorgan lysosomal storage disorder caused by mutations in the alfa-galactosidase A (GLA ) gene. Pathogenic GLA mutations lead to impaired or even lost enzyme activity, which causes the accumulation of sphingolipids, e.g., globotriaosylceramide, in cells and tissues. The majority of FD patients experience triggerable pain, mainly acral and burning, which often begins in early childhood. While small fiber pathology is assumed to be the basis of FD pain, the underlying molecular mechanisms are not well understood. This review summarizes the clinical characteristics of neuropathy and neuropathic pain in FD, presents current treatment options, and gives an overview of the latest findings from experimental and human model systems on the pathomechanisms contributing to small fiber pathology and FD-associated pain.

Rapid body colouration changes in Oryzias celebensis as a social signal influenced by environmental background.

Rapid body colouration changes in some animals, such as chameleons and octopuses, serve dual functions: camouflage and intraspecific communication. It has been hypothesized that these colouration changes originally evolved to provide camouflage and subsequently were co-opted as social signals; however, experimental model systems that are suitable for studying such evolutionary processes are limited. Here, we investigated the relationship between rapid colouration changes of the blackened markings and aggressive behaviours in male Oryzias celebensis, an Indonesian medaka fish, under triadic relationships (two males and one female) or three males conditions with two different environmental backgrounds. In an algae-covered tank, mimicking the common laboratory rearing conditions, males with blackened markings exhibited more frequent attacks towards different conspecific individuals compared with non-blackened males and females. The blackened males were seldom attacked by non-blackened males and females. By contrast, neither aggressive behaviours nor black colouration changes were observed in the transparent background condition with a brighter environment. These indicated that the blackened markings in O. celebensis serve as a social signal depending on the environmental backgrounds. Considering that such colouration changes for camouflage are widely conserved among teleost fishes, the traits are likely to be co-opted for displaying social signals in O. celebensis.

Controlled creation of point defects in three-dimensional colloidal crystals.

Crystal defects crucially influence the properties of crystalline materials and have been extensively studied. Even for the simplest type of defect-the point defect-however, basic properties such as their diffusive behavior, and their interactions, remain elusive on the atomic scale. Here, we demonstrate in situ control over the creation of isolated point defects in a three-dimensional (3D) colloidal crystal allowing insight on a single-particle level. Our system consists of thermoresponsive microgel particles embedded in a crystal of nonresponsive colloids. Heating this mixed-particle system triggers the shrinking of the embedded microgels, which then vacate their lattice positions, creating vacancy-interstitial pairs. We use temperature-controlled confocal laser scanning microscopy to verify and visualize the formation of the point defects. In addition, by reswelling the microgels we quantify the local lattice distortion around an interstitial defect. Our experimental model system provides a unique opportunity to shed light on the interplay between point defects, on the mechanisms of their diffusion, on their interactions, and on collective dynamics.

ФЛУОРИМЕТРИЧЕСКАЯ ОЦЕНКА ВЛИЯНИЯ РИБОКСИНА, МЕДНОГО ХЛОРОФИЛЛИНА, ТРОЛОКСА И РАСТВОРИМОЙ ФОРМЫ ИНДРАЛИНА НА РОСТОВЫЕ СВОЙСТВА КЛЕТОК А549 В КУЛЬТУРЕ

Currently, cell cultures are most often used as an experimental model system in biological research. However, to correctly plan studies using this model system, many aspects must be taken into account. Thus, to be able to correctly assess the effect of radioprotective drugs on cells, it is first necessary to study the effect of these substances on the properties of cells in culture. And the main property for work using plate readers is the ability of cells to adhere to the bottom of the plate and the rate of cell proliferation. This work is devoted to the study of human riboxin (inosine), copper chlorophyllin, trolox and indralin, the growth properties of cells in a culture based on the A549 cell line - human lung adenocarcinoma. Riboxin, chlorophyllin and trolox are promising compounds that make it possible to study their radioprotective properties. Indralin is a reference classic radioprotector. This process used a water-soluble form containing tartaric acid to allow indralin to dissolve in water. The experiment consisted of incubating A549 cells for 24 hours in a solution of riboxin (inosine), copper chlorophyllin, Trolox or tartaric acid at a concentration of 2 mM or in a mixture of 2 mM tartaric acid and 1.9 mM indralin, followed by assessment of the cell content in the samples compared to cells incubated without the addition of these substances, based on the fluorescence of the Hoechst-33342 dye. An additional experiment with chlorophyllin consisted in incubating cells with chlorophyllin already attached to the bottom of the tablet in the concentration range of 50–500 μM for 2.5 hours, followed by an assessment of the content of the remaining cells in the tablet. All studied substances statistically significantly reduced the cell content in the samples compared to the control. The greatest decrease in cell content was observed in the sample with chlorophyllin, and the least – with riboxin. As a result of additional experience in incubating already attached cells in chlorophyllin solution with concentrations of 50–500 μM, it was shown that this substance dose-dependently inhibits the adhesive properties of cells of the A549 line. At the same time, chlorophyllin already at a concentration of 50 μM statistically significantly reduced the cell content in the sample after washing the wells of the tablet compared with the control sample. The decrease in cell content in the sample containing a mixture of indralin and tartaric acid was statistically significantly more pronounced than that caused by incubation in a solution of tartaric acid alone. That is, the recognized radioprotector indralin demonstrated a pronounced inhibition of the growth properties of the A549 cell line. Based on the information obtained, we can conclude that when planning future studies of the substances studied in this work on a cell culture model, it is necessary to take into account the fact that they inhibit the growth of the cell culture.

Protocol Standardization of Homeopathic Medicines' Preparation and Dosing for Experimental Model Systems In Vitro.

Protocol Standardization of Homeopathic Medicines' Preparation and Dosing for Experimental Model Systems In Vitro.

Deciphering the role of FUS::DDIT3 expression and tumor microenvironment in myxoid liposarcoma development

BackgroundMyxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene, however, the precise functional contributions of these two elements remain enigmatic in tumor development.MethodsTo study the cell-free microenvironment in MLS, we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently, scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing.ResultsCharacterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions, as well as chromatin remodeling, immune response, and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS.ConclusionsOur experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.

Abstract LB072: Tumor-infiltrating lymphocytes (TIL) engineered with membrane-bound IL15 (cytoTIL15 cells) exhibit pharmacologically regulatable signal transduction in cis and trans

Abstract Introduction: We have previously shown the successful engineering of membrane-bound IL15 (mbIL15)-expressing tumor-infiltrating lymphocytes (TIL; cytoTIL15™ cells) from solid tumors such as melanoma, which are CD8+ enriched TIL exhibiting enhanced persistence and anti-tumor activity compared with unengineered TIL (SITC 2022, 2023; clinical candidate OBX-115: NCT05470283). The mbIL15 expressed by cytoTIL15 cells is regulated via a carbonic anhydrase 2 (CA2) drug-responsive domain, which allows expression to be induced upon delivery of the FDA-approved small molecule ligand acetazolamide (ACZ) and was designed to engage the IL2Rβ and IL2Rγ receptors in both cis and in trans. Using natural killer (NK) cells, which are potently responsive to soluble, trans-presented IL15, as an experimental model system, we examined the potential of mbIL15 on cytoTIL15 cells to potentiate activity in both cis and trans. Methods: cytoTIL15 cells and unengineered TIL were generated as described previously (SITC 2022, 2023). Cells were assessed for expression of mbIL15 and phosphorylation of downstream signaling molecules STAT5 and S6 by flow cytometry, and co-cultured with allogeneic healthy donor NK cells for 1-10 days with and without transwell inserts. Functional effects of cytoTIL15 cells were compared to those of soluble recombinant IL15. Supernatant was obtained from co-cultures for assessment of cytokine production. Results: cytoTIL15 cells demonstrated robust ACZ-dependent mbIL15 expression, and ACZ dosing regulated mbIL15 activity in cis by potentiating the phosphorylation of STAT5 (geoMFI with 0 vs. 25 µM ACZ, p=0.012) and S6 (geoMFI with 0 vs. 25 µM ACZ, p=0.016). Upon removal of ACZ, mbIL15 expression and pSTAT5 and pS6 returned to basal levels within 24 hours. mbIL15 expressed on the surface of cytoTIL15 cells could also activate cells in trans in an ACZ-dependent titratable manner. Specifically, co-culture of cytoTIL15 cells with NK cells led to an ACZ-dependent increase in pSTAT5 (geoMFI with 0 vs. 25 µM ACZ, p=0.038) in NK cells, which was not seen in co-cultures of unengineered TIL with NK cells. Additionally, NK cells showed a similar cytokine production profile when exposed to mbIL15 by cytoTIL15 cells as with soluble, trans-presented IL15. NK cell transactivation relied on direct cell contact with cytoTIL15 cells, as magnitudes of phosphorylation were decreased in co-cultures with NK and cytoTIL15 cells separated by a transwell membrane (pSTAT5 geoMFI p=0.008). Conclusions: cytoTIL15 cells demonstrate functional engagement of cytokine receptors via mbIL15 in both cis and in trans, including phosphorylation of STAT5 and S6. Taken together, these data highlight the potential for robust activity of cytoTIL15 cells through potentiation of ACZ-dependent mbIL15 activity in both cis and trans. Citation Format: Rachel Burga, Gauri Kulkarni, Zheng Ao, Dhruv K. Sethi, Jan ter Meulen, Michelle Ols. Tumor-infiltrating lymphocytes (TIL) engineered with membrane-bound IL15 (cytoTIL15 cells) exhibit pharmacologically regulatable signal transduction in cis and trans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB072.

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair.

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

Abstract 5129: Subcellular MLH1 protein as first in class biomarker for CDK4/6 response in endocrine resistant ER+/HER2- breast cancer

Abstract Over 175,000 diagnoses of estrogen receptor-positive breast cancer are made every year in the US. Of these, >40,000 patients will go on to die of their disease. We recently identified defects in a principal mismatch repair (MMR) gene, MLH1, as a driver of resistance to standard-care treatment and poor patient outcomes. Moreover, we demonstrated increased sensitivity to palbociclib, a CDK4/6 inhibitor, in experimental model systems in vitro and in vivo and in clinical trial data. MLH1 is mutated in 5-10% of treatment-resistant tumors, i.e., 4,000 patient tumors every year. However, most of these mutations are missense and variants of unknown significance (VUS). The impact of these mutations on mismatch repair capability and on treatment resistance remains uncertain. Here, we use experimental model systems to phenotype MLH1 VUS´ in vitro and in vivo using xenograft systems.Specifically, we use nuclear localization of MLH1 and repair of mismatches through a mismatch repair deficiency assay to test for mismatch repair capability in ER+ breast cancer cells engineered to express mutations in MLH1 that are found in patient tumors associated with poor survival. Second, we assess the ability of variants of MLH1 to induce activation of G1/S cell cycle checkpoints by activating Chk2. Lastly, we directly test the ability of variants in MLH1 to induce resistance to standard-care endocrine therapies and sensitivity to palbociclib, using 2D and 3D growth assays and xenograft experiments. Results from these experiments serve to systematically catalog VUS´ in MLH1 based on their predictive potential. We identified for the first-time specific mutation in MLH1 that induces resistance to endocrine therapy by stalling nuclear translocation of MLH1 which inhibits Chk2 activation. Cells with impaired Chk2 activation maintain a high CDK4/6 levels and are therefore more sensitive to CDK4/6 inhibition. Currently, there are few tests available in the clinic to predict response to therapies early in the timeline of ER+ breast cancer patients. Due to the nature of the disease, this can result in years of overtreatment. Overtreatment comes at great financial, physical, and emotional cost to patients and their families. Simultaneously, a lack of predictive markers can result in missed opportunities of presenting patients likely to relapse on standard care with personalized alternative therapeutic strategies. Based on our findings, cytoplasmic MLH1 may serve as first in-class biomarker for CDK4/6 response in ER+/HER2- patients. Citation Format: Aloran Mazumder, Elena Oropeza, Nindo Punturi, Svasti Haricharan. Subcellular MLH1 protein as first in class biomarker for CDK4/6 response in endocrine resistant ER+/HER2- breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5129.