Drug delivery to solid tumors is challenged by multiple physiological barriers arising from the tumor microenvironment, including dense extracellular matrix, cellular heterogeneity, hypoxic gradients, and elevated interstitial fluid pressure. These features hinder the uniform distribution and accumulation of therapeutics, reducing treatment efficacy. Despite their widespread use, conventional two-dimensional monolayer cultures fail to reproduce these complexities, contributing to the poor translational predictability of many preclinical candidates. Three-dimensional multicellular tumor spheroids have emerged as more representative in vitro models that capture essential features of tumor architecture, stromal interactions, and microenvironmental resistance mechanisms. Spheroids exhibit spatially organized regions of proliferation, quiescence, and hypoxia, and can incorporate non-tumor cells to mimic tumor-stroma crosstalk. Advances in spheroid analysis now enable detailed evaluation of drug penetration, cellular migration, cytotoxic response, and molecular gradients using techniques such as optical and confocal imaging, large-particle flow cytometry, biochemical viability assays, and microfluidic integration. By combining physiological relevance with analytical accessibility, spheroid models support mechanistic studies of drug transport and efficacy under tumor-like conditions. Their adoption into routine preclinical workflows has the potential to improve translational accuracy while reducing reliance on animal models.

Advanced 3D heterospheroids, composed of cancer, fibroblast, and immune cells, serve as more physiologically relevant models for anticancer drug screening and immunotherapy research compared to traditional 2D cultures. This study aimed to optimize the culturing, dissociation, and analysis of heterospheroids, addressing limitations that restrict their broader use in immunotherapy research. Our study revealed significant effects of Human Plasma-Like culture medium on cell viability, necrotic core formation, and the spatial organization of cancer and fibroblast cells within heterospheroids compared to DMEM and RPMI media. In HT-29 heterospheroids, cell viability decreased from 75 % in DMEM to 20 % in HPLM, which was accompanied by increased necrotic core formation and elevated PD-L1 expression. TrypLE™ effectively dissociated heterospheroids but compromised immune cell viability and surface marker detection. In comparison, Accutase™ significantly reduced cell yield, while collagenase I preserved immune cell markers but affected those on cancer cells. Furthermore, we developed a luciferase-based assay to measure immune-mediated cancer cell killing in heterospheroids, excluding signals from non-target cells, such as dying fibroblasts and immune cells, without requiring spheroid lysis or dissociation. Our findings highlight the importance of tailoring experimental conditions to reflect specific tumor characteristics, thus enhancing the utility of heterospheroids in drug discovery and immunotherapy research.

Recent advancements in three-dimensional (3D) cell culture technologies, such as cell spheroids, organoids, and 3D bioprinted tissue constructs, have significantly improved the physiological relevance of in vitro models. These models better mimic tissue structure and function, closely emulating in vivo characteristics and enhancing phenotypic analysis, critical for basic research and drug screening in personalized cancer therapy. Despite their potential, current 3D cell culture platforms face technical challenges, which include user-unfriendliness in long-term dynamic cell culture, incompatibility with rapid cell encapsulation in biomimetic hydrogels, and low throughput for compound screening. To address these issues, we developed a 144-pillar plate with sidewalls and slits (144PillarPlate) and a complementary 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for dynamic 3D cell culture and predictive compound screening. To accelerate biomimetic tissue formation, small Hep3B liver tumor spheroids suspended in alginate were printed and encapsulated on the 144PillarPlate rapidly by using microsolenoid valve-driven 3D bioprinting technology. The microarray bioprinting technology enabled precise and rapid loading of small spheroids in alginate on the pillar plate, facilitating reproducible and scalable formation of large tumor spheroids with minimal manual intervention. The bioprinted Hep3B spheroids on the 144PillarPlate were dynamically cultured in the 144PerfusionPlate and tested with anticancer drugs to measure drug effectiveness and determine the concentration required to inhibit 50% of the cell viability (IC50 value). The perfusion plate enabled the convenient dynamic culture of tumor spheroids and facilitated the dynamic testing of anticancer drugs with increased sensitivity. It is envisioned that the integration of microarray bioprinting of tumor spheroids onto the pillar plate, along with dynamic 3D cell culture in the perfusion plate, could more accurately replicate tumor microenvironments. This advancement has the potential to enhance the predictive drug screening process in personalized cancer therapy significantly.

This pipeline presents a refined approach for deriving personalized neurobiological insights from iPSC-derived neurospheres. By employing Tandem Mass Tag (TMT) labeling, we optimized sample pooling and multiplexing for robust comparative analysis across experimental conditions, maximizing data yield per sample. Through single-patient-derived neurospheres-composed of neural progenitor cells, early neurons, and radial glia-this study explores proteomic profiling to mirror the cellular complexity of neurodevelopment more accurately than traditional 2D cultures. Given their enhanced relevance, these 3D neurospheres serve as a valuable model for elucidating neurogenesis, differentiation, and neuropathological mechanisms, contributing to the advancement of in vitro neural models and reducing dependency on animal models. SIGNIFICANCE: This study evaluates ten protein extraction protocols using TMT 10-plex labeling to optimize proteomic analysis from single neurospheres. It compares cost, protein yield, and the ability to detect differentially expressed proteins, identifying methods like SPEED and S-Trap as efficient for high-throughput studies, while FASP excels in peptide yield. TMT labeling enhances protein identification, particularly for low-abundance proteins, and allows pre-fractionation to maximize analysis from limited samples. However, challenges such as limited PTM analysis and the potential loss of minor proteins highlight the importance of selecting protocols based on specific research goals. This work contributes to optimizing proteomic workflows for in vitro neural models, advancing single-cell analysis with minimal reliance on animal models.

Multicellular spheroids (MCSs) are increasingly employed as 3D cell culture models in biomedical research due to their ability to effectively replicate in vivo cell interactions, making them suitable for high-throughput drug screening. Accurate cell counting is critical for data normalization, therapeutic evaluation, and exploration of culture conditions; however, affordable software solutions for 3D cell counting using microscopic images are limited. To fill this gap, we created SUNSHINE, an innovative on-chip analytical workflow that uniquely merges optical clearing, histogram matching (HM)-assisted fluorescence calibration, and simple linear iterative clustering (SLIC) supervoxel segmentation. This tool offers an efficient method for analyzing the characteristics and counts of fluorescence-labeled nuclei within MCSs. While optical clearing improves the penetration depth of microscopic imaging, deeper regions of thicker samples often yield faint fluorescence signals. SUNSHINE resolves this issue through the HM image post-processing algorithm. Moreover, SLIC is an effective alternative to traditional contour-wise segmentation, enabling the identification of irregularly shaped fluorescent nuclei. We found that SUNSHINE generated results comparable to commercial software like Imaris and machine learning (ML)-based tools, such as StarDist and Cellpose, in our analysis of the effects of seeding density and cell type on spheroid growth. We also used it to measure the volume and spatial distribution of nuclei, focusing on the hypoxic and peripheral regions of spheroids. Overall, this study finds that SUNSHINE serves as a valuable and economical approach for characterizing cellular activity and interactions in 3D, diminishing the reliance on costly proprietary software.

Cerebral organoid cultures from human-induced pluripotent stem cells are widely used to study complex human brain development; however, there is still limited ultrastructural information regarding the development. In this study, we examined the structural details of cerebral organoids using various microscopy techniques. Two protocols were chosen as representative methods for the development of brain organoids: the classic whole-cerebral organoid (Whole-CO) culture technique, and the air-liquid interface-cerebral organoid (ALI-CO) culture technique. Immunostained confocal laser scanning microscopy (CLSM) revealed the formation of the CTIP2- and TBR1-positive cortical deep layer on days 90 and 150, depending on the developmental progress of both methods. Furthermore, the presence of astrocytes and oligodendrocytes was verified through immunostained CLSM utilizing two-dimensional and three-dimensional reconstruction images after a 150-day period. Transmission electron microscopy analysis revealed nanometer-resolution details of the cellular organelles and neuron-specific structures including synapses and myelin. Large-area scanning electron microscopy confirmed the well-developed neuronal connectivity from each culture method on day 150. Using those microscopy techniques, we clearly showed significant details within two representative culture protocols, the Whole-CO and ALI-CO culture methods. These multi-level images provide ultrastructural insight into the features of cerebral organoids depending on the developmental stage.

A three-dimensional cell culture called a spheroid serves as a foundational entity in a wide variety of modern tissue engineering applications, including 3D-bioprinting and preclinical drug testing. Lack of oxygen within tissue spheroids hinders metabolism of cells and eventually leads to cell death. Prevention of necrosis is crucial to success of tissue engineering methods and such prevention requires estimation of cell viability in the spheroid. We propose a novel approach for numerical modeling of diffusion in tissue spheroids during their fusion. The approach is based on numerical solutions of partial differential equations and the application of Function Representation (FRep) framework for geometric modeling. We present modeling of oxygen diffusion based on meshes derived from the geometry of fusing spheroids, a method for selecting optimal spheroid size, and several statistics for estimating cellular viability. Our findings provide insights into oxygen diffusion in three-dimensional cell cultures thus improving the robustness of biotechnological methods that employ tissue spheroids.

Blackcurrant press cake (BPC) anthocyanins were recovered using ultrasound-assisted extraction, and the optimal BPC extract was tested for its antioxidant capacity using chemical and biological assays and applied in a functional food model. Extraction at 400 W for 10 min followed by freeze-drying rendered an extract rich in polyphenols (47.83 mg GAE/g), where delphinidin-3-rutinoside, delphinidin-3-glucoside, cyanidin-3-rutinoside, and cyanidin-3-glucoside accounted for 75 % of total phenolics. When the reverse process of protonation/deprotonation was performed (pH 10 to pH 2), anthocyanins exhibited 79.3 % reversibility. The BPC extract inhibited human plasma oxidation (5052 mg AAE/g) and decreased intracellular reactive oxygen species generation by 54 % in erythrocytes and 45 % in LPS-stimulated THP-1 macrophage-like cells. BPC extract exhibited antiproliferative activity and cytostatic effect on HepG2 cells in monoculture at 100-250 μg/mL. Gummies added with BPC extract had a sensory acceptability of 75 %, but bioactive compounds had a low bioaccessibility.

Spheroids and other 3D cellular models more accurately recapitulate physiological responses when compared to 2D models and represent potential alternatives to animal testing. The cryopreservation of spheroids remains challenging, limiting their wider use. Standard DMSO-only cryopreservation results in supercooling to low subzero temperatures, reducing viability, shedding surface cells, and perforating spheroid interiors. Here, cocultured spheroids with differentially labeled outer cell layers allow spatial evaluation of the protective effect of macromolecular ice nucleators by microscopy and histology. Extracellular nucleation is shown to reduce damage to both interior and exterior regions of the spheroids, which will support the development of "off-the-shelf" 3D models.

Multicellular tumor spheroids (MCTS) are advanced cell culture systems for assessing the impact of combinatorial radio(chemo)therapy as they exhibit therapeutically relevant in vivo-like characteristics from 3-dimensional cell-cell and cell-matrix interactions to radial pathophysiological gradients. State-of-the-art assays quantify long-term curative endpoints based on collected brightfield image time series from large treated spheroid populations per irradiation dose and treatment arm. This analyses require laborious spheroid segmentation of up to 100,000 images per treatment arm to extract relevant structural information from the images (e.g., diameter, area, volume, and circularity). While several image analysis algorithms are available for spheroid segmentation, they all focus on compact MCTS with a clearly distinguishable outer rim throughout growth. However, they often fail for the common case of treated MCTS, which may partly be detached and destroyed and are usually obscured by dead cell debris.

To address these issues, we successfully train 2 fully convolutional networks, UNet and HRNet, and optimize their hyperparameters to develop an automatic segmentation for both untreated and treated MCTS. We extensively test the automatic segmentation on larger, independent datasets and observe high accuracy for most images with Jaccard indices around 90%. For cases with lower accuracy, we demonstrate that the deviation is comparable to the interobserver variability. We also test against previously published datasets and spheroid segmentations.

The developed automatic segmentation can not only be used directly but also integrated into existing spheroid analysis pipelines and tools. This facilitates the analysis of 3-dimensional spheroid assay experiments and contributes to the reproducibility and standardization of this preclinical in vitro model.

Due to its high heterogeneity and significant impact on women's health globally, breast cancer necessitates robust preclinical models to understand tumor biology and guide personalized treatment strategies. Three-dimensional (3D) in vitro tumor models hold immense promise in this regard. These tumor models not only mimic the spatial structure and growth environment of tumors in vivo, but also retain the pathological and genetic characteristics of solid tumors. This fidelity makes them powerful tools for accelerating advancements in fundamental research and translational medicine. The diversity, modularity, and efficacy of 3D tumor models are driving a biotechnological revolution. As these technologies become increasingly sophisticated, 3D tumor models are poised to become powerful weapons in the fight against breast cancer. This article expounds on the progress made in utilizing 3D tumor models for breast cancer research.

Precision medicine in oncology aims to identify an individualized treatment plan based on genomic alterations in a patient's tumor. It helps to select the most beneficial therapy for an individual patient. As it is now known that no patient's cancer is the same, and therefore, different patients may respond differently to conventional treatments, precision medicine, which replaces the one-size-fits-all approach, supports the development of tailored treatments for specific cancers of different patients. Patient-specific organoid or spheroid models as 3D cell culture models are very promising for predicting resistance to anti-cancer drugs and for identifying the most effective cancer therapy for high-throughput drug screening combined with genomic analysis in personalized medicine. Because tumor spheroids incorporate many features of solid tumors and reflect resistance to drugs and radiation, as in human cancers, they are widely used in drug screening studies. Testing patient-derived 3D cancer spheroids with some anticancer drugs based on information from molecular profiling can reveal the sensitivity of tumor cells to drugs and provide the right compounds to be effective against resistant cells. Given that many patients do not respond to standard treatments, patient-specific treatments will be more effective, less toxic. They will affect survival better compared to the standard approach used for all patients.

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.

Cancer cell monolayers are commonly used for preclinical drug screening. However, monolayers do not begin to mimic the complexity of the tumor microenvironment, including hypoxia and nutrient gradients within the tumor. To more accurately mimic solid tumors, we developed and drug-tested an anaplastic lymphoma kinase (ALK)-positive (H3122) non-small-cell lung cancer 3D (three-dimensional) culture model using light-activated gelatin methacryloyl hydrogels. We previously demonstrated that the combination of alectinib, an ALK inhibitor, and SHP099, an SHP2 inhibitor, had synergistic efficacy in ALK-positive cell monolayers. We aimed to test this drug combination in our novel ALK-positive 3D cancer model. We first validated the 3D cultures by comparing the distribution of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the 3D cultures with sections from time-matched mouse xenografts, finding a comparable percentage of TUNEL-positive cells in the 3D culture and xenograft inner cores at each time point. When we investigated the effect of the combination of alectinib and SHP099 in these novel 3D cultures, we found a comparable cellular response compared with our two-dimensional experiments especially with the drugs in combination. We suggest that 3D cultures be used as preclinical screening platforms to ensure that only the most efficacious drug candidates move on to in vivo testing.

The tumor microenvironment features dynamic biomechanical interactions between extracellular matrix physics and tumor progression. Tumor growth compresses the supportive matrix, and the stiffness-gradient guides tumor invasion. From the mechanical perspective, the complexity of the matrix topology involving durotaxis-driven metastasis remains lacking in a comprehensive description. In this study, A549 adenocarcinoma spheroids were exposed to a stiffness-and fiber-adjusted collagen matrix to examine the influence of collective motility. Centrifugated compression on the collagen constructs was adopted to mimic the matrix deformation in response to solid tumor development. Centrifugated compression physically stiffened and condensed collagen constructs simultaneously. Cultured with A549 spheroids for 7 days, compressed collagen constructs disadvantaged spheroid expansion without the effect of tumor proliferation potency but promoted matrix metalloproteinase activity corresponding to softened rigidity. Results suggested that the fibrous structure may counterbalance the matrix stiffness-induced motility.

Multicellular spheroids embedded in 3D hydrogels are prominent in vitro models for 3D cell invasion. Yet, quantification methods for spheroid cell invasion that are high-throughput, objective and accessible are still lacking. Variations in spheroid sizes and the shapes of the cells within render it difficult to objectively assess invasion extent. The goal of this work is to develop a high-throughput quantification method of cell invasion into 3D matrices that minimizes sensitivity to initial spheroid size and cell spreading and provides precise integrative directionally-dependent metrics of invasion. By analyzing images of fluorescent cell nuclei, invasion metrics are automatically calculated at the pixel level. The initial spheroid boundary is segmented and automated calculations of the nuclear pixel distances from the initial boundary are used to compute common invasion metrics (i.e., the change in invasion area, mean distance) for the same spheroid at a later timepoint. We also introduce the area moment of inertia as an integrative metric of cell invasion that considers the invasion area as well as the pixel distances from the initial spheroid boundary. Further, we show that principal component analysis can be used to quantify the directional influence of a stimuli to invasion (e.g., due to a chemotactic gradient or contact guidance). To demonstrate the power of the analysis for cell types with different invasive potentials and the utility of this method for a variety of biological applications, the method is used to analyze the invasiveness of five different cell types. In all, implementation of this high-throughput quantification method results in consistent and objective analysis of 3D multicellular spheroid invasion. We provide the analysis code in both MATLAB and Python languages as well as a GUI for ease of use for researchers with a range of computer programming skills and for applications in a variety of biological research areas such as wound healing and cancer metastasis.

Pitavastatin is commonly prescribed to treat hypercholesterolemia through the regulation of cholesterol biosynthesis. Interestingly, it has also demonstrated a great potential for treating brain tumors, although the detailed cytotoxic mechanism, particularly in glioblastoma, remains incompletely understood. This work explores the activity of pitavastatin in 2D and 3D glioblastoma models, in an attempt to provide a more representative and robust overview of its anticancer potential in glioblastoma. The results show that not only is pitavastatin 10-1000 times-fold more effective in reducing tumoral metabolic activity than temozolomide, but also demonstrate a synergistic activity with this alkylating drug. In addition, low micromolar concentrations of this statin strongly impair the growth and the invasion ability of multicellular tumor spheroids. The obtained qRT-PCR and proteomics data highlight the modulation of cell death via apoptosis (BAX/BCL2, CASP9) and autophagy (BECN1, BNIP3, BNIP3L and LC3B), as well as an epithelial to mesenchymal transition blockage (HTRA1, SERPINE1, WNT5A, ALDH3B1 and EPHA2) and remodeling of the extracellular matrix (VCAN, SERPINE1 and TGFBI). Overall, these results lay the foundation for further investigations on the potential combinatory clinical treatment with temozolomide.

This study introduces a novel method for the fabrication of concave microwells involving water vapor permeation through polydimethylsiloxane (PDMS). This method leverages the exceptional water vapor permeability of PDMS to enable a scalable and cost-effective fabrication process, addressing the limitations of existing techniques such as photolithography that are resource-intensive and complex. PDMS is more permeable to water vapor than to other gas molecules, resulting in the formation of microwells. Smooth-sloped concave microwells are formed by depositing droplets of 10% ethylene glycol on a PDMS substrate followed by curing at 70 °C and evaporation of water vapor. These microwells exhibit a unique structural gradient that is highly conducive for biological applications. Concave microwells were further used as a platform to generate animal cell spheroids, demonstrating their potential for three-dimensional cell culture. Unlike conventional methods, this approach allows precise control over microwell morphology by simply adjusting droplet size and curing conditions, offering enhanced tunability and reproducibility. The formation yield of these microwells is dependent on the volume of the water droplets, demonstrating the importance of droplet size in controlling microwell morphology. This approach provides a simple and effective method for creating microwells without complex lithographic processes, making it a highly promising tool for a range of biomedical applications, including tissue engineering, cancer research, and high-throughput drug screening.

The anti-cancer potential of eugenol (EUG) is well recognized, whereas that of spermidine (SPD) is subject to dispute and requires further research. The anti-tumorigenic potential of wheat germ SPD (150 µM) and clove EUG (100 µM), alone, in combination as SPD+EUG (50 µM + 100 µM) and, as a supplement (SUPPL; 0.6 µM SPD + 50 µM EUG), was investigated on both metastatic SW620 and primary Caco-2 colorectal cancer (CRC) spheroids. Compared to untreated controls, all treatments significantly reduced the vitality and spheroid area, increased the necrotic area, and induced apoptosis on both cell-type spheroids after 96 h, with a reduced migration evident in 2D (two-dimensional) cultures after 48 h. The comparable anti-CRC effects of the SPD+EUG and the SUPPL reflected a wide-range dose efficacy of SPD and EUG. It is of note that SPD+EUG induced a synergistic effect on the increased caspase-3 expression and reduced the migration percentage in SW620. In more physiologically relevant intestinal equivalents (healthy enterocytes [NCM460], fibroblasts [L929], and monocytes [U937]) containing embedded SW620/Caco-2 spheroids, SPD+EUG administration significantly reduced the spheroid CEA marker and proliferation, whilst simultaneously increasing occludin, autophagy LC3-II expression, and monocyte differentiation, compared to the control models. Exogenous SPD, alone and in combination with EUG, displayed an anti-CRC potential on tumor growth and metastasis, and warrants further investigation.

The widespread use of pesticides, particularly in combinations, has resulted in enhanced hazardous health effects. However, little is known about their molecular mechanism of interactions. The aim of this study was to assess the neurotoxicity effect of pesticides in mixtures by adopting a 3D in vitro developed neurospheroid model, followed by treatment by increased concentrations of pesticides for 24 h and analysis by a shotgun proteomic-based approach with high-resolution tandem mass spectrometry. Three proteins, namely, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), α-enolase, and phosphoglycerate-kinase-1, were selected as key targets in the metabolic process. Only high doses of pesticides mitigated cell-density proliferation with the occurrence of apoptotic cells, which unlikely makes any neurological alterations in environmental regulatory exposures. The proteomic analysis showed that majority of altered proteins were implicated in cell metabolism. De novo peptide sequencing revealed ion losses and adduct formation, namely, a trityl-post-translational modification in the active site of 201-GAPDH protein. The study also highlights the plausible role of pyrethroids to be implicated in the deleterious effects of pesticides in a mixture. To the best of our knowledge, our finding is the first in toxicoproteomics to deeply elucidate pesticides' molecular interactions and their ability to adduct proteins as a pivotal role in the neurotoxicity mechanism.

The goal of this study is to explore the role of the LMNB1 gene in glioma. A cohort of 160 patients who underwent glioma surgery were randomly selected of this study. The LMNB1 expression was assessed employing immunohistochemical and real-time quantitative polymerase chain reaction methods. Initially, RNA interference technology was applied to suppress gene expression, followed by the evaluation of tumor cell proliferation, apoptosis, cell cycle dynamics, and migration. The underlying molecular mechanisms of LMNB1 function were examined by a human phospho-kinase array and immunoblotting. And we established the xenograft models to determine the effect of tumor growth as well as the degree of invasion in shLMNB1 mice. Elevated LMNB1 expression correlated with unfavorable overall survival and disease-free survival. A substantial inhibition in cell growth was observed subsequent to LMNB1 knockdown in SHG-44 and U251 glioma cells. SHG-44-shLMNB1 cells exhibited a reduction in the S phase population, along with an increase in cells in G1 and G2 phases. Similarly, shLMNB1 U251 cells showed fewer cells in the S phase and an elevation in cells in G1 phase. Notably, increased apoptosis was observed in U251-shLMNB1 cells and SHG-44-shLMNB1 cells. Wound healing and Transwell migration assays demonstrated a significant decrease in the migration rate of both SHG-44-shLMNB1 and U251-shLMNB1 cells. The phosphorylation levels of Akt1/2/3, as well as the expressions of PI3K, AKT, and p-AKT proteins, were reduced in the shLMNB1 group. Downregulation of LMNB1 repressed tumor progress in vivo. The silencing of LMNB1 was found to significantly reduce the proliferation of human glioma cells, induce apoptosis in tumor cells, impede the progression of the cell cycle, and inhibit the migration of tumor cells. Consequently, we hypothesize that LMNB1 promotes glioma cell proliferation through mechanisms involving the inhibition of tumor cell apoptosis, acceleration of the cell cycle, and enhancement of tumor cell migration. We found that LMNB1 exert critical roles in glioma progression may via regulation of PI3K/Akt signaling pathway. These observations suggest that LMNB1 holds clinical potential for diagnostic and prognostic applications in glioma, presenting novel targets for drug development.

In the attempts to mitigate uncertainties in the results of monolayer culture for the identification of cancer therapeutic targets and compounds, there has been a growing interest in using 3D cancer spheroid models, which include tumorospheres (TSs), tissue-derived tumor spheres (TDTSs), organotypic multicellular tumor spheroids (OMSs), and multicellular tumor spheroids (MCTSs). The MCTSs, either Mono-MCTSs or Hetero-MCTSs, with or without scaffold, in particular, offer numerous advantages over other spheroid models, including easy cultivation, high reproducibility, accessibility, high throughput, controllable size, well-rounded shape, simplicity of genetic manipulation, economical and availability of various biological methods for their development. In this review, we have attempted to discuss the role of MCTSs concerning various aspects of translational cancer research, such as drug response and penetration, cell-cell interaction, and invasion and metastasis. However, the Mono-MCTSs, either scaffold-free or scaffold-based, may not adequately represent the cellular heterogeneity and complexity of clinical tumors, limiting their utility in translational cancer research. Conversely, Hetero-MCTS models, both scaffold-free and scaffold-based, show better suitability due to the presence of a similar in vivo type tumor microenvironment. Nonetheless, scaffold-based Hetero-MCTS models show batch variability and challenges in performing quantitative assays due to difficulties extracting spheroids and cells from scaffolds. Further, incorporating stromal cells with cancer cells in a more precise ratio to develop Hetero-MCTSs can enhance the model's relevance, yielding more clinically reliable outcomes for drug candidates and improving insights into tumor biology.

Drug repositioning of approved drugs offers advantages over de novo drug development for a rare type of cancer. To efficiently identify on-target drugs from clinically successful kinase inhibitors in cancer drug repositioning, drug screening and molecular profiling of cell lines are essential to exclude off-targets. We developed a pharmacoproteogenomic approach to identify on-target kinase inhibitors, combining molecular profiling of genomic features and kinase activity, and drug screening of patient-derived cell lines. This study examined eight patient-derived giant cell tumor of the bone (GCTB) cell lines, all of which harbored a signature mutation of H3-3A but otherwise without recurrent copy number variants and mutations. Kinase activity profiles of 100 tyrosine kinases with a three-dimensional substrate peptide array revealed that nine kinases were highly activated. Pharmacological screening of 60 clinically used kinase inhibitors found that nine drugs directed at 29 kinases strongly suppressed cell viability. We regarded ABL1, EGFR, and LCK as on-target kinases; among the two corresponding on-target kinase inhibitors, osimertinib and ponatinib emerged as on-target drugs whose target kinases were significantly activated. The remaining 26 kinases and seven kinase inhibitors were excluded as off-targets. Our pharmacoproteomic approach enabled the identification of on-target kinase inhibitors that are useful for drug repositioning.

The tumour microenvironment (TME) significantly influences tumour formation and progression through dynamic interactions. Cholangiocarcinoma (CCA), a highly desmoplastic tumour, lacks early diagnostic biomarkers and has limited effective treatments owing to incomplete understanding of its molecular pathogenesis. Investigating the role of the TME in CCA progression could lead to better therapies. RNA sequencing was performed on seven CCA patient-derived xenografts (PDXs) and their corresponding patient samples. Differential expression analysis was conducted, and Qiagen Ingenuity Pathway Analysis was used to predict dysregulated pathways and upstream regulators. PDX- and cell line-derived spheroids, with and without immortalised mesenchymal stem cells, were grown and analysed for morphology, growth and viability. Histological analysis confirmed biliary phenotypes. RNA sequencing indicated upregulation of extracellular matrix-receptor interaction and PI3K-AKT pathways in the presence of mesenchymal cells, with several genes linked to poor survival. Mesenchymal cells restored the activity of inhibited cancer-associated kinases. Thus, adding mesenchymal cells to CCA spheroid models restored key paracrine signalling pathways lost in PDXs, enhancing tumour growth and viability. These findings highlight the importance of including stromal components in cancer models to improve pre-clinical studies.

Angiogenesis is a key player in drug resistance to targeted therapies for breast cancer. The average expression of angiogenesis-related cytokines is widely associated with the treatments of target therapies for a population of cells or spheroids, overlooking the distinct responses for individuals. In this work, a highly integrated microfluidic platform is developed for the generation of monodisperse multicellular tumor spheroids (MTSs), drug treatments, and the measurement of cytokines for individual MTSs in a single chip. The platform allows the correlation evaluation between cytokine secretion and drug treatment at the level of individual spheroids. For validation, quantities of six representative proangiogenic cytokines are tested against treatments with four model drugs at varying times and concentrations. By applying a linear regression model, significant correlations are established between cytokine secretion and the treated drug concentration for individual spheroids. The proposed platform provides a high-throughput method for the investigation of the molecular mechanism of the cytokine response to targeted therapies and paves the way for future drug screening using predictive regression models at the single-spheroid level.

Glioblastomas exhibit remarkable heterogeneity at various levels, including motility modes and mechanoproperties that contribute to tumor resistance and recurrence. In a recent study using gridded micropatterns mimicking the brain vasculature, glioblastoma cell motility modes, mechanical properties, formin content, and substrate chemistry are linked. Now is presented, SP2G (SPheroid SPreading on Grids), an analytic platform designed to identify the migratory modes of patient-derived glioblastoma cells and rapidly pinpoint the most invasive sub-populations. Tumorspheres are imaged as they spread on gridded micropatterns and analyzed by this semi-automated, open-source, Fiji macro suite that characterizes migration modes accurately. SP2G can reveal intra-patient motility heterogeneity with molecular correlations to specific integrins and EMT markers. This system presents a versatile and potentially pan-cancer workflow to detect diverse invasive tumor sub-populations in patient-derived specimens and offers a valuable tool for therapeutic evaluations at the individual patient level.

Glioblastoma multiforme (GBM) is among the most aggressive brain cancers, and it contains glioma stem cells (GSCs) that drive tumor initiation, progression, and recurrence. These cells resist conventional therapies, contributing to high recurrence rates in GBM patients. Developing in vitro models that mimic the tumor microenvironment (TME), particularly the GSC niche, is crucial for understanding GBM growth and therapeutic resistance. Three-dimensional (3D) spheroid models provide a more physiologically relevant approach than traditional two-dimensional (2D) cultures, recapitulating key tumor features like hypoxia, cell heterogeneity, and drug resistance. This review examines scaffold-free and scaffold-based methods for generating 3D GBM spheroids, focusing on their applications in studying the cancer stem cell niche. The discussion encompasses methods such as the hanging drop, low-adhesion plates, and magnetic levitation, alongside advancements in embedding spheroids within extracellular matrix-based hydrogels and employing 3D bioprinting to fabricate more intricate tumor models. These 3D culture systems offer substantial potential for enhancing our understanding of GBM biology and devising more effective targeted therapies.

Cell spheroids, including organoids, serve as a valuable link between in vitro systems and in vivo animal models, offering powerful tools for studying cell biology in a three-dimensional environment. However, existing methods for generating cell spheroids are time consuming or difficult to scale up for large-scale production. Our recent study has revealed that transcytotic peptide assemblies, which transform from nanoparticles to nanofibers by enzymatic reactions, can create an intercellular fibril/gel, accelerating cell spheroid formation from a 2D cell culture or a cell suspension. While this finding presents an alternative approach for generating cell spheroids, the specific structural features required for efficient cell spheroid formation remain unclear. Based on our observation that a phosphotetrapeptide with a biphenyl cap at its N-terminus enables cell spheroid formation, we produced 10 variants of the original peptide. The variants explored modifications to the peptide backbone, length, electronic properties of the biphenyl capping group, and the type of phosphorylated amino acid residue. We then evaluated their ability for inducing cell spheroid formation. Our analysis revealed that, among the tested molecules, peptides with C-terminal phosphotyrosine, low critical micelle concentration, and dephosphorylation-guided nanoparticle to nanofiber morphological transition were the most effective in inducing the formation of cell spheroids. This work represents the first example to correlate the thermodynamic properties (e.g., self-assembling ability) and kinetic behavior (e.g., enzymatic dephosphorylation) of peptides with the efficacy of controlling intercellular interaction, thus offering valuable insights into using enzymatic self-assembly to generate peptide assemblies for biological applications.

Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by Aspergillus and Penicillium genera present mainly in fruit, cereals and nuts. This study compares the cytotoxicity produced by CPA after 24, 48 and 72 h of exposure using both monolayers and 3D spheroids in human neuroblastoma SH-SY5Y cells. Furthermore, CPA toxicokinetics was evaluated using in silico models. Cytotoxicity increased dose- and time-dependently, as shown by the MTT assay. The lowest CPA IC50 values were found in the monolayer study compared to the 3D spheroids at all exposure times (24 h: 864.01 vs. 1132; 48 h: 437 vs. 1069; 72 h: 392 vs. 567 nM). The CPA exposure on SH-SY5Y spheroid organization and morphology was also studied. Morphological changes, including spheroid disaggregation, were observed after mycotoxin exposure. The in silico methods, SwissADME and admetSAR, were used for short and full ADMEt profiles of CPA. The ADMEt predictive profile shows high gastrointestinal absorption and ability to penetrate the blood-brain barrier. Including in silico studies emphasizes the comprehensive approach to understanding mycotoxin toxicity and risk assessment. By combining in vitro 3D spheroid models with computational simulations, this study aims to provide a holistic perspective on the effects of CPA, enhancing the accuracy and relevance of our findings.

This review centers on the synthesis and characterization of both natural and synthetic hydrogels, highlighting their diverse applications across various fields. We will delve into the evolution of hydrogels, focusing on the importance of polysaccharide-based and synthetic variants, which have been particularly chosen for 3D spheroid development in cancer research and drug delivery. A detailed background on the research and specific methodologies, including the in-situ free radical polymerization used for synthesizing these hydrogels, will be extensively discussed. Additionally, the review will explore various applications of these hydrogels, such as their self-healing properties, swelling ratios, pH responsiveness, and cell viability. A comprehensive literature review will support this investigation. Ultimately, this review aims to clearly outline the objectives and significance of hydrogel synthesis and their applications.

Brain organoids provide a unique opportunity to model organ development in a system similar to human organogenesis in vivo. Brain organoids thus hold great promise for drug screening and disease modeling. Conventional approaches to organoid characterization predominantly rely on molecular analysis methods, which are expensive, time-consuming, labor-intensive, and involve the destruction of the valuable three-dimensional (3D) architecture of the organoids. This reliance on end-point assays makes it challenging to assess cellular and subcellular events occurring during organoid development in their 3D context. As a result, the long developmental processes are not monitored nor assessed. The ability to perform non-invasive assays is critical for longitudinally assessing features of organoid development during culture. In this paper, we demonstrate a label-free high-content imaging approach for observing changes in organoid morphology and structural changes occurring at the cellular and subcellular level. Enabled by microfluidic-based culture of 3D cell systems and a novel 3D quantitative phase imaging method, we demonstrate the ability to perform non-destructive high-resolution quantitative image analysis of the organoid. The highlighted results demonstrated in this paper provide a new approach to performing live, non-destructive monitoring of organoid systems during culture.

Developing accurate in vitro models that replicate the in vivo tumor environment is essential for advancing cancer research and therapeutic development. Traditional 2D cell cultures often fail to capture the complex structural and functional heterogeneity of tumors, limiting the translational relevance of findings. In contrast, 3D culture systems, such as spheroids, provide a more physiologically relevant context by replicating key aspects of the tumor microenvironment. This study aimed to compare the metabolism of three intrahepatic cholangiocarcinoma cell lines in 2D and 3D cultures to identify metabolic shifts associated with spheroid formation. Cells were cultured in 2D on adhesion plates and in 3D using ultra-low attachment plates. Metabolic exchange rates were measured using NMR, and intracellular metabolites were analyzed using LC-MS. Significant metabolic differences were observed between 2D and 3D cultures, with notable changes in central carbon and glutathione metabolism in 3D spheroids. The results suggest that 3D cultures, which more closely mimic the in vivo environment, may offer a more accurate platform for cancer research and drug testing.

The synthesis and characterization of nine Schiff bases of pyrazolone ligands HLn (n = 1-9) and the corresponding zinc(II) complexes 1-9 of composition [Zn(Ln)2] (n = 1-9) are reported. The molecular structures of complexes 2, 3, 4, 8, and 9 were determined by single-crystal X-ray diffraction analysis, highlighting in all cases a distorted tetrahedral geometry around the Zn(II) ion. Density functional theory studies are performed on both the HLn ligands and the derived complexes. A mechanism of dissociation and hydrolyzation of the coordinated Schiff base ligands is suggested, confirmed experimentally by powder X-ray diffraction study and photophysical studies. Complexes 1-9 were investigated in vitro as anticancer agents, along with mutant p53 (mutp53) protein levels in human cancer cell lines carrying R175H and R273H mutp53 proteins. Only those complexes with the highest Zn(II) ion release via dissociation have shown a significant cytotoxic activity with reduction of mutp53 protein levels.

Sixteen patient-derived xenografts (PDXs) were analyzed using a mass spectrometry (MS)-based kinase inhibitor pull-down assay (KIPA), leading to the observation that death-associated protein kinase 3 (DAPK3) is significantly and specifically overexpressed in the triple-negative breast cancer (TNBC) models. Validation studies confirmed enrichment of DAPK3 protein, in both TNBC cell lines and tumors, independent of mRNA levels. Genomic knockout of DAPK3 in TNBC cell lines inhibited in vitro migration and invasion, along with down-regulation of an epithelial-mesenchymal transition (EMT) signature, which was confirmed in vivo. The kinase and leucine-zipper domains within DAPK3 were shown by a mutational analysis to be essential for functionality. Notably, DAPK3 was found to inhibit the levels of desmoplakin (DSP), a crucial component of the desmosome complex, thereby explaining the observed migration and invasion effects. Further exploration with immunoprecipitation-mass spectrometry (IP-MS) identified that leucine-zipper protein 1 (LUZP1) is a preferential binding partner of DAPK3. LUZP1 engages in a leucine-zipper domain-mediated interaction that protects DAPK3 from proteasomal degradation. Thus, the DAPK3/LUZP1 heterodimer emerges as a newly discovered regulator of EMT/desmosome components that promote TNBC cell migration.

Magnetic Fe3O4 nanoparticles (MNPs) functionalized with (3-aminopropylo)trietoksysilan (APTES) or N-carboxymethylchitosan (CMC) were proposed as nanocarriers of methotrexate (MTX) to target ovarian cancer cell lines. The successful functionalization of the obtained nanostructures was confirmed by FT-IR spectroscopy. The nanoparticles were characterized by transmission electron spectroscopy (TEM) and dynamic light scattering (DLS) techniques. Their potential zeta, magnetization, and hyperthermic properties were also explored. MTX was conjugated with the nanocarriers by ionic bonds or by amide bonds. The drug release kinetics were examined at different pH and temperatures. The MTT assay showed no toxicity of the MNPs[APTES] and MNPs[CMC]. Finally, the cytotoxicity of the nanostructures with MTX attached towards the ovarian cancer cells was measured. The sensitivity and resistance to methotrexate was determined in simplistic 2D and spheroid 3D conditions. The cytotoxicity tests of the tested nanostructures showed similar values for inhibiting the proliferation of ovarian cancer cells as methotrexate in its free form. Conjugating MTX with nanoparticles allows the drug to be directed to the target site using an external magnetic field, reducing overall toxicity. Combining this approach with hyperthermia could enhance the therapeutic effect in vivo compared to free MTX, though further research on advanced 3D models is needed.

Ovarian cancer has the highest mortality rate and lowest survival rate among female reproductive system malignancies. There are treatment options of surgery and chemotherapy, but both are limited. In this study, we developed and evaluated micelles composed of D-α-tocopheryl polyethylene-glycol (PEG) 1000 succinate (TPGS) and Soluplus® (SOL) loaded with olaparib (OLA), a poly(ADP-ribose)polymerase (PARP) inhibitor, and rapamycin (RAPA), a mammalian target of rapamycin (mTOR) inhibitor in ovarian cancer.

We prepared micelles containing different molar ratios of OLA and RAPA embedded in different weight ratios of TPGS and SOL (OLA/RAPA-TPGS/SOL) were prepared and physicochemical characterized. Furthermore, we performed in vitro cytotoxicity experiments of OLA, RAPA, and OLA/RAPA-TPGS/SOL. In vivo toxicity and antitumor efficacy assays were also performed to assess the efficacy of the mixed micellar system.

OLA/RAPA-TPGS/SOL containing a 4:1 TPGS:SOL weight ratio and a 2:3 OLA:RAPA molar ratio showed synergistic effects and were optimized. The drug encapsulation efficiency of this formulation was >65%, and the physicochemical properties were sustained for 180 days. Moreover, the formulation had a high cell uptake rate and significantly inhibited cell migration (**p < 0.01). In the in vivo toxicity test, no toxicity was observed, with the exception of the high dose group. Furthermore, OLA/RAPA-TPGS/SOL markedly inhibited tumor spheroid and tumor growth in vivo.

Compared to the control, OLA/RAPA-TPGS/SOL showed significant tumor inhibition. These findings lay a foundation for the use of TPGS/SOL mixed micelles loaded with OLA and RAPA in the treatment of ovarian cancer.

Protein-protein interaction studies using proximity labeling techniques, such as biotin ligase-based BioID, have become integral in understanding cellular processes. Most studies utilize conventional 2D cell culture systems, potentially missing important differences in protein behavior found in 3D tissues. In this study, we investigated the protein-protein interactions of a protein, Bcl-2 Agonist of cell death (BAD), and compared conventional 2D culture conditions to a 3D system, wherein cells were embedded within a 3D extracellular matrix (ECM) mimic. Using BAD fused to the engineered biotin ligase miniTurbo (BirA*), we identified both overlapping and distinct BAD interactomes under 2D and 3D conditions. The known BAD binding proteins 14-3-3 isoforms and Bcl-XL interacted with BAD in both 2D and 3D. Of the 131 BAD-interactors identified, 56% were specific to 2D, 14% were specific to 3D, and 30% were common to both conditions. Interaction network analysis demonstrated differential associations between 2D and 3D interactomes, emphasizing the impact of the culture conditions on protein interactions. The 2D-3D overlap interactome encapsulated the apoptotic program, which is a well-known role of BAD. The 3D unique pathways were enriched in ECM signaling, suggestive of hitherto unknown functions for BAD. Thus, exploring protein-protein interactions in 3D provides novel clues into cell behavior. This exciting approach has the potential to bridge the knowledge gap between tractable 2D cell culture and organoid-like 3D systems.