The influence of oxygen on viability and proliferation in cellular spheroids.
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Animals↗
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Proliferation and viability in cellular spheroids of human origin.
The capacity of cells to form growing tumor-like colonies in culture was tested by a new and unlaborious suspension technique. Six of nine tested cell lines formed spheroids, five of which started to grow. The growing spheroids reached a maximal size determined by balanced cell gain in a proliferative, superficial layer and cell death, particularly at larger depth. Sections of spheroids of different origin showed large variations in the thickness of the viable cell layers and in the shape of the proliferative gradients. The data, taken together with earlier published information, indicate that a difference between rodent and human cells generally exists, the former showing thinner viable and proliferative layers.
Animals↗
3D Cell Culture Models as a Platform for Studying Tumor Progression, Testing Treatment Responses, and Discovering Biomarkers.
In this chapter, we present a detailed protocol for establishing a three-dimensional (3D) multicellular tumor spheroids (MCTSs) model to simulate the tumor microenvironment (ME) associated with metabolic dysfunction-associated steatotic liver disease (MASLD) for the study of hepatocellular carcinoma (HCC) and colorectal cancer (CRC) cell aggressiveness, growth, and metastasis potential. The MASLD microenvironment (MASLD-ME) is recreated by embedding hepatic stellate cells in a collagen I matrix within a Boyden chamber system. The metabolic medium mimics MASLD conditions, enriched with high glucose, fructose, insulin, and fatty acids, to simulate metabolic stresses associated with the disease.In the protocol, cancer cells are loaded in the upper compartment to analyze their migration toward the MASLD-ME, thereby facilitating studies on cancer cell invasiveness and metastatic capacity. This method offers an adaptable, reproducible model to research disease progression and investigate therapeutic interventions, contributing to preclinical research on MASLD-related liver cancer pathophysiology and potential drug responses.
Humans↗
Screening of Estrogenic and Antiestrogenic Effects of Estradiol, Bisphenol A, and Fulvestrant Using 2D and 3D Breast Cancer Cell Systems With a Luciferase Reporter Gene Assay.
Endocrine-disrupting chemicals (EDCs) like bisphenol A (BPA) pose health risks by interfering with hormones. This study develops and utilizes in vitro 2D and 3D cell models to evaluate the estrogenic and antiestrogenic properties of compounds. Human breast cancer cell lines T47D and MCF7, stably transfected with a luciferase reporter gene (ERE-LUC), were first compared in 2D. Due to the significantly higher sensitivity and responsiveness observed in the T47D line during preliminary 2D screenings, this cell line was exclusively selected for the development of the 3D spheroid model. Cells were treated with 17β-estradiol (E2), BPA, and Fulvestrant (FUL) to assess cell viability and luciferase activity. In 2D models, T47D ERE-LUC cells showed higher responsiveness than MCF7 ERE-LUC, which failed to show significant luciferase induction with E2. In the 3D T47D model, cells exhibited significant and robust changes in luciferase activity in response to E2 and BPA, highlighting the enhanced fidelity of 3D cultures in replicating tissue conditions compared to their 2D counterparts. The study highlights the effectiveness of 3D models over 2D in evaluating estrogenic activity. Specifically, the 3D T47D ERE-LUC system serves as a superior, sensitive, and reliable platform for screening EDCs, offering benefits in cost, data speed, and reduced in vivo reliance.
Humans↗
ERBB3 overexpression due to miR-205 inactivation confers sensitivity to FGF, metabolic activation, and liability to ERBB3 targeting in glioblastoma.
In glioblastoma (GBM), the most frequent and lethal brain tumor, therapies suppressing recurrently altered signaling pathways failed to extend survival. However, in patient subsets, specific genetic lesions can confer sensitivity to targeted agents. By exploiting an integrated model based on patient-derived stem-like cells, faithfully recapitulating the original GBMs in vitro and in vivo, here, we identify a human GBM subset (∼9% of all GBMs) characterized by ERBB3 overexpression and nuclear accumulation. ERBB3 overexpression is driven by inheritable promoter methylation or post-transcriptional silencing of the oncosuppressor miR-205 and sustains the malignant phenotype. Overexpressed ERBB3 behaves as a specific signaling platform for fibroblast growth factor receptor (FGFR), driving PI3K/AKT/mTOR pathway hyperactivation, and overall metabolic upregulation. As a result, ERBB3 inhibition by specific antibodies is lethal for GBM stem-like cells and xenotransplants. These findings highlight a subset of patients eligible for ERBB3-targeted therapy.
Antibodies↗
Genome-wide CRISPR screens in spheroid culture reveal that the tumor suppressor LKB1 inhibits growth via the PIKFYVE lipid kinase.
The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor-suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1-mediated growth suppression, we developed a spheroid-based cell culture assay to study LKB1-dependent growth. We then performed genome-wide CRISPR screens in spheroidal culture and found that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase. Finally, we used chemical inhibitors and a pH-sensitive reporter to determine that LKB1 impairs growth by promoting the internalization of wild-type EGFR in a PIKFYVE-dependent manner.
Humans↗
CTGF/CCN2 Promotes Invasive Growth in Cervical Cancer Spheroids and Is Associated With Metastatic Cervical Cancer Tissue.
BACKGROUND/AIM: Metastatic spread defines the lethality of cervical cancer (CC). Connective tissue growth factor (CTGF/CCN2) regulates cell- extracellular matrix interactions but its role in CC is not well-defined. This study investigates the role of CTGF in driving CC invasive growth and its prevalence in patient tissues. MATERIALS AND METHODS: CC spheroids (C33A, HT3) were treated with recombinant human CTGF (rhCTGF) or a function-blocking antibody (IgG CTGF). Invasive growth was assessed via 3D spheroid assay using a Celigo imaging cytometer. Cancer stem cell (CD133, CD44) and epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin) were analyzed by immunofluorescence. CTGF expression was evaluated using a tissue microarray containing 69 cases in triplicate from pre-invasive, invasive (FIGO I-III), and metastatic cervical lesions, quantified via immunofluorescence scoring. RESULTS: Functional blockade of CTGF significantly reduced 3D spheroid invasive growth in C33A and HT3 cells (p<0.0001). Immunofluorescence revealed that CTGF modulation altered spatial distribution of key proteins: rhCTGF induced surface clustering of CD133 and peripheral N-cadherin enrichment, while CTGF blockade was associated with apparent nuclear/perinuclear enrichment of CD133 and E-cadherin and reduced N-cadherin signal. In patient tissue cores, metastatic samples exhibited the highest CTGF fluorescence intensity. High CTGF expression [immunoreactivity score (IRS) ≥ 6] was most prevalent in FIGO stage I (35.5%) compared to stage III (10.0%). Kaplan-Meier analysis revealed that high CTGF mRNA expression was associated with significantly reduced recurrence-free survival (log-rank p=0.0032). CONCLUSION: In 3D models of CC, CTGF appears to regulate an invasive phenotype, presumably by controlling aberrant localization of stemness and EMT markers. Its apparently elevated expression in early-stage cervical carcinomas and metastases, combined with its prognostic value for recurrence-free survival, suggests that CTGF may be involved in triggering the potential for metastasis and could therefore serve as an early prognostic biomarker.
Humans↗
Evaluation of 3D Spheroids for AAV Transduction Studies.
Adeno-associated viruses (AAVs) are potent vectors used for gene delivery in gene therapy products. Their development requires in vitro systems that can reliably detect differences in vector design, serotype performance, regulatory element strength, and expression kinetics. These systems must also support applications such as potency assessment and vector optimization. Here, we describe a streamlined three-dimensional spheroid platform optimized for evaluating AAV potency, transgene expression kinetics, and serotype-specific transduction efficacy across diverse cell lines. Uniform spheroids are generated using ultra-low attachment plates and maintained under conditions that support stable architecture and long-term imaging. Following AAV transduction, fluorescent or luminescent readouts are monitored in real time using live-cell imaging systems. This enables quantitative assessment of reporter signal, dose responsiveness, regulatory element activity, and onset time through continuous kinetic imaging. The platform effectively discriminates between potent and weak vector genome designs and among multiple AAV serotypes. This method demonstrates robust performance across both slowly and rapidly dividing cell lines. These results establish its utility as a scalable and physiologically relevant system for preclinical gene therapy evaluation and development.
Dependovirus↗
Matrix Mechanics Governs Mechano-Metabolic Adaptation across Cancer Grades in Bladder Spheroids.
Extracellular matrix (ECM) mechanics is pivotal regulators of tumor progression, yet how viscoelasticity and matrix architecture converge to shape metabolic and invasive adaptation remains insufficiently defined. We postulate that mechanical stimuli from the ECM induce coordinated changes in adhesive and metabolic pathways, and that the nature of this independent mechano-metabolic pathway is conserved across benign, low-invasive, and high-invasive bladder cancer phenotypes. Therefore, we engineered collagen-hyaluronan hydrogels with tunable stiffness to recapitulate soft and rigid tumor microenvironments and profiled bladder cancer spheroids representing benign, low-invasive, and highly invasive states. Integrating hydraulic force spectroscopy, rheology, and molecular phenotyping, we show that matrix stiffening differentially reprograms spheroid architecture, motility, and adhesion- and metabolism-related gene expression. Spheroid behavior emerged from the interplay between intrinsic mechanical properties, matrix rheology, and molecular adaptation. HCV29 spheroids formed rigid, compact structures, relying on cell-matrix adhesion rather than metabolic or proteolytic remodeling. HT1376 spheroids activated glycolysis (HK2) and MMP-2-dependent ECM remodeling in soft matrices, but remained largely nonmigratory, indicating decoupling of invasive priming from motility. T24 spheroids were soft, deformable, and highly migratory in compliant matrices, integrating metabolic reprogramming, adhesion remodeling (E-/N-cadherin, SDC4), and radial collagen fiber alignment to drive invasion. Notably, canonical FAK/AKT/mTOR signaling was absent across all spheroids, while pS6 ribosomal protein and ILK indicated noncanonical, SDC4/integrin-ILK-dependent mechanotransduction supporting cytoskeletal dynamics, metabolism, and ECM remodeling. Collagen organization further differed across spheroid types, with dense, radially aligned fibers in HT1376, intermediate architecture in HCV29, and loose, disorganized networks in T24, closely matching their distinct migratory behaviors and cell-ECM interactions. These findings reveal stage-specific mechanometabolic strategies in bladder cancer, demonstrating how ECM mechanics and architecture jointly guide invasion, metabolic adaptation, and local immune modulation, including the regulation of immune cell infiltration and tumor immune evasion.
Humans↗
MicroRNAs and predicted targets in the switch from monolayered to spheroids of cholangiocarcinoma cells.
BACKGROUND: Extrahepatic cholangiocarcinoma (eCCA) is characterized by marked molecular heterogeneity and limited therapeutic options. MicroRNAs (miRNAs) are key post-transcriptional regulators of cancer-related pathways, but their contribution to tumor adaptation in physiologically relevant models remains poorly understood. Three-dimensional (3D) tumor spheroids better mimic in vivo conditions than conventional two-dimensional (2D) cultures. METHODS: We compared miRNA expression profiles in two eCCA cell lines (Sk-ChA-1 and Mz-ChA-1) grown as monolayers (2D) or multicellular tumor spheroids (3D). MiRNA profiling was performed using NanoString technology. Predicted targets were analyzed by over-representation analysis, and selected miRNAs and genes were validated by RT-qPCR and ELISA-based assays. RESULTS: 3D growth induced extensive miRNA remodeling, with distinct (54 deregulated in Sk-ChA-1 and 29 in Mz-ChA-1 cells) and partially overlapping signatures (miR-1283, miR-577, and miR-2113). Among the shared miRNAs, predicted targets included DUSP10 and RBFOX1, while in spheroids, cell-specific multiple miRNAs converged on shared targets (TNRC6B, SMARCAD1, ATG14, HMGA2, and CLOCK) displaying inverse expression patterns. The transcriptional program impacted MAPK signaling, enhanced EMT, and activated stress-adaptive networks but attenuated proliferation in 3D Sk-ChA-1 cells, while Mz-ChA-1 cells retained a more epithelial and proliferative profile. In this context, we point out the involvement of miR-19b-3p using anti-miR transfection experiments. CONCLUSION: Our findings reveal a miRNA-driven regulatory landscape associated with 3D growth in eCCA, linking tumor architecture to signaling rewiring and cellular plasticity, and highlight potentially druggable candidate targets and pathways to investigate as candidates using inhibitors or gene therapy-based interventions.
Humans↗
Extracellular Vesicles From Glioblastoma Cells Reflect 2D vs. 3D Culture Adaptation and Resistance to Temozolomide.
Glioblastoma (GBM) is an aggressive brain tumor marked by extensive heterogeneity, resistance to therapy, and dismal prognosis. Extracellular vesicles (EVs) have emerged as key players in GBM biology, mediating intercellular communication and therapy adaptation. However, the exact functions and molecular impact of EVs in GBM remain incompletely understood. In this study, we performed a comparative proteomic analysis of U87MG GBM cells grown in two-dimensional (2D) monolayers and three-dimensional (3D) spheroids following temozolomide (TMZ) treatment, alongside characterization of EVs derived from both culture systems. 3D-spheroids secreted more EVs of smaller size and exhibited a more TMZ-resistant, stem-like proteome under TMZ-induced genotoxic stress. In contrast, 2D cell cultures demonstrated greater proteome remodeling, with EVs enriched in protein families involved in DNA repair, oxidative stress adaptation, and methylation processes. Notably, several methyltransferases were decreased intracellularly but selectively retained in EVs, suggesting active sorting to influence the tumor microenvironment or modulate epigenetic states in recipient cells. EVs also carried adhesion molecules and signaling proteins linked to migration, invasion, and Wnt pathway activation, as well as metabolic enzymes connecting serine metabolism and redox control to TMZ resistance. Mapping EV and cellular proteomes onto The Cancer Genome Atlas (TCGA) dataset identified prognostic protein families associated with either poor or favorable patient outcomes. Our data demonstrate that EV cargo composition mirrors TMZ-induced phenotypic adaptation and reveals molecular mechanisms underlying therapeutic resistance. These EV-associated signatures may serve as clinically actionable biomarkers for patient stratification and offer potential targets to overcome chemoresistance in GBM.
Humans↗
[Indirect hemagglutination test at the ultrastructural level].
A study on ultra-thin sections was made of the preparations of agglutinate produced during the reaction of the immunoglobulin erythrocytic diagnostic agent with dry corpuscular Rickettsia prowazeki antigen, fluoresceine isothiocyanate labeled, and also SRBC used for the preparation of the diagnostic agent after formalinization, tannin treatment, sensitization with hyperimmune horse serum immunoglobulins and lyophilization, respectively. Formalin and tannin treatment of erythrocytes failed to be reflected on the ultrastructure of their cellular membranes; the treatment with hemosensitin was accompanied by the appearance of spheroid protrusions of the erythrocyte cytoplasmic membrane with the preservation of its three-layer structure. Specific interaction of sensitized erythrocytes with the antigen corpuscles was expressed morphologically in their apposition or connection through a gap of 20--30 nm.
Animals↗
The multicellular spheroid as a model tumor allograft. II. Characterization of spheroid-infiltrating cytotoxic cells.
Alloimmune lymphoid cells infiltrating multicellular spheroids of EMT6 mammary sarcoma cells (a solid tumor allograft model) have been characterized according to their morphological and functional properties. Both lymphocytes and macrophages were found within spheroids at the time of peak tumor cell damage. Cytotoxic cells specific for allograft antigens were also present. Using a short-term 51-Cr release assay, the cells responsible for cytotoxicity were characterized as a nonadherent, nonphagocytic T cell population. Velocity sedimentation cell separation further demonstrated that these cytotoxic cells had the physical properties of small lymphocytes. Some evidence for selective spheroid infiltration by specifically alloimmune cells was also obtained. The possible relationship of this cellular infiltrate to graft damage is discussed.
Animals↗
A novel 2D and 3D model for primary adrenocortical carcinoma of advanced and metastasized stage co-secreting cortisol, aldosterone, testosterone, 18-oxocortisol and 18-hydroxycortisol.
Adrenocortical carcinoma (ACC) is a highly aggressive malignancy with poor survival rates and few treatment options. Preclinical models are indispensable to further strengthen our understanding of disease progression and development of novel therapeutic treatments. Here, we report the establishment of a new cell line named ZUC-1 originating from the resection of an advanced primary ACC and its characterization at the genomic, cellular and molecular level. ZUC-1 cells were successfully propagated as monolayer cultures and three-dimensional spheroids. LC-MS/MS analysis revealed for ZUC-1 cells co-secretion of cortisol, aldosterone and testosterone, and the model represented in direct comparison with other current ACC pre-clinical models furthermore significantly elevated expression of SF-1, CYP11B1 and CYP11B2 genes. Whole genome sequencing identified various mutations in genes linked to DNA repair/stress response, stemness, and also steroidogenesis. Interestingly, ZUC-1 represents genotypic and phenotypic variations that might be of interest beyond ACC, including congenital adrenal hyperplasia (CAH) and polycystic ovary syndrome (PCOS). Moreover, 18-oxocortisol and 18-hydroxycortisol release was detected in ZUC-1, conditions which are often linked to hyperaldosteronism, but forskolin, potassium and, at higher concentration, angiotensin II modulability of CYP11B2 for this model is retained. ZUC-1 spheroids exhibited furthermore an intra-spheroidal heterogeneous mix of canonical and non-canonical Wnt pathway activation. We conclude that due to its origin and unique geno- and phenotypes, ZUC-1 represents an intriguing model to further gain a basic understanding of adrenal function, the pathogenesis of ACC, but it might be also of interest in the context of CAH and PCOS.
Humans↗
Understanding proneural-mesenchymal transition using patient-derived glioma stem-like cell (GSC) organoids and engineered extracellular matrix.
Glioblastoma multiforme (GBM) is a highly aggressive, angiogenic WHO grade IV glioma marked by rapid progression, therapeutic resistance, and poor prognosis. A defining feature of GBM is the presence of glioma stem-like cells (GSCs), which reside in specialized perivascular niches and drive tumor progression, recurrence, and therapeutic resistance. The blood-brain barrier, coupled with the complex and dynamic tumor microenvironment, poses significant challenges for both treatment and mechanistic investigation. Current in vitro GBM models inadequately recapitulate the structural and biochemical cues of the native perivascular niche due to the absence of functional vasculature and brain-mimetic extracellular matrix (ECM), limiting their physiological relevance and predictive power. To address the limitations of existing in vitro GBM models, we developed a patient-derived glioma stem cells (GSC) derived Matrigel spheroid system that transitions into organoids and enables integration into engineered microenvironments. Our model incorporates GSC organoids representing proneural and mesenchymal GBM subtypes, a synthetic engineered extracellular matrix (eECM), and endothelial cells (ECs) seeded on the matrix surface. We evaluated the expression of subtype-specific, pro-angiogenic, stemness, and differentiation markers under increasingly complex co-culture conditions. Our results show that Matrigel-derived GSC spheroids progressively differentiate into organoids over two weeks, with significantly enhanced expression of cell-specific markers in the presence of ECs. Encapsulation of these organoids within eECM, combined with EC co-culture, further promoted cellular invasion and induction of GBM associated genes. This in situ encapsulation strategy enables real-time observation of GSC behavior in a tunable microenvironment that mimics key features of the native tumor niche. Together, this platform provides a physiologically relevant and modular in vitro system for investigating GBM pathophysiology. It holds promise for uncovering tumor-specific cellular dependencies, studying GSC-vascular interactions, and conducting high-throughput drug screening under controlled, biomimetic conditions.
Engineered extracellular matrix↗
Live-cell transcriptomics with engineered virus-like particles.
Transcriptomic profiling is widely applied to characterize cellular gene expression, yet existing approaches lyse cells and preclude direct analysis of transcriptional dynamics in the same sample over time. We addressed this limitation by engineering mammalian cells to "self-report" their transcriptional states via mRNA export in virus-like particles (VLPs). Repeated sampling of culture media from VLP-producing cell populations faithfully captured evolving transcriptional states in complex biological settings, including acute inflammatory stimulation of primary cell spheroids and multi-day differentiation of pluripotent stem cells. We engineered VLP components for multiplexed readouts from distinct cell types in co-culture and for tuning self-reported RNA profiles. Finally, we demonstrated the unique utility of self-reporting for selective longitudinal tracking of endothelial cell dynamics within the enclosed architecture of a microphysiological co-culture system to identify perivascular stroma-dependent temporal gene programs underlying vasculogenesis. Altogether, this work establishes cellular self-reporting as a broadly enabling technology for live-cell transcriptome-wide gene expression profiling.
RNA↗
Effects of sensitizers on cell respiration: III. The effects of hypoxic cell radiosensitizers on oxidative metabolism and the radiation response of an in vitro tumour model.
Physiological factors are important when considering the effects of radiosensitizers on the radiation response of complex systems such as multicellular spheroids. In this system, under conditions of unlimited nutrient supply, cells are rendered hypoxic by metabolism. Thus, using the spheroid system as an in vitro model of the tumour-cell microenvironment, we have determined the relative contribution of radiosensitization and respiratory effects of a number of electron-affinic sensitizers having potential clinical use. These studies are indicative of physiological responses at the cellular level, and suggest optimal drug administration schemes for obtaining maximal radiation response in vivo hypoxic cell sensitizers.
Cell Survival↗
The cytoskeletal framework and poliovirus metabolism.
The cytoskeletal framework prepared by detergent lysis of suspension-grown HeLa cells is compared to the structure obtained from poliovirus-infected cells. This framework, which retains major features of cell morphology and carries the cellular polyribosomes as well as the major structural filaments, is profoundly reorganized following virus infection. This reorganization underlies, at least in part, the morphological changes termed the "cytoplasmic effect." These cytoskeletal changes appear related to the involvement of the framework with viral-specific metabolism. Extensive cytoskeleton alterations occur even when guanidine inhibits viral replication, and thus result from small amounts of early viral products. The normally spheroidal nucleus deforms, allowing a modified region of the cytoplasm to occupy a central position in the cell, and many membrane-enclosed vesicles peculiar to the infected cell are elaborated here. The skeleton preparation reveals that this region contains intermediate filaments arranged in a pattern unique to infected cells. Further changes occur when viral replication is permitted. The central region filaments become coated with darkly staining material which may be viral RNA. Numerous small particles appear on the filaments which resemble partially assembled virions. Mature virions, however, have no affinity for the cytoskeleton and appear to be free in the cytoplasm. Host cell messenger RNA, normally attached to the skeletal framework, is released in infected cells and is replaced by the viral-specific polyribosomes. The trabecular network which carries polyribosomes appears to be rearranged; the viral polyribosomes are located principally at the cell periphery and are excluded from the central region. The viral replication complex with its double-stranded RNA is also attached to the skeletal framework and may comprise the dark staining material coating the filaments of the central cell region.
Cell Nucleus↗