By combining hanging-drop self-assembly with melt infiltration and selective inversion, we fabricate millimetric and free-standing curved photonic heterostructures that integrate infiltrated-opal, inverse-opal, embossed, and white-scattering 2.5D metasurface domains within a single continuous body. These architectures enable configurations inaccessible to planar fabrication, including naturally formed concavities within convex inverse-opal films and alternating ordered/single-layer regions that preserve local coherence while introducing disorder at larger scales. Across these heterogeneous curved landscapes, we observe optical phenomena absent in flat photonic structures-spectrally selected lateral collimation, geometry-shifted ghost images, and transmission-derived valleys shaped by curvature-mediated Bragg extraction. Their origin lies in the geometric constraints inherent to curved assemblies, where spatially varying normals, non-parallel lattice orientations, and topologically required defects couple order and disorder into a distributed-coherence regime. This coupling expands the accessible photonic state space, establishing curvature as an active functional degree of freedom rather than a geometric constraint, positioning the self-assembled photonic heterostructures as a scalable route toward multifunctional 3D metasurfaces and new regimes of light-matter interaction.

Background/Objectives: Metabolic dysfunction-associated steatotic liver disease (MASLD) involves the interplay of hepatic lipid accumulation and immune-mediated inflammatory signaling, yet human-relevant in vitro systems that capture both processes simultaneously in a scalable format remain limited. The objective of this study was to develop and characterize a matrix-free 3D hepatocyte-macrophage co-culture model enabling simultaneous assessment of lipid accumulation and NF-κB-mediated inflammatory activation under glucolipotoxic stress. Methods: A 3D liver co-culture model was established by combining HepG2 hepatocyte-like cells with phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophage-like cells stably expressing a NF-κB-Luc2 reporter. Spheroids were generated using a hanging-drop method in standard 96-well plates and matured for 8-10 days. Mature spheroids were subjected to acute 24 h glucolipotoxic challenge combining high glucose and palmitic acid and assessed for neutral lipid accumulation, NF-κB reporter activation (luciferase), and macrophage marker expression (qPCR). Results: Time-course characterization demonstrated progressive hepatocyte marker remodeling (albumin, alpha-fetoprotein, CYP3A4) and dynamic macrophage phenotype shifts (CD14, CD206, MARCO, TREM2). Acute glucolipotoxic challenge induced dose-dependent increases in neutral lipid accumulation and NF-κB reporter activation, accompanied by coordinated macrophage-associated transcriptional changes consistent with lipid-handling and tissue-remodeling programs. Post-challenge metabolic activity was retained under the selected stress conditions. As a proof-of-concept demonstration, three botanical extracts showed distinct attenuation profiles across the lipid and inflammatory endpoints. Conclusions: This 3D hepatocyte-macrophage co-culture model provides orthogonal readouts of steatosis and NF-κB-mediated inflammatory activation under glucolipotoxic stress, offering a reproducible, fit-for-purpose screening tool for investigating early glucolipotoxic hepatic responses and evaluating candidate compounds in a defined in vitro setting.

ABSTRACT Objective: Cancer, characterized by uncontrolled cell proliferation and invasion into surrounding tissues, is a leading cause of global mortality. Traditional two-dimensional (2D) cell culture systems fail to adequately replicate the tumor microenvironment (TME). In contrast, three-dimensional (3D) culture systems, which better simulate cell–cell and cell–extracellular matrix (ECM) interactions, have become powerful tools in biomedical research. This study aims to compare the spheroid formation capacity of A549 lung cancer cells using three different 3D culture methods: ultra-low attachment (ULA) plates, agarose hydrogel, and the hanging drop technique. The primary objective is to identify the most effective spheroid formation method for A549 cells and to provide findings that can guide future biomedical research, particularly in cancer modeling, drug screening studies, and investigations of the tumor microenvironment.Materials and Methods: A549 cells were cultured using three different 3D culture methods: ultra-low attachment plates, agarose hydrogel, and the hanging drop method. In the ultra-low attachment method, spheroid formation was observed at cell densities of 5,000, 10,000, and 30,000 cells/ml. In the agarose hydrogel method, agarose concentrations of 1%, 1.5%, and 2% were used to evaluate cell aggregation and spheroid stability. In the hanging drop method, cells aggregated under the influence of gravity. Spheroid diameter and area were analyzed using ImageJ software.Results: In this study, the spheroid formation capacity of A549 lung cancer cells was evaluated using three different three-dimensional (3D) culture methods. The ultra-low attachment (ULA) plate method allowed cell aggregation; however, the resulting structures were not large or compact enough to be classified as spheroids. The hanging drop method showed that cells formed small clusters by day 3 but failed to develop a compact and stable spheroid structure by day 7. The agarose hydrogel method, particularly at a 2% agarose concentration, demonstrated the highest spheroid formation capacity compared to the other methods. In this method, spheroid formation began at 72 hours depending on cell density, with significant growth observed at a density of 30,000 cells/ml (p < 0.0001). Trypan Blue staining results indicated that 2% agarose and cell densities of 10,000–30,000 cells/ml provided the highest cell viability. Specifically, 4,800 viable cells were counted at a density of 30,000 cells/ml, while 3,600 viable cells were observed at 10,000 cells/ml. These findings suggest that the agarose hydrogel method, especially at 2% agarose concentration and higher cell densities, offers optimal spheroid formation and cell viability for A549 lung cancer cells.Conclusion: This study demonstrated that the agarose hydrogel method effectively promoted stable and organized spheroid formation in A549 lung cancer cells. Notably, the 2% agarose concentration was identified as the most effective condition for maintaining cell viability and optimizing spheroid size. In contrast, the ultra-low attachment (ULA) plate and hanging drop methods exhibited limited spheroid formation capacity, resulting in less compact and disorganized structures. These findings emphasize the critical role of three-dimensional (3D) cell culture methods in biomedical research, particularly for experimental tumor modeling and drug screening studies. In this context, the agarose hydrogel method, with its high spheroid formation capacity and ability to support cell viability, emerges as a promising 3D culture model that warrants further exploration in cancer research.

Glioblastoma (GBM), a rare, highly aggressive and chemoresistant brain cancer, exhibits profound metabolic plasticity that relies, in part, on aberrant transforming growth factor-β (TGF-β) signaling. Such plasticity was recently associated with TGF-β-regulated apoptosis and autophagy. Here, we questioned whether TGF-β-regulated apoptotic/autophagic phenotypes are recapitulated in a preclinical in vitro 3D spheroid culture model of human U87 GBM-derived cells, and how metabolic alterations affect such phenotypes. 3D U87 spheroids were cultured using the hanging drop method. Western blotting was used to assess protein expression, while RT-qPCR was used to assess gene expression levels. 3D spheroids exhibited decreased AKT phosphorylation, and increased TGF-β, fibronectin, and Smad2 phosphorylation, indicative of both cell death signaling and epithelial-mesenchymal transition molecular signatures. 2-Deoxy-D-glucose (2DG), a glycolytic inhibitor, depleted ATP dose-dependently (30-300 μM) and prevented those increases both at the protein and transcriptional levels. This was also observed in 3D spheroids upon TGF-β transient siRNA-mediated silencing or when TGF-βR1 kinase activity was inhibited by galunisertib. Transcriptomic profiling revealed shared upregulation of apoptosis-related (BCL2, CASP7, FAS, FASLG, GADD45A) and autophagy-related (ATG7, ATG16L1, IRGM, PIK3C3, ULK1) genes in response to TGF-β or upon 3D spheroid formation. 2DG, transient silencing of TGF-β, or galunisertib treatment prevented these increases. 3D spheroids require ATP and a TGF-β/TGF-βR1 autocrine signaling axis to recapitulate the apoptosis/autophagy phenotypes. Combining glycolysis inhibition with TGF-β signaling inhibition could offer a promising therapeutic strategy for this rare and lethal brain cancer.

Glioblastoma multiform (GBM) exhibit heterogeneity. Persistence of glioma stem cells (GSCs) are the root cause of tumor recurrence and drug resistance. So, targeting GSCs can be a better therapeutic strategy to tackle GBM. To mimic the tumor microenvironment, we have developed tumor spheroids by hanging drop method. Compared to monolayer cells spheroids had higher expression of stemness markers like CD133, CD44, PAX6 and reduced expression of differentiation marker. Cancer cells modulate the metabolic pathways to sustain high proliferation. Among the metabolic pathways, cholesterol biosynthetic pathways are mostly dysregulated in cancers, including GBM. The spheroids showed high expression of cholesterol biosynthetic genes (HMGCR, DHCR24), and Caveolin1 (CAV1). Targeting cholesterol metabolism by lovastatin resulted in depletion of cellular cholesterol levels, including in plasma membrane. Lowering of cholesterol affected membrane fluidity and hampered Hh signaling by lowering Gli1; consequently, causing downregulation of HMGCR, DHCR24, CAV1, and IDH3A, along with the loss of the stemness factors. However, there is enhanced expression of epigenetic chromatin modification enzymes, including DNMT1 and KDM5A. Tracking into the root cause of silencing of CAV1 gene, we found that CAV1 gene promoter is methylated by DNMT1, and H3K4me3 level was depleted due to enhanced KDM5A mediated demethylation. CAV1 gene silencing by siRNA validated its role in maintenance of stem-like phenotype and metabolic alterations of GBM spheroids. Collectively, this study demonstrated the regulatory role of Caveolin1 and cholesterol in maintaining stem-like characteristics of GBM spheroids and the importance of tumor models in better understanding of the molecular mechanism of GBM.Key words: Glioblastoma, cholesterol biosynthesis, stemness, Caveolin1, DNMT1, KDM5A, Gli1.

Spheroids offer significant advantages over two-dimensional (2D) cell cultures by providing a three-dimensional (3D) microenvironment that enhances cell-cell interactions and better mimics native architecture of the original tissue. C2C12 myoblasts, a well-established model for skeletal muscle regeneration, have been widely studied in 2D cultures, but systematic investigations into their spheroid formation and therapeutic potential for muscular injuries remain limited. In this study, hanging drop method was used for the preparation of C2C12 spheroids with different range of initial seeding densities (500–5000 cells per 30 µL drop). Compact, uniform spheroids formed at 2500 and 5000 cells per drop, with diameters around 100 µm as confirmed by optical microscopy and F-actin/DAPI staining. Immunostaining revealed that higher-density spheroids (5000 cells/drop) exhibited stronger and more localized expression of myogenic markers (MyoD and myogenin), indicating advanced differentiation compared to lower-density counterparts. The study results demonstrated that cell density and culture duration are important for the successful generation and myogenic differentiation of C2C12 spheroids. This spheroid model can be considered a potential cellular therapy platform for skeletal muscle regeneration studies.

Protodioscin (PD) is a natural saponin with anti-inflammatory, anticancer, and metabolic regulatory properties. Senolytic agents, which selectively eliminate senescent cells, are gaining attention for enhancing cancer therapy. This study evaluated the senolytic potential of PD in 3D HepG2 spheroids subjected to Methotrexate (MTX)-induced senescence. Spheroids were generated using the hanging drop method with a 1:1 mixture of HepG2 medium and HFF1-conditioned medium. Senescence was induced with 6 µM MTX, followed by 24-h PD treatment at 1.6, 4, and 8.14 µM (10%, 25%, and 50% of IC₅₀). Cell viability was assessed via MTT; ROS, NO, and TGF-β levels via ELISA; apoptosis via Annexin V/PI; and cell death via Calcein-AM. Expression of p16, p21, p27, p53, and p-Smad2/4 was measured by Western blot, and β-Galactosidase activity was used as a senescence marker. PD dose-dependently reduced spheroid viability and, combined with MTX, significantly enhanced apoptosis, cell death, ROS, NO, β-Gal activity, and expression of p16, p27, and p53, while downregulating p-Smad2/4 and TGF-β compared to MTX treatment alone. These findings suggest that PD potentiates MTX-induced senescence through modulation of cell cycle regulators and inhibition of the TGF-β/p-Smad2/4 pathway, highlighting its potential as a senolytic adjuvant in liver cancer therapy.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-27926-6.

Diffuse infiltrating gliomas (DIGs) account for approximately 80% of all malignant gliomas, making them the second most common primary tumor of the central nervous system (CNS). Among them, glioblastoma (GBM) is the most aggressive form, associated with poor prognosis. These features are attributed to the interplay between clonogenic glioblastoma stem cells (GSCs) and the tumor microenvironment (TME). In fact, the secretion of growth factors and cytokines supports GSCs, collectively altering the tumor secretome that maintains cancer cell pluripotency, drives invasiveness, facilitates intra-brain tumor dissemination, and conferrers resistance to chemotherapy. The goal of this study is to develop a novel approach to investigate the interplay between GSCs and secretome through an in vitro microfluidic system, through the MIVO platform organ-on-chip. To this aim, tumor spheres of different glioma cell lines were cultured using the hanging drop method and moved to the upper chamber of MIVO support: a compartment divided from the fluidic channel by a porous membrane, simulating the microcirculation of secreted cells in the TME. A volume of 150-200 μL of medium was added into the chamber to balance the liquid level inside and outside the insert, to ensure proper flow. The plate with the inserts was placed in the specific support, then the cartridge was secured inside the pump head. To validate the platform’s potential, deferoxamine (DFX, a hypoxiamimetic agent) was added to the flow of the microfluidic system, worsening the tumor microenvironment. In this contest, DFX effects on the secretome were evaluated by ELISA assay. Preliminary results revealed a significant increase of vascular endothelial growth factor (VEGF) and interleukin-1β (IL-1β) in glioma secretomes following DFX exposure, observing some differences among glioma cell lines. These findings support our hypothesis by this microfluidic platform is able to offer a valuable tool for studying GSC behavior during therapeutic interventions. Furthermore, it holds promise for use in preliminary screening of therapeutic agents targeting DIGs, potentially accelerating drug discovery and improving treatment strategies. This study was supported by Piano di Incentivi per la Ricerca di Ateneo 2024/2026. Linea di intervento 1.

BACKGROUND:Ischemic diseases have become a major threat to global health, with endothelial cell (EC) damage closely associated with their pathogenesis and progression. Cell therapies targeting endothelial repair have thus become a treatment approach of great interest, yet the procurement of clinically approved ECs for these applications has not been fully established. Modulating the expression of Atf3 (activating transcription factor 3) represents a potential strategy for deriving ECs from stem cells; however, its precise function in the development and differentiation of ECs from stem cells remains elusive. In the present study, we sought to elucidate the potential role of Atf3 in the differentiation of embryonic stem cells into ECs.METHODS:CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) system was used to knockout Atf3 (Atf3KO [Atf3 knockout]) in mouse embryonic stem cells. EC differentiation was initially induced using the hanging drop method to promote embryoid bodies formation, followed by embryoid bodies attachment onto culture slides. The expression changes of EC markers during differentiation were assessed by RNA sequencing, Western blotting, immunofluorescence staining, flow cytometry, and reverse transcription quantitative polymerase chain reaction. Functional comparisons of differentiated ECs were performed by assessing LDL (low-density lipoprotein) uptake and NO production. Potential molecular mechanisms were further explored via bioinformatic analysis of RNA sequencing data.RESULTS:Atf3KO led to a significant upregulation in the expression levels of progenitor and mesoderm cell markers on days 3 and 6 of differentiation. By day 9, the expression of mature EC markers also exhibited a notable increase. Moreover, Atf3KO enhanced the functional properties of differentiated Atf3KO ECs. In addition, our findings revealed that the activation of the Rap1 (Ras-related protein 1) signaling pathway, triggered by Atf3KO, contributed to ECs development and maturation.CONCLUSIONS:Atf3KO directs embryonic stem cells toward the mesodermal lineage and activates the Rap1 signaling pathway, thereby promoting ECs development. These findings highlight a key role of Atf3 in regulating early stage of vascular endothelial development.

ABSTRACTBackgroundMucoepidermoid carcinoma (MEC) is the most prevalent salivary gland malignancy, with a poor prognosis in high‐grade tumors at diagnosis. This highlights the need for effective antitumor agents for treating MEC. Therefore, we aimed to investigate the antineoplastic efficacy of pyrimethamine (PYR), a Food and Drug Administration‐approved antiparasitic medicine, to repurpose it as an alternative therapeutic option for treating human MEC.MethodsThe trypan blue exclusion assay, cell counting kit‐8 assay, and live/dead assay were performed to assess the antiproliferative efficacy of PYR. PYR‐induced apoptosis was confirmed with 4′,6‐diamidino‐2‐phenylindole staining, cell cycle analysis, and annexin V/propidium iodide staining. A western blot assay was conducted to measure changes in cleaved caspase 8 and myeloid cell leukemia‐1 (Mcl‐1) expression after PYR treatment. Mcl‐1 overexpression was used to further confirm the apoptosis‐inducing activity of PYR. The hanging drop method was employed to assess the efficacy of PYR in a three‐dimensional culture system.ResultsPYR‐induced apoptotic cell death in the YD‐15 high‐grade MEC cell line by promoting apoptosis, as evidenced by MCl‐1 proteasomal degradation and increased cleaved caspase 8 expression.ConclusionOur results indicate that PYR can effectively target human MEC by inducing both intrinsic and extrinsic apoptotic pathways.

Source: Hanafiah, A., et al. Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells. J. Vis. Exp. (2020). The video demonstrates the differentiation of mouse embryonic stem cells (mESCs) to embryoid bodies (EBs) by the hanging drop method. Upon suspending the mESCs in hanging drops and incubating, the cells aggregate to form EBs, which are further differentiated into neuronal progenitor cells (NPCs) using a differentiation medium supplemented with retinoic acid (RA).

Angora goats are a distinct breed that differs significantly from common goats and shares a similar appearance to sheep. In Angora goats, only the level of glutathione (GSH) is elevated during under-stimulated conditions, as well as after the period of hypoxic stress; however, no changes are found in 2,3-diphosphoglycerate (2,3-DPG) levels, which are commonly present in the red blood cells (RBCs) of most mammals. We chose the Angora goat for our investigation because no previous studies have been conducted on the structural and functional aspects of hemoglobin (Hb). In addition, no sequence or structural information is currently available in any database. Angora goat Hb was isolated and purified by anion-exchange chromatography, followed by crystallization using various methods. X-ray data collection for Angora goat Hb was performed under a liquid nitrogen cryo-stream using a Bruker D8 Venture Bio Photon III 28-pixel array area detector system. Good diffracting crystals were obtained using the hanging-drop vapor-diffusion method with polyethylene glycol (PEG) 3350 as the precipitant in water, without the addition of any salt or buffer. The Angora goat Hb diffracted to a resolution of 1.85 Å, and the structure solution was obtained by the molecular replacement method, using the structure of domestic goat Hb as the starting model. The solved structure of Angora goat crystallized in the monoclinic space group P21, consisting of one whole biological molecule in the asymmetric unit, with unit cell dimensions of a = 52.08 Å, b = 76.70 Å, c = 74.08 Å, and β = 91.77 °. The solvent content and Matthews coefficient (Vm) for the Angora goat Hb are 49.05% and 2.41 Å3/Da, respectively, and are within the normal range for protein crystals. Purification, crystallization, and preliminary X-ray diffraction studies of Angora goat Hb were performed successfully. Structural refinement and biophysical characterization of Angora goat Hb are in progress in the absence and presence of GSH and 2,3-DPG.

Background Three-dimensional (3D) cell culture is gaining attention for replicating in vivo conditions better than traditional two-dimensional (2D) cultures. The hanging drop method is a simple 3D culture technique with significant potential due to its ease of use. This study compares mesenchymal stem cells’ morphology, viability, surface markers and pluripotency gene of Umbilical Cord Mesenchymal Stem Cells (UC-MSCs) from the umbilical cord in 2D and 3D cultures. Methods Umbilical cord MSCs were cultured using the hanging drop technique for 3D culture and 2D culture. Morphology and viability were observed while surface marker analysis was performed using flow cytometry. The harvested cells were then extracted and underwent RNA isolation, cDNA synthesis, and examination of the expression of the OCT4 and SOX2. Results Significant differences between 2D and 3D cultures regarding morphology, viability, and CD expression were observed. Spheroids in 3D culture exhibited a significant increase in diameter over time ( p <0.0001), while CD73 expression was higher in 2D cultures compared to 3D ( p = 0.0225). In 2D culture cells, the OCT4 gene was expressed more than in 3D culture ( p = 0.0286). Conclusions These findings suggest that 3D culture provides valuable insights into stem cell behavior, with important implications for regenerative medicine.

Salmonella enterica subsp. enterica serovar Typhi is endemic in Nigeria where serovar Gallinarum is rarely reported. Following routine blood culture and identification, three patients with probable S. Gallinarum infections were reported in Ibadan within 10-days, precipitating an outbreak investigation. All three isolates were re-identified using VITEK2, motility-tested using Sulphide-Indole-Motility (SIM) medium and whole genome sequenced (WGS) on Illumina and Oxford Nanopore platforms. Short reads were used to determine sequence type, serotype and phylogenetic relationships to previously characterized S. Typhi from Nigeria. Chromosomal configurations were deduced from hybrid assemblies using socru v2.2.4. The three isolates, initially identified as S. Gallinarum, re-identified as gamma glutamyl transferase-positive S. Typhi genotype 3.1.1 by VITEK-2 and WGS. Two individuals in the same household yielded isolates with no single nucleotide polymorphisms, suggesting a point-source and the third case was an independent infection event. Motility was negative by hanging drop method and in SIM medium at 24h, but positive after 48h incubation. The three isolates formed smaller colonies than typical Salmonella strains. Hybrid genome assemblies revealed chromosomal fragments arrangements with imbalances on either side of ori and ter, recently been shown to slow S. Typhi growth. We have shown that slow-growing S. Typhi can be mistaken for S. Gallinarum and that a cluster of blood culture-derived S. Typhi, with imbalanced chromosome rearrangements, was thereby mistaken for a zoonotic S. Gallinarum outbreak. Suspected S. Gallinarum isolates in typhoid endemic areas should be evaluated biochemically and for motility after extended incubation, and verified by serological or molecular methods.

Salmonella enterica subsp. enterica serovar Typhi is endemic in Nigeria where serovar Gallinarum is rarely reported. Following routine blood culture and identification, three patients with probable S. Gallinarum infections were reported in Ibadan within 10-days, precipitating an outbreak investigation. All three isolates were re-identified using VITEK2, motility-tested using Sulphide-Indole-Motility (SIM) medium and whole genome sequenced (WGS) on Illumina and Oxford Nanopore platforms. Short reads were used to determine sequence type, serotype and phylogenetic relationships to previously characterized S. Typhi from Nigeria. Chromosomal configurations were deduced from hybrid assemblies using socru v2.2.4. The three isolates, initially identified as S. Gallinarum, re-identified as gamma glutamyl transferase-positive S. Typhi genotype 3.1.1 by VITEK-2 and WGS. Two individuals in the same household yielded isolates with no single nucleotide polymorphisms, suggesting a point-source and the third case was an independent infection event. Motility was negative by hanging drop method and in SIM medium at 24h, but positive after 48h incubation. The three isolates formed smaller colonies than typical Salmonella strains. Hybrid genome assemblies revealed chromosomal fragments arrangements with imbalances on either side of ori and ter, recently been shown to slow S. Typhi growth. We have shown that slow-growing S. Typhi can be mistaken for S. Gallinarum and that a cluster of blood culture-derived S. Typhi, with imbalanced chromosome rearrangements, was thereby mistaken for a zoonotic S. Gallinarum outbreak. Suspected S. Gallinarum isolates in typhoid endemic areas should be evaluated biochemically and for motility after extended incubation, and verified by serological or molecular methods.

Background: Proton therapy is considered one of the most promising methods in the treatment of complex localized tumors, but still has some shortcomings, which requires the development of new approaches to improve its effectiveness. One of the most promising approaches is the use of radiosensitizers that can enhance the radiation-induced effects of a proton beam. However, the use of 2D tumor cell models for screening potential radiosensitizers is insufficient for the effective translation of the experimental data to the in vivo level. 3D cellular spheroids are a convenient and relevant model for studying new approaches in the therapy of solid tumors, since they allow simulating the conditions of the microenvironment of tumor cells and simulating in vivo conditions, including the presence of an intercellular matrix and the formation of a certain zonality. Purpose: To create an experimental model of a tumor spheroid based on 4T1 tumor cells irradiated with a proton beam for screening potential nanoradiosensitizers. Material and methods: In vitro biological activity was assessed using a 4T1 cell line (mouse carcinoma) culture. The hanging drop method was used to form cell spheroids. The spheroids were irradiated with a proton beam at the Bragg peak on at a dose of 0–12 Gr using the “Prometheus” therapeutic proton complex . The clonogenic test was used to analyze the viability and mitotic activity of the cells after irradiation. The growth dynamics of irradiated 3D spheroids has been assessing by analyzing micromorphometry for 8 days after irradiation.

Triple-negative breast cancer (TNBC) is highly aggressive and lacks targeted therapies, posing a major challenge in oncology. Traditional two-dimensional (2D) cell cultures fail to capture the tumor microenvironment’s complexity, whereas three-dimensional (3D) cultures provide a more accurate model of tumor biology. We developed an advanced 3D culture system for TNBC cell lines BT-20 and MDA-MB-231, enhancing the hanging-drop method with Matrigel to restore essential extracellular matrix interactions. Confocal imaging showed MDA-MB-231 cells forming clusters typical of aggressive carcinoma, while BT-20 cells organized into duct-like structures. Molecular analysis of PI3K and β-catenin target genes revealed distinct expression patterns, with PI3K overexpressed and β-catenin downregulated in 3D cultures. Moreover, β-catenin distribution in the 3D cell culture closely resembles its pattern in tissue. These findings underscore the value of 3D models in understanding TNBC progression and in supporting the exploration of novel therapeutic strategies.

Abstract Glioblastoma (GBM) is the most aggressive primary brain cancer with current treatments achieving an average overall survival of less than 2 years. This is due in part to the highly immunosuppressive tumor microenvironment (TME), a characteristic of many solid tumors that thwarts immunotherapy. However, the complex interactions between cancer cells and immune cells are difficult to recapitulate in culture models. Microgravity offers key advantages for cancer organoid development, including the ability to generate more robust, larger, complex, uniform, and reproducible spheroids that recapitulate cell-cell interactions more accurately than ground-based 3-D models, which are subject to gravitational forces leading to spheroid settling, disassociation, and heterogeneity. We sent individual small-scale, static experiments contained within Space Tango's High Sample Capacity CubeLab to the International Space Station (ISS) in March 2024 aboard the SpX-30 commercial resupply mission. We first developed tumor-myeloid organoids to recreate the 3D spatial interactions in the TME. These combine human GBM cells with monocytes and macrophages and can be treated to polarize the macrophages into pro-inflammatory (M1-like) or anti-inflammatory (M2-like) phenotypes. Spheroids of uniform size and tunable cell content were generated using the hanging drop method. We then optimized spheroid size and media conditions to support long-term spheroid growth in the absence of gas or media exchange to be compatible with the carrier system. We observed intact spheroids with viable cells beyond two months in sealed vials. Over time, the organoids reside in an acidic and hypoxic environment, with live cells at the organoid edges and a necrotic central core. This recapitulates the microenvironment of GBM and other tumors, which is known to be acidic, hypoxic, and necrotic. The launched experiment contained spheroids with GBM cells alone, GBM cells + macrophages, GBM cells + monocytes, and GBM + macrophages with M1-like or M2-like polarizing agents. These were maintained in microgravity aboard the ISS for 40 days and were processed within 24 hours of their return to Earth. We collected RNA for bulk sequencing, media for secreted proteomics, and fixed intact spheroids for spatial sequencing, spatial proteomics, and histology. While analysis is ongoing to determine the impact of microgravity on GBM-immune interactions, this experiment has already laid the groundwork for future flights by revealing successful organoid culture conditions for future experiments. We anticipate that these microgravity immune-tumoroids will provide valuable insight into the role of macrophages in GBM biology that would not be possible to obtain in Earth’s gravity. Upcoming microgravity experiments will include more complex organoids from patient-derived glioma cells and the potential for drug screening. Citation Format: Alice Burchett, Maksym Zarodniuk, Fionn Lay, Ina Satpathy, Wenjun Zhao, Karyme Hernandez Torrens, Haley Marco, Maria Mendez, Julian Najera, Shelby Giza, Jason Rexroat, Paul Kuehl, Twyman Clements, Meenal Datta. Leveraging the International Space Station to study glioblastoma-immune interactions in microgravity-grown organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5190.

The process the organism individual development implies its adaptation, the patterns and principles of which, among other things, are reflected in the implementation of the male gametophyte. The forest stand natural renewal primarily depends on the indicators of the seed formation success, determined among other things by the pollen characteristics. For species threatened with extinction the problem of renewal stability is especially acute. The paper is aimed to determine the biometric characteristics and the viability level of J. deltoides’s pollen grains in the Mountainous Crimea. In that regard, the following tasks were set: to determine the morphometric parameters of J. deltoides pollen grains, to assess their potential and real viability, and to identify the degree of environmental factors influence on the development of the male reproductive system of J. deltoides in the Mountainous Crimea. Within each trial area, 10 model trees were identified to collect their pollen grains during the flight period. The diameter of pollen grains in two mutually perpendicular planes was measured on temporary acetocarmine preparations. Pollen germination was carried out using the hanging drop method. A new methodology was developed to determine the integral assessment of pollen quality, including a comprehensive analysis of its histochemical and morphological features characterizing viability. The sizes of J. deltoides pollen grains in Mountainous Crimea differ within the error limits. The average diameter of fertile pollen for the species, under the conditions of the peninsula, is 27.08 ± 0.49 μm. The average percentage of fertile (colored) pollen is 55.19 ± 3.84%. At the beginning of the 21st century, this figure ranged from 87.1% to 98.2%. It can be assumed that within the past two decades, the amount of fertile pollen has decreased by 1.7 times. The greatest influence on the fertility of J. deltoides pollen is almost equally exerted by the altitude above sea level and the edaphic conditions of the places of growth of individuals (6.38% and 6.50%, respectively). During the integral assessment of the pollen quality of the Crimean population of J. deltoides, it was found that the overwhelming majority of individuals (64.71%) are characterized by good quality pollen grains. The proportion of germinated pollen of J. deltoides in Mountainous Crimea is low and amounts to 20.16±1.12%. The main factor influencing the viability of pollen grains is the anthropogenic load of the territory (the strength of influence is 51.63%). The average proportion of germinated pollen in significantly anthropogenically disturbed areas is 12.0±1.26%, which is 2.3 times lower than that of individuals growing in undisturbed areas.

Ex vivo studies with first trimester placental tissues are crucial for understanding human placental development, and culturing placental villi in floating conditions is vital to mimic the natural setting. Moreover, being able to cryopreserve these scarce materials for ex vivo studies would open unprecedented avenues, as they are very limited to research and the need to culture freshly adds further hurdles while limiting efficient use. Here we showed that hanging drop method is a simple yet effective approach to culture placental floating villi. We also revealed that these functional units of the early-stage human placenta can be cryopreserved for culturing, and that frozen-thawed villi can regenerate the syncytial layer following denudation. We further illustrated the utility of frozen-thawed tissues to study syncytialization, while validating the importance of HtrA4 in the process which was shown previously in cell models. These represent significant new knowledge to the field of placental biology.