ABSTRACT This study aimed to develop an accessible, adjustable cell culture model utilizing varying liquid column heights to evaluate the combined microenvironmental impacts of hydrostatic pressure (HP), nutrient availability, and depth‐dependent oxygen gradients on NIH/3T3 fibroblasts. The liquid column medium (LCM) system was constructed by integrating customized glass pipes with standard 48‐well cell culture plates, achieving a maximum column height of 23.3 cm (equivalent to 18 mmHg of base HP). Twenty‐four hours post‐seeding, fibroblasts were exposed to four distinct LCM heights: 0.3 cm (control), 7.7, 15.5, and 23.3 cm, and analyzed after 1 and 3 days of exposure. First‐order mathematical modeling of oxygen diffusion predicted that increasing medium depth concurrently restricts pericellular oxygen availability over extended culture durations. Exposure to LCM heights induced marked cell spreading, elevated cell density along the well margins, and significantly reduced cell proliferation in a height‐dependent manner. By day 3, the highest cell count was observed in the control group (16 × 10 3 ± 850 cells/well), whereas total cell counts decreased progressively under increasing LCM heights (13 × 10 3 ± 720, 10 × 10 3 ± 610, and 6.5 × 10 3 ± 430 cells/well at 7.7, 15.5, and 23.3 cm, respectively). Furthermore, LCM exposure expanded mean nuclear area and increased the proportion of binucleated cells. Overall, these findings demonstrate that manipulating medium column height provides a versatile, low‐cost in vitro model for exploring cell behaviors under coupled mechanical pressure and fluid depth conditions.
Background Breast cancer (BrCa) has become the most common malignant tumor among women worldwide. Inducing tumor cell pyroptosis has emerged as a potential anti-tumor strategy, although the biological effects of pyroptosis are context-dependent and may be dual-edged. The NLRP3 inflammasome is a major regulator of canonical pyroptosis. However, the role of ZDHHC7-mediated NLRP3 palmitoylation in BrCa remains unclear.Methods ZDHHC7 expression was analyzed using UALCAN, Kaplan-Meier plotter, qPCR, and Western blotting. The growth and metastasis ability of cells were detected and evaluated, respectively, using the CCK-8 method and the Transwell assay. Pyroptosis was evaluated through Western blot, ELISA, and flow cytometry. NLRP3 palmitoylation was examined by acyl-biotin exchange (ABE) assay. Tumor growth was evaluated in xenograft models.Results Compared with normal controls, ZDHHC7 is characterized by low expression in BrCa, and its low expression level is related to a decreased survival rate of patients. ZDHHC7 overexpression suppressed proliferation, migration, and invasion. Furthermore, ZDHHC7 overexpression promoted NLRP3 palmitoylation, activating the NLRP3 inflammasome, which increased levels of cleaved Caspase-1 and GSDMD-N, elevated secretion of IL-1 beta, IL-18, and LDH, and induced pyroptosis. These effects were partially reversed by NLRP3 inhibitor. Functional rescue experiments confirmed that NLRP3 inhibition restored the malignant phenotypes suppressed by ZDHHC7. In vivo, ZDHHC7 or NLRP3 overexpression inhibited tumor growth, reduced Ki67 expression, and increased pyroptosis markers.Conclusion ZDHHC7 may function as a tumor suppressor in BrCa by promoting NLRP3 palmitoylation, thereby activating NLRP3 inflammasome-mediated pyroptosis and inhibiting malignant progression in experimental models.
Tumor-derived extracellular vesicles (EVs) facilitate metastatic progression by transferring bioactive cargo to recipient cells, thereby increasing endothelial permeability and promoting angiogenesis. The harsh conditions of the tumor microenvironment, such as extracellular acidosis, can alter the content of EVs, leading to changes in recipient cells, including endoplasmic reticulum (ER) stress. This study aimed to investigate whether EVs released by tumor cells adapted to acidosis or ER stress can alter endothelial functional properties. ER stress was induced in MDA-MB-231 breast cancer cells with tunicamycin, and adaptation to acidosis was achieved by culturing at pH 6.5 for 30 days. Large extracellular vesicles (LEVs) in these conditions were isolated by centrifugation. LEVs were characterized by nanoparticle tracking analysis (NTA), immunogold, Western blot, and high-performance liquid chromatography (HPLC). ER stress and autophagy markers were quantified by Western blot; cancer cells' adhesion to endothelial cells was assessed by adhesion assay; barrier permeability by dextran diffusion in an organ-on-a-chip model; migration by wound healing assay, and angiogenesis by tube formation assay. Tunicamycin was not detected in LEV from tunicamycin-treated cells (LEV T). LEV T increased the migration of endothelial cells, while not inducing ER stress, nor altering adhesion, angiogenesis, or permeability. Acidosis-adapted cells did not have greater UPR activation, but their LEVs (LEV 6.5) induced ER stress, reduced adhesion and angiogenesis, and increased permeability and migration. These findings indicate that LEVs from acidosis-adapted tumor cells reprogram endothelial physiology, supporting a potential role in metastatic progression.
ABSTRACT Extracellular vesicles (EV) secreted by living cells are important players in cell‐to‐cell communication, particularly in the diffuse immune system, where they contribute to local and systemic coordination. Indeed, EV are in the ideal size range for diffusion in interstitial and lymphatic fluids, which is the main route of tissue EV trafficking towards secondary lymphoid organs that orchestrate immune responses. In the lymph nodes, EV interact with multiple immune and stromal cell populations sensitive to antigens and co‐delivered tissue messages via degradative and non‐degradative pathways. Natural conveyers of bioactive compounds inherited from their mother cells and their surroundings, EV modulate innate and adaptive immune responses in health and disease. The aim of this scoping review is to aggregate information from the literature on cellular effectors and molecular pathways of EV‐mediated immune responses in the lymph node environment and to gain insight into practices using EV for immune intervention .
ABSTRACT Lamin A is a major contributor to nuclear mechanics and an important mediator of mechanotransduction. While its role in regulating nuclear architecture is well established, considerably less attention has been given to how mechanical cues regulate lamin A itself. Here, we review current evidence demonstrating that lamin A homeostasis is dynamically controlled by mechanical stimulation through transcriptional, translational, and post‐translational mechanisms regulating protein abundance, stability, and turnover. We further discuss emerging evidence suggesting that prelamin A maturation, a highly conserved multistep processing pathway, may represent a previously unrecognized mechanosensitive regulatory node. Recent studies challenge the canonical model of prelamin A processing and raise the possibility of alternative maturation pathways with important implications for lamin A homeostasis. We highlight current evidence, unresolved questions, and future directions, proposing that mechanical regulation of lamin A biogenesis represents an underexplored dimension of lamin mechanobiology with broad implications for development, aging, laminopathies, and mechanically driven diseases.
