Search Results (785)

Background/Objectives: PI3K/AKT/mTOR is a key pathway in cell proliferation, metabolism, and survival. Activating PIK3CA mutations are seen in up to 20% of colorectal cancers and are associated with increased cyclo-oxygenase-2 (COX-2) expression. Recent studies demonstrated a significant survival benefit from taking low-dose aspirin, a nonselective COX inhibitor, supporting further exploration of the synergistic effects of combined PI3Kα inhibitor (inavolisib) and COX-2 inhibitor (celecoxib) therapy. Methods: The effects of celecoxib–inavolisib combination treatment were tested on human colorectal cancer cell lines and patient-derived organoid models. Experiments included cell viability and colony formation assays, immunoblotting, and immunofluorescence. Results: We found that celecoxib and inavolisib demonstrated synergy in suppressing the growth of colorectal cancer cell lines, grown in both 2D and 3D cell culture, regardless of PIK3CA mutation status. In patient-derived organoid models, while synergy was seen in both organoids, growth of the PIK3CA mutated organoid was more potently suppressed. Immunoblotting of cells after combination treatment showed decreased expression of mitogenic signaling marker p-AKT across all 2D cell lines and in both cell lines grown as 3D spheroids, as well as increased expression of apoptotic marker cPARP in four out of five 2D cell lines and in both cell lines grown as 3D spheroids. Immunofluorescence staining of organoids after combination treatment, however, showed no significant increase in expression of apoptotic marker Cas-3 nor in mitogenic marker Ki-67 in either organoid. Furthermore, an apoptosis assay performed on two cell lines showed no significant increase in Annexin V or phosphatidylserine staining. Conclusions: Celecoxib and inavolisib demonstrated synergy in suppressing the growth of both colorectal cancer cell lines and patient-derived organoids, though PIK3CA mutation status did not appear to affect drug efficacy in cell lines as it did in patient-derived organoids. Potential compensatory or resistance mechanisms might include oncogene drivers in the MAPK/ERK pathway. When compared to monotherapy, combination therapy was the only drug condition to significantly increase the percentage of apoptotic cells based on Annexin V and phosphatidylserine staining, and this effect was only seen in the PIK3CA mutated cell line. Ultimately, our findings provide preliminary support for celecoxib–inavolisib combination treatment as a rational therapeutic avenue warranting further preclinical investigation. Full article

Introduction: Glioblastoma (GBM) is the most aggressive primary tumor of the central nervous system and is highly resistant to temozolomide (TMZ). Rutin is a potent antioxidant with immunomodulatory and anti-glioma effects in vitro, although its mechanisms of action remain incompletely understood. This study investigated the effects of rutin on morphology, viability, redox balance, and pro-tumoral signaling in GBM 2D cultures and 3D spheroids, as well as its association with TMZ sensitivity. Methods: GL15 and U343 human GBM cell lines and primary astrocytes were treated with rutin (5–30 μM) and/or TMZ (125–4000 μM). Cell metabolic activity and viability were assessed by MTT, PI/DiOC18(3) or PI/Hoechst. Cell migration was assessed from spheroid-derived cells, and extracellular matrix (ECM) components (fibronectin and laminin) were evaluated by immunofluorescence. Intracellular reactive oxygen species (ROS) were measured by DCFH-DA fluorescence. IL-6, STAT3, NOS2, and IDO1 gene expression were determined by RT-qPCR, and protein expression of MMP2, fibronectin, STAT3, PI3K, and AKT by Western blotting. Nitric oxide (NO) and L-kynurenine levels were quantified in the supernatant by colorimetric assays. Results: Rutin reduced cell viability and enhanced TMZ cytotoxicity in both 2D and 3D cultures, while exerting selective effects by increasing metabolic activity and attenuating TMZ-induced effects in non-tumoral primary astrocytes. In 3D spheroids, rutin affected structural organization and reduced spheroid-derived cell migration, accompanied by changes in ECM components, including MMP2, fibronectin, and laminin. Rutin decreased intracellular ROS levels and suppressed the TMZ-induced increase in ROS and NOS signaling. These effects were accompanied by modulation of IL-6/STAT3 signaling, along with reduced STAT3, PI3K, and AKT protein levels. Rutin also modulated immunometabolic parameters, including extracellular L-kynurenine and nitric oxide levels, and enhanced TMZ responsiveness following pre-sensitization. Conclusions: Rutin enhances TMZ responsiveness by modulating interconnected pro-tumoral mechanisms, including redox balance, pro-survival signaling, ECM remodeling and migratory behavior, and immunometabolic pathways linked to chemoresistance, supporting its potential as an adjuvant therapeutic strategy. Full article

Epithelial ovarian cancer (EOC) remains a lethal malignancy requiring novel therapeutic strategies due to high recurrence and chemoresistance. This study evaluated the combined antitumor effect of metformin and the co-transfection of tumor-suppressor microRNAs miR-145 and miR-23b in A2780 and OV90 EOC cell lines using both 2D and 3D models. In monolayer cultures, our approach significantly reduced the expression of proliferation markers Ki-67 and c-MYC, and decreased cell migration and invasion in both cell lines compared to controls. In 3D spheroid models, the treatment reduced VEGF secretion and relative spheroid area in A2780 cells, significantly increasing cytotoxicity; however, OV90 spheroids exhibited marked resistance. Fluorescent miRNA tracking revealed that this resistance occurs despite successful intracellular delivery, indicating an intrinsic biological resistance conferred by the 3D microenvironment. Overall, these findings suggest that the combined administration of metformin and miRs effectively limits tumor progression, but also strongly underscore the importance of using complex 3D models to accurately evaluate therapeutic efficacy and intrinsic resistance mechanisms. Full article

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a widespread condition with a complex pathogenesis. Cell-based models are important tools for studying the mechanisms underlying its development and progression. The aim of this review is to analyze the HepaRG, Huh-7, immortalized human hepatocyte (IHH), and primary human hepatocyte (PHH) cell lines for modeling and studying MASLD. HepaRG represents the most metabolically competent immortalized hepatocyte model with preserved biotransformation activity and a physiological bioenergetic response to lipid loading, making it valuable for pharmacological and toxicological studies. Huh-7 is distinguished by its accessibility and suitability for studying steatosis, lipotoxicity, insulin resistance, and paracrine mechanisms of fibrogenesis; however, its use is limited by its tumor origin, impaired carbohydrate metabolism, and low activity of xenobiotic-metabolizing enzymes. The IHH model occupies an intermediate position because of its non-tumor origin and is of interest for studies of senescence, epigenetic regulation, and signaling pathways involved in steatosis, although interpretation of results requires consideration of immortalization-related effects and specific metabolic limitations. PHH remains the most physiologically relevant platform for MASLD modeling, particularly in three-dimensional (3D) and microphysiological formats; however, its use is limited by high cost, interindividual variability, and the limited duration of the differentiated phenotype. Increasing model complexity—from two-dimensional (2D) monocultures to co-cultures, spheroids, and organ-on-chip systems—enhances physiological relevance and enables reproduction not only of steatosis but also of the inflammatory and fibrogenic components of MASLD progression, yet it reduces reproducibility and complicates standardization. Overall, none of the existing models is universal, and the optimal strategy is to select models according to the specific research question. A key direction for future research is the standardization of steatosis induction protocols and the unification of criteria for evaluating results. Full article

Human endometrial mesenchymal stem/stromal cells (eMSCs) are widely used in laboratories and clinical applications to study various aspects of tissue engineering and regenerative medicine. Three-dimensional (3D) cultivated MSCs have a higher therapeutic efficacy compared to 2D culture. Ion channels are involved in maintaining many physiological cell functions, including proliferation, differentiation, apoptosis, and migration. This study describes the functional expression of voltage-gated sodium channels (NaV) in eMSCs and the role of these channels in cell migration. Using RT-PCR analysis and immunofluorescent microscopy, we identified the expression of almost all pore-forming alpha (NaV 1.1, 1.2, 1.4–1.9) and channel-modulating beta-NaV subunits (except beta2) in eMSCs. In the whole-cell patch-clamp configuration, channels activated by membrane depolarization of eMSC were detected. The channels were blocked by the selective NaV antagonist TTX in nanomolar concentrations. The NaV agonist veratridine at a concentration of less than 40 μM inhibited voltage-gated sodium currents, while 100 μM and above prevented channel inactivation. The wound healing assay showed that both TTX (10 μM) and veratridine (100 μM) reduced the migration properties (the wound healing rate) of eMSCs cultivated in 2D conditions compared to the control. An opposite effect by both agents was shown on the motility of eMSCs cultivated in 3D conditions, increasing the cell spreading rate from spheroids. Our data suggest that NaV channels are expressed in human eMSCs and play an important role in the regulation of stem cell migration; this regulatory mechanism significantly depends on the culture conditions of MSCs. Full article

Background/Objectives: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and lethal forms of malignant neoplasm of the endocrine system, and osteopontin (OPN) has been shown to be aberrantly expressed in this tumor type. Among the five OPN splicing isoforms (OPN-SI), OPN-4 has been recently reported in several tumor types, including ATC, but its functional role(s) have not yet been elucidated. Methods: To characterize OPN-4 roles in ATC cells, OPN-4 was ectopically overexpressed in the c643 ATC cell line, generating the c643/OPN-4 cells. OPN-roles were evaluated by cell functional assays, including cell proliferation and viability, using Carboxyfluorescein Succinimidyl Ester (CFSE), crystal violet, and trypan blue assays. For migration, clonogenicity, cell cycle and apoptosis assays were used. For assessment, c643/OPN-4 cells were cultured in two-dimensional (2D) monolayers or three-dimensional (3D) spheroids with the latter being maintained in a bespoke microfluidic system. Results: OPN-4 overexpression led to a significant reduction in cell proliferation, viability, migration and clonogenicity. c643/OPN-4 cells displayed a significant accumulation in the G0/G1 phase and a decrease in the S phase of the cell cycle; however this did not affect cell death or the expression levels of other OPN-SI. In a spheroid model of c643/OPN-4 cells, no significant differences were found in spheroid size or viability when compared to those formed by control cells. Notably, OPN-4 overexpression enhanced the effects of sorafenib on cell viability under dynamic treatment conditions involving continuous perfusion. Conclusions: These early findings point to the fact that OPN-4 may reduce some aspects of tumor progression features in ATC cells and open new avenues for investigating OPN-4 as a biomarker of therapeutic response in personalized treatment strategies. Full article

Background/Objectives: Peritoneal micrometastases (micromets) remain a major barrier to durable cytoreduction in ovarian and other intra-abdominal cancers because lesions are difficult to visualize and are often resistant to systemic therapy. Liposomal doxorubicin (Dox) improves pharmacokinetics but can be limited by slow intratumoral release. Porphyrin-phospholipid (PoP) liposomes enable near-infrared light–triggered release of Dox (chemophototherapy (CPT)), creating an opportunity for intraoperative fluorescence-guided treatment planning and monitoring. Here, we evaluate a laparoscopic fluorescence imaging platform for quantifying light-triggered drug delivery. Methods: LC-Dox-PoP was applied to SCC2095sc and SKOV-3 cultures in 2D monolayers and 3D spheroid clusters. Dox fluorescence was quantified using a laparoscopic fluorescence imaging system over 1–9 μg/mL concentrations and compared with standard well-plate reader measurements. Porphyrin fluorescence was monitored to assess spheroid localization and photobleaching after activation light exposure. Results: For both cell lines, Dox fluorescence exhibited an approximate 4-fold increase at the maximum administered LC-Dox-PoP concentration, following a linear trend in both SCC2095sc and SKOV-3 cultures (R² = 0.97, 0.98 for 2D and R² = 0.98, 0.98 for spheroids). Laparoscope-derived fluorescence measurements agreed with well-plate reader measurements (R² = 0.89–0.96). Porphyrin fluorescence provided stronger complementary contrast for localizing spheroid constructs and decreased after activation light exposure, consistent with photobleaching during triggered release. Conclusions: These results support a quantitative imaging framework for fluorescence-guided monitoring of light-triggered liposomal drug release and may enable individualized CPT dosimetry for peritoneal micrometastases. Findings in SCC2095sc additionally suggest potential relevance of fluorescence-guided CPT for head and neck/oral cancer, where localized post-resection adjuvant treatment may improve control of residual disease. Full article

Human adipose biology is strongly influenced by three-dimensional (3D) architecture, cell–cell interactions, and local oxygen availability maintained over a long-term culture period, features that are not reproduced in traditional two-dimensional (2D) culture systems. To address this gap, we established a long-term human adipose-derived stem cell (hASC) spheroid model using elastin-like polypeptide–polyethyleneimine (ELP-PEI) coating. The ELP-PEI coating facilitated stable spheroid formation and sustained adipogenic differentiation over 56 days. As spheroids enlarged and matured, they exhibited hallmark features of adipocytes, including lipid accumulation, morphological compaction, and transition out of the proliferative state. Glucose uptake increased during maturation and declined as spheroids became larger. This reduction coincided with a marked rise in hypoxia-inducible factor-1α (HIF-1α) expression, indicating the emergence of a hypoxic microenvironment within larger spheroids. Notably, inhibiting HIF-1α restored insulin-stimulated glucose uptake, demonstrating that hypoxia was the primary driver of impaired insulin responsiveness in late-stage spheroids. These findings position ELP-PEI-supported hASC spheroids as a practical and physiologically relevant platform for studying human adipocyte biology, particularly the development and reversibility of hypoxia-associated metabolic dysfunction. This model offers new opportunities for mechanistic studies and for evaluating therapeutic strategies targeting insulin resistance and adipose tissue pathology. Full article

Background/Objectives: Endometrial cancer frequently develops resistance to apoptosis-based therapies, highlighting the need for alternative strategies that control tumor growth independently of cell death induction. Three-dimensional (3D) tumor models more accurately recapitulate tumor architecture, cellular interactions, and treatment resistance compared to conventional two-dimensional (2D) cultures. This study aimed to investigate whether imatinib-based combination treatments can enforce sustained cytostatic responses in a 3D endometrial cancer model. Methods: Ishikawa spheroids were treated with imatinib alone or in combination with lithium chloride or medroxyprogesterone acetate. Proliferation was assessed by bromodeoxyuridine incorporation, cell cycle distribution by flow cytometry, and apoptosis by Annexin V/propidium iodide staining over 96 h. Results: Imatinib monotherapy produced modest antiproliferative effects, whereas combination treatments resulted in sustained suppression of DNA synthesis, increased G₀/G₁ accumulation, and reduced S-phase entry. Despite strong growth inhibition, apoptotic fractions remained low across all groups. Conclusions: Imatinib-based combinations suppress 3D endometrial cancer growth predominantly through sustained cell cycle arrest rather than apoptosis induction. Targeting apoptosis-resistant proliferation through cytostatic mechanisms may represent a complementary therapeutic strategy for hormone-responsive endometrial cancer and warrants further translational evaluation. Full article

Breast cancer is classified into distinct molecular subtypes, including Luminal A, Luminal B, HER2-enriched, Basal-like, and Claudin-low. While traditional studies mostly use 2D cell cultures, 3D models better mimic in vivo tumor conditions. In this study, we generated and characterized 3D spheroids from breast cancer cell lines representing different molecular subtypes. Morphologically, spheroids were either compact (MCF-7/AZ, T47D, BT474, MDA-IBC-3, BT-20, SUM149PT) or loosely adhered (MDA-MB-468, SK-BR-3, MDA-MB-231), while retaining key parental subtype biomarkers. Cell viability decreased with increasing spheroid size, but apoptotic cCasp3 staining was restricted to Basal-like spheroids. Compact spheroids expressed E- and/or P-cadherin, indicating epithelial or epithelial–mesenchymal transition (EMT) hybrid traits, while loose spheroids showed vimentin expression linked to a mesenchymal phenotype. In conclusion, EMT-associated features, rather than intrinsic molecular subtype, may contribute to 3D spheroid architecture of breast cancer cell lines. Full article

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease (NAFLD), is the most common chronic liver disease in the world. The disease progresses from steatosis to metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, cirrhosis, and hepatocellular carcinoma. The modern concept of “multiple parallel hits” interprets disease progression as the result of the synergistic action of lipotoxicity, oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, proinflammatory signals, and gut–liver axis dysfunction. Against the background of the limited translation of preclinical data from animal models due to interspecies differences, the importance of human-oriented in vitro platforms compatible with controlled design and high-throughput screening is increasing. The current review analyzes MASLD models based on the HepG2 cell line, systematizing steatosis induction protocols, evaluating the metabolic characteristics and limitations of this cell, and comparing 2D monocultures, 3D systems, and co-cultures. HepG2 has been shown to demonstrate a predictable steatogenic response to free fatty acids (FFAs) and is convenient for reproducing early stages of pathogenesis and primary pharmacological selection of compounds. At the same time, key limitations of the model are highlighted, namely tumor origin, glycolytic shift (Warburg effect), reduced β-oxidation, impaired very-low-density lipoprotein (VLDL) assembly and secretion, and sharply reduced cytochrome P450 (CYP450) activity, as well as limited reproducibility of fructose-induced de novo lipogenesis (DNL). Comparative analysis demonstrates an increase in physiological relevance with the transition from 2D to 3D and multicomponent co-cultures, accompanied by increased complexity and cost, but allowing for the modeling of inflammation and fibrogenesis. The review justifies approaches to selecting the appropriate platform based on the specific research task. Full article

Bone formation requires a substantial energy supply to sustain extracellular matrix production and mineralization, yet the temporal contribution of lipid metabolism during osteoblast maturation remains incompletely characterized. This study investigated the molecular and transcriptional remodeling of lipid metabolism. Intracellular lipid distribution was analyzed by confocal microscopy using Nile Red staining. Transcriptional modulation of lipid synthesis, storage, lipolysis, genes associated with mitochondrial fatty acid oxidation, and osteogenic markers were assessed by quantitative real-time PCR, and the biochemical composition was evaluated by Raman spectroscopy. Early stages of spheroid development showed higher expression of genes involved in lipid synthesis and storage (FASN, DGAT2, and PLIN2) together with intracellular lipid accumulation, whereas later stages displayed increased expression of lipolytic and β-oxidation markers (PNPLA2/ATGL, CPT1A, and HADHA), accompanied by the redistribution of lipid droplets. The Raman analysis revealed a time-dependent variation of lipid-associated CH₂/CH₃ bands and modulation of protein-related Amide I–III signals, consistent with biochemical remodeling during maturation. Overall, the data indicate a coordinated transcriptional shift from lipid accumulation-associated pathways toward lipid mobilization during osteogenic progression in a 3D culture. This model provides a controlled experimental platform for investigating metabolic regulation during bone formation and for studying metabolic alterations associated with skeletal disorders. Full article

Glioblastoma (GBM) is one of the most aggressive and therapy-resistant primary brain tumors, mainly due to its pronounced intratumoral heterogeneity and highly invasive phenotype. Patient-derived three-dimensional (3D) culture models, including tumor spheroids, represent valuable tools to preserve the cellular complexity, phenotypic plasticity, and microenvironmental features of GBM ex vivo. However, standardized and reproducible protocols for the generation and maintenance of GBM spheroids from surgical specimens are still limited. Here, we describe a detailed and robust protocol for the isolation, 3D cultures, and expansion of primary GBM cells obtained from patient biopsies, leading to the formation of stable and morphologically consistent spheroids. The protocol provides step-by-step instructions for tissue dissociation, cell seeding under low-adhesion conditions, optimization of culture density, and long-term spheroid maintenance. In addition, we include guidelines for the morpho-phenotypical characterization of the resulting 3D structures. This methodological workflow offers a reproducible platform for modeling GBM in vitro, enabling the study of tumor biology and supporting translational applications such as drug screening, biomarker validation, and patient-specific therapeutic testing in a 3D context. Full article

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. Full article

Three-dimensional (3D) spheroid cancer models provide enhanced physiological relevance relative to traditional monolayer cultures but often demonstrate restricted drug responsiveness due to their dense architecture, hypoxic gradients, and diminished therapeutic penetrance. This study overcomes these limitations by establishing a sensitized 3D spheroid cancer cell model that employs the adenovirus-mediated gene expressions of tumor-suppressor and pro-apoptotic genes consisting of MOAP-1, BAX, and RASSF1A. The optimization of adenoviral infectivity led to the discovery of an intermediate multiplicity of infection (MOI) that facilitated effective and uniform transduction while reducing cytotoxicity. Adenovirus-infected 3D spheroid cells demonstrated enhanced apoptotic activities, evidenced by increased cell death relative to untreated spheroids. When exposed to the anti-cancer compound such as piperonal and pyrazole, the sensitized spheroids exhibited significantly enhanced drug responsiveness and synergistic effects over a five-day treatment period, surpassing the effects of adenovirus or anti-cancer drug treatment alone. Notably, similar responses were noted between low- and high drug doses, suggesting an enhancement of therapeutic efficacy at lower concentrations. This sensitized 3D spheroid model constitutes a more predictive in vitro system for anti-cancer drug discovery, facilitating enhanced mechanistic evaluation and the identification of potent drug candidates with greater translational significance. Full article