Search Results (194)

Despite significant advancements in understanding the pathogenesis and treatment of hematological malignancies, including leukemia and multiple myeloma, the majority of patients continue to experience poor long-term outcomes. This is partly due to the difficulty of accurately recapitulating the malignant microenvironment in vitro, particularly the bone marrow niche. The complexity of the bone marrow microenvironment poses a challenge for the in vitro examination of hematological malignancies. Traditionally, 2D culture and animal models have been utilized, but these representations are limited and have been criticized for their lack of human physiological relevance. In an attempt to overcome this, 3D models have been developed that more accurately recapitulate the in vivo microenvironment. Herein, we present an overview of recent developments in 2D and 3D models used for studying the bone marrow niche in hematological malignancies, highlighting their advantages and limitations. Full article

Background/Objectives: VitroScreenORA^® (by VitroScreen srl) Dermo Papilla spheroids, based on two micro-physiological systems (non-vascularized DP and vascularized VASC-DP), were used to study the molecular mechanisms behind hair cycle regression. Methods: Dermal papilla cells (HFDPC) were cultured to develop both models. Hair cycle regression was induced by exposing DP spheroids to TGF-β1 for 72 h and/or FGF-18 for an additional 24 h. Catagen phase entrance was evaluated by modulating specific genes (FGF7, CCND1, and WNT5B). The VASC-DP model was obtained by sequentially co-culturing HFDPC and primary dermal microvascular endothelial cells (HMDEC), mimicking the surrounding capillary loop. The vascular system’s impact was assessed at 5 and 10 days using IF on CD31 (micro-vessels) and Fibronectin (FN). Nanostring nCounter^® technology was applied to investigate the transcriptional signature based on the WNT pathway. Extended culture time up to 11 days simulated natural hair cycle regression, monitored by versican and FN expression (IF). Minoxidil, Doxorubicin, and Retinol-based products were used to modify physiological aging over time. Results: Data shows that the vascular system improves tissue physiology by modulating the associated genes. Extended culture time confirms progressive DP structure degeneration that is partially recoverable with Retinol-based treatments. Conclusions: Both models provide a reliable platform to investigate the hair cycle, paving the way for new testing systems for personalized therapies. Full article

Due to the complexity of modeling tumor-host interactions within the tumor microenvironment in vitro, we developed a 3D heterotypic cellular breast cancer (BC) model. We generated spheroid models using MCF7, MDA-MB-231, and SK-BR-3 cell lines alongside cancer-associated (BrC4f) and normal (BN120f) fibroblasts in ultra-low attachment plates. Stromal spheroids (3Df) were formed using a liquid overlay technique (graphical abstract). The YT cell line and peripheral blood NK (PB-NK) cells were used as immune components in our 3D model. In this study, we showed that stromal cells promoted tumor cell aggregation into spheroids, regardless of the initial proliferation rates, with NK cells accumulating in fibroblast-rich regions. The presence of CAFs within the model induced alterations in the expression levels of MICA/B and PD-L1 by tumor cells within the 3D-2 model. The feasibility of utilizing a 3D cell BC model in combination with cytokines and PB-NKs was evaluated. We observed that IL-15 and IL-2 enhanced NK cell activity within spheroids, whereas TGFβ had varying effects on proliferation depending on the cell type. Stimulation with IL-2 and IL-15 or TGFβ1 altered PB-NK markers and stimulated their differentiation into ILC1-like cells in 3D models. These findings underscore the regulatory function of CAFs in shaping the response of the tumor microenvironment to immunotherapeutic interventions. Full article

Glioblastoma (GBM) is the most aggressive form of malignant gliomas, with the eicosanoid-synthesizing enzyme cyclooxygenase-2 (COX-2) playing a pivotal role in its progression via the COX-2/prostaglandin E2/4 axis. COX-2 upregulations in tumor cells induces a pro-inflammatory tumor microenvironment (TME), affecting the behavior of invading bone marrow-derived macrophages (Mϕ) and brain-resident microglia (MG) through unclear autocrine and paracrine mechanisms. Using CRISPR/Cas9 technology, we generated COX-2 knockout U87 glioblastoma cells. In spheroids and in vivo xenografts, this resulted in a significant inhibition of tumorigenic properties, while not observed in standard adherent monolayer culture. Here, the knockout induced a G1 cell cycle arrest in adherent cells, accompanied by increased ROS, mitochondrial activity, and cytochrome c-mediated apoptosis. In spheroids and xenograft models, COX-2 knockout led to notable growth delays and increased cell death, characterized by features of both apoptosis and autophagy. Interestingly, these effects were partially reversed in subcutaneous xenografts after co-culture with Mϕ, while co-culture with MG enhanced the growth-suppressive effects. In an orthotopic model, COX-2 knockout tumors displayed reduced proliferation (fewer Ki-67 positive cells), increased numbers of GFAP-positive astrocytes, and signs of membrane blebbing. These findings highlight the potential of COX-2 knockout and suppression as a therapeutic strategy in GBM, particularly when combined with suppression of infiltrating macrophages and stabilization of resident microglia populations to enhance anti-tumor effects. Full article

Background/Objectives: Three-dimensional (3D) cell culture models more accurately simulate the in vivo tissue environments as compared to conventional two-dimensional (2D) monolayer cultures. Among these, spheroid cultures are particularly valuable for pharmaceutical research, as they allow for the study of tumor growth, drug responses, and cell–cell interactions in a physiologically relevant manner. Superhydrophobic surfaces (SHSs) have shown a promise in enhancing spheroid formation by reducing cell–substrate adhesion and promoting cell–cell aggregation. This study aims to evaluate the effectiveness of two different SHS coatings (SHS1: fluorinated; SHS2: silicone-based) in generating co-culture spheroids composed of non-tumoral fibroblasts (3T3) and tumoral epidermoid carcinoma cells (A431), thereby mimicking aspects of the tumor microenvironment. Methods: Co-cultures of 3T3 and A431 cells were seeded at varying ratios onto SHS1 and SHS2 substrates to assess their ability to support 3D spheroid formation. Spheroids were characterized by measurements of circularity and size distribution, viability through live/dead staining, and surface topography using 3D profilometry. Results: Spheroid formation was significantly influenced by both the surface properties and the fibroblast-to-carcinoma cell ratio. The fluorinated SHS1 surface facilitated superior cell viability and promoted the formation of well-rounded, uniform spheroids. In contrast, the silicone-based SHS2 surface resulted in less defined spheroidal structures and lower overall viability. Profilometry confirmed more consistent and compact 3D architectures on SHS1. Conclusions: This study demonstrates that SHS1, a fluorinated superhydrophobic coating, is more effective than SHS2 in supporting the formation of viable and structurally coherent 3D co-culture spheroids. These findings underscore the potential of SHS1 as a low-cost, tunable platform for developing in vitro cancer models and advancing the study of tumor–stroma interactions. Full article

Background/Objectives: Non-melanoma skin cancer (NMSC) is among the most common cancers in the United States, with solar ultraviolet (UV) radiation being a primary etiological factor. T-LAK cell-originated protein kinase (TOPK), a serine/threonine kinase activated by solar UV, has been implicated in skin carcinogenesis. This study aimed to investigate the mechanistic role of TOPK in solar UV-induced skin damage and tumor development. Methods: RNA sequencing (RNA-seq) was performed on skin tissues from wild-type (WT) and TOPK knockout (KO) mice, with or without solar UV exposure, to identify TOPK-regulated genes and pathways. Follow-up experiments using Western blotting, immunofluorescence, and luciferase assays were conducted in vitro and in vivo. Functional assays included 3D spheroid and Transwell co-culture systems involving cutaneous squamous cell carcinoma (cSCC) and fibroblast cells. Results: TOPK deletion altered gene expression profiles and inhibited solar UV-induced activation of multiple signaling pathways, including cytokine–cytokine receptor interaction, PI3K/AKT, MAPKs, PKG, cAMP, and calcium signaling. RNA-seq and protein analyses identified interleukin-19 (IL19) as a key downstream effector suppressed by TOPK deletion. In cSCC and fibroblast cells, TOPK knockdown reduced IL19 expression and secretion. IL19 promoted cSCC growth and activated PI3K/AKT, ERK, and TOPK pathways. Additionally, chronic TGFβ exposure increased IL19 expression and activated fibroblasts, as indicated by elevated αSMA and FAPα levels. Conclusions: These findings establish TOPK as a central regulator of solar UV-induced skin carcinogenesis, partially via modulation of IL19 signaling and fibroblast activation. Targeting TOPK may offer a novel strategy for the prevention and treatment of NMSC. Full article

The present study compared the free and encapsulated photosensitizer hypocrellin B (HB) in terms of photophysical-chemical properties, cellular uptake, subcellular distribution, and phototoxicity. The hydrophobic HB was encapsulated into liposomes (HB@Lipo) or poly (lactic-co-glycolic acid) nanoparticles (HB@PLGA). Encapsulation into nanocarriers exerted no obvious influence on the photophysical-chemical properties of HB, including UV-visible absorbance, fluorescence spectra, singlet oxygen (¹O₂) production capacity, and photostability. Free and encapsulated HB revealed some disparities in cellular uptake and subcellular localization patterns. In 2D-cultured B16 cells and tumor spheroids, free HB exhibited the fastest cellular uptake, while HB@PLGA had the lowest, as evidenced. Subcellular localization analysis first revealed a significant colocalization of free HB, HB@Lipo, and HB@PLGA within lipid droplets, with minimal colocalization in mitochondria and the endoplasmic reticulum. Unlike free HB and HB@Lipo, HB@PLGA exhibited strong lysosomal colocalization, indicating a unique intracellular trafficking pathway for PLGA-encapsulated HB. Upon laser irradiation, both free and encapsulated HB induced pronounced phototoxicity with substantial ROS production, confirming the robust PDT effect of HB. The photodynamic killing effect correlated with the intracellular HB content. These findings highlighted the impact of nanoformulation on HB’s cellular behavior and therapeutic performance. Full article

Perfusion offers unique benefits to tissue-engineered systems, enhancing oxygen and nutrient transport, which improves tissue formation and growth. In this study, we present a novel and integrated portable perfusion system. Weighing < 10 lbs, the system can maintain continuous flow in a standard incubation environment (37 °C, 5% CO₂), effectively functioning as a portable perfusion and tissue culturing system. To characterize the perfusion system’s flow parameters, we measured the volumetric flow rate across a range of pressures and found that the system could achieve flow velocities between 1.69 to 4.6 $\mathsf{\mu }$m/s, which is similar to in vivo interstitial flow. Computational fluid dynamics revealed fully developed laminar flow within the sample-containing region of the perfusion system, helping ensure even fluid and nutrient distribution. To study the system’s compatibility with live tissues, bioengineered tissue patches were created and perfused. After 24 h of perfusion, no significant difference in cell viability was observed between the perfused samples and static controls, indicating no adverse effects on cell health. Perfusion also facilitated enhanced spatial organization within tissue patches, reducing the inter-spheroids distance. Furthermore, perfusion strengthened the tissue matrix and reduced the degradation rate of the hydrogel scaffold. Complemented by its ability to provide mobile perfusion and incubation, this novel integrated portable perfusion system holds promise for promoting tissue maturation and advancing tissue bioengineering studies. Full article

Background: Celastrol (Cela), a phytochemical extracted from Tripterygium wilfordii, has been extensively investigated for its potential anti-inflammatory, anti-psoriatic, antioxidant, neuroprotective, and antineoplastic properties. However, its clinical translation is limited due to poor bioavailability, low solubility, and nonspecific toxicity. This study aimed to develop and evaluate an inhalable Cela-loaded nanoemulsion (NE) formulation to enhance targeted drug delivery and therapeutic efficacy in non-small cell lung cancer (NSCLC). Methods: The NE formulation was optimized using Capmul MCM (25%), Tween 80 (20%), Transcutol HP (5%), and water (50%) as the oil, surfactant, co-surfactant, and aqueous phase, respectively. Physicochemical characterization included globule size, zeta potential, and drug release in simulated lung fluid. In vitro aerosolization performance, cytotoxicity in NSCLC cell lines (A549), scratch and clonogenic assays, and 3D tumor spheroid models were employed to assess therapeutic potential. Results: The NE showed a globule size of 201.4 ± 3.7 nm and a zeta potential of −15.7 ± 0.2 mV. Drug release was sustained, with 20.4 ± 5.5%, 29.1 ± 10%, 64.6 ± 4.1%, and 88.1 ± 5.2% released at 24, 48, 72, and 120 h, respectively. In vitro aerosolization studies indicated a median aerodynamic particle size of 4.8 ± 0.2 μm, confirming its respirability in the lung. Cell culture studies indicated higher toxicity of NE-Cela in NSCLC cells. NE-Cela significantly reduced A549 cell viability, showing a ~6-fold decrease in IC₅₀ (0.2 ± 0.1 μM) compared to Cela alone (1.2 ± 0.2 μM). Migration and clonogenic assays demonstrated reduced cell proliferation, and 3D spheroid models supported its therapeutic activity in tumor-like environments. Conclusions: The inhalable NE-Cela formulation improved Cela’s physicochemical limitations and demonstrated enhanced anti-cancer efficacy in NSCLC models. These findings support its potential as a targeted, well-tolerated therapeutic option for lung cancer treatment. Full article

Hydroxyapatite nanowires (HAW) can effectively improve the bone repair ability in bone engineered tissue. However, due to their single function, the application of HAWs in biological tissue engineering materials is limited. In this study, strontium-doped hydroxyapatite nanowires (SrHAW) were synthesized by a hydrothermal method and coated with polydopamine (PDA) to improve the function of HAWs. The material structure, biocompatibility evaluation, and differentiation capability testing of PDA-coated strontium-doped hydroxyapatite (SrHAW@PDA) nanowires were conducted. Then, the nanowires were co-cultured with rat bone marrow mesenchymal stem cells (BMSCs) and rat umbilical vein endothelial cells (UVECs) to prepare cell spheroids. Compared with the undoped and uncoated HAW, the SrHAW@PDA nanowires enhanced the cell activity and their angiogenesis and osteogenesis abilities. In addition, their performance in the three-dimensional spheroid also played a positive role in the cells in the spheroid. Due to the presence of PDA, the adhesion between the cells in the three-dimensional spheroid and the nanowires were enhanced. In summary, these results show that SrHAW@PDA has the potential to be used as an alternative material to regulate cell biological activity in three-dimensional cell spheroids. Full article

Three-dimensional (3D) spheroid cultures are crucial for modeling salivary gland (SG) morphogenesis and advancing regenerative medicine. This study evaluated the effects of varying ratios of mouse SG-derived epithelial cells co-cultured with human dermal fibroblasts (hDFs), identifying a 2:1 ratio (spheroids containing 67% EpCAM^pos cells with 33% hDFs) as optimal for preserving native SG-derived epithelial cell phenotypes. At this ratio, 67% EpCAM^pos spheroids maintained structural integrity and demonstrated a significant reduction in apoptosis and senescence markers, specifically, cleaved caspase-3 (Cc3) and Serpine1, alongside an enhanced expression of the progenitor marker Keratin 5 (KRT5). This highlights the pivotal role of fibroblasts in supporting epithelial cell function in 3D cultures. These spheroids provide a useful model for developing SG tissues that closely mimic physiological properties. Despite promising results, these findings are preliminary and require further validation under diverse conditions and across different SG models. Full article

Pancreatic ductal adenocarcinoma (PDAC) presents significant treatment challenges due to its desmoplastic reaction, which impedes therapeutic effectiveness, highlighting the need for advanced vitro models to better mimic the complex tumor environment. The current three-dimensional co-culture models of fibroblasts and endothelial cells are lacking, which presents a challenge for performing more comprehensive in vitro research. Our study developed triple co-culture spheroid models using MiaPaCa-2 and BxPC-3 cancer cell lines, with RLT-PSC and hPSC21 pancreatic stellate cell lines and the endothelial cell line HMEC-1. These models were assessed through growth assays, multicolor flow cytometry to optimize cell ratios, cell viability assays to evaluate drug responses, and a tube formation assay with a spheroid-conditioned medium to examine angiogenesis. Our triple co-culture spheroids effectively replicate the PDAC microenvironment, showing significant variations in drug responses influenced by cellular composition, density, and spatial arrangement. The tube formation assay showcased the potential of our models to quantitatively assess a treatment-induced angiogenic response. These cost-effective triple-co-culture in vitro spheroid models provide vital insights into the PDAC microenvironment, significantly improving the quality of the in vitro evaluation of treatment responses. Full article

The engraftment of transplanted islets depends on the rapid establishment of a novel vascular network. The present study evaluated the effects of cord blood-derived blood outgrowth endothelial cells (BOECs) on the viability of neonatal porcine islets (NPIs) and the post-transplant outcome of grafted NPIs. Dispersed NPIs and human BOECs were reaggregated on microwell cell culture plates and tested for their anti-apoptotic and pro-angiogenic capacity by qRT-PCR and immunohistochemistry. The in vivo functionality was analyzed after transplantation into diabetic NOD-SCID IL2rγ^−/− (NSG) mice. The spheroids, which contained reaggregated neonatal porcine islet cells (REPIs) and BOECs, exhibited enhanced viability and a significantly elevated gene expression of VEGFA, angiopoetin-1, heme oxygenase-1, and TNFAIP3 (A20) in vitro. The development of normoglycemia was significantly faster in animals transplanted with spheroids in comparison to the only REPI group (median 51.5 days versus 60 days) (p < 0.05). Furthermore, intragraft vascular density was substantially increased (p < 0.01). The co-transplantation of prevascularized REPI-BOEC spheroids resulted in superior angiogenesis and accelerated in vivo function. These findings may provide a novel tool to enhance the efficacy of porcine islet xenotransplantation. Full article

Lipotoxicity, resulting from the buildup of excess lipids in non-adipose tissues, is increasingly recognized as a major contributor to the progression of kidney disease, highlighting the need for alternative models to assess its effects on renal cells. The main aim of this study was to investigate the usefulness of Caki-1, a human proximal tubule (PT) and renal cell carcinoma (RCC) representative cell line, as a 3D model system for studying free fatty acid-induced PT lipotoxicity. Caki-1 spheroids were generated and maintained on ultra-low attachment plates and characterized regarding time-dependent morphology changes. In optimal 3D culture conditions, Caki-1 cells formed well-defined large compact spheroids with uniform morphology, good circularity, and increased diameter from days 4–12. Chronic exposure to saturated palmitate resulted in dose- and time-dependent spheroid disintegration and cell death, including dispersed and flattened spheroid morphology, with increased dead cells in the peripheral layers and decreased spheroid core. Moreover, palmitate-treated spheroids showed a significant increase in cleaved poly(ADP-ribose) polymerase (PARP) and active caspase-3. Palmitate-induced PARP cleavage, as well as endoplasmic reticulum (ER) stress and autophagy dysfunction, were blunted by triacsin C, an inhibitor of long-chain acyl-CoA synthetases. In addition, co-incubation with unsaturated oleate prevented palmitate-induced spheroid disintegration and apoptotic cell death in Caki-1 3D culture. While fatty acid overload upregulated lipid droplet protein perilipin 2 in Caki-1 cells, knockdown of perilipin 2 by siRNAs resulted in an exacerbation of palmitate-induced cell death. Together, these results indicate that the 3D Caki-1 spheroid model is a simple and reproducible in vitro system for studying renal lipotoxicity and lipid metabolism that gives useful readouts at the molecular, cellular, and multicellular levels. Full article

Pancreatic cancer is a malignant tumor with one of the worst prognoses among solid tumors, characterized by resistance to treatment. Therefore, there is an urgent need for new methods of targeted therapy. Previous studies have shown that the overexpression of receptor tyrosine kinases such as c-KIT or PDGFR can increase proliferation, migration, and invasion of cancer cells. The aim of our study was to analyze aggregates between a supramolecular carrier (Congo red, CR) and a tyrosine kinase inhibitor (BLU-258) as well as to investigate the effect of the free inhibitor and its aggregate with Congo red (CR-BLU-258) on selected properties of pancreatic cells, including these cells’ viability and three-dimensional cell spheroid cultures. To better understand the interactions between Congo red and BLU-258, we used molecular modeling in addition to biophysical methods. These attempts allowed us to determine the optimal molar ratio, which we used for in vitro studies on pancreatic cancer cell lines. A significantly greater decrease in the viability of the tested 3D cultures was observed after 48 h of incubation with CR-BLU-258, which resulted in a lower IC₅₀ value for the tested co-aggregate compared with BLU-258 alone. Moreover, a higher resistance of PANC-1 and BxPC3 spheroid cells to the tested compounds was noted compared with the 2D culture model. A significantly lower response was observed in 3D cell cultures (BxPC3 and PANC-1) treated with BLU-258 alone compared with the 2D culture. Thus, our results showed that both BLU-258 (alone) and in its co-aggregate with Congo red exhibit anticancer activity, inhibiting the growth of pancreatic cancer cells and reducing their viability, survival, and migration. Both tested compounds also affected the phosphorylation of the selected signaling proteins. We conclude that the selected tyrosine kinase inhibitor (alone) and in its co-aggregate with Congo red exhibit anticancer activity and should be considered as a novel effective therapy against pancreatic cancer. Full article