Biology subjects

Ovsianikov, A.

Publications and source records attributed to Ovsianikov, A..

3 recordsLinked to original sources

bioRxiv2025

Mevalonate pathway activation in Ewing sarcoma reveals a 3D-specific synergy between statins and BCL-xL inhibition

Bone sarcomas are rare and aggressive pediatric cancers with limited progress in targeted therapy development, in part due to the poor physiological relevance of conventional two-dimensional (2D) culture systems used for preclinical testing. To address this gap, we developed a standardized three-dimensional (3D) culture and drug-testing platform for Ewing sarcoma (ES) and osteosarcoma (OS) that more accurately recapitulates in vivo tumor biology. Across 3D spheroids, bioprinted constructs, and patient-derived xenograft (PDX) cultures, we observed a consistent activation and dependency on the mevalonate pathway in ES. Leveraging this platform, we identified a selective therapeutic synergy between statins, which inhibit mevalonate pathway flux, and BCL-xL inhibitors, a vulnerability that was not detectable in 2D cultures. These findings highlight the mevalonate pathway as a targetable metabolic dependency in ES and demonstrate how physiologically grounded 3D models can uncover clinically actionable treatment strategies that remain hidden in traditional 2D systems. Our findings show that 3D tumor models can expose actionable metabolic vulnerabilities obscured by traditional approaches, supporting their use in rational combination therapy discovery for aggressive pediatric sarcomas.

cancer biology↗

bioRxiv2025

Cryo-SEM Reveals Native Architecture and Matrix Complexity in P. aeruginosa Biofilms

Pseudomonas aeruginosa (PA) biofilms resist antibiotics and immune clearance through their multicellular community organization. Yet, the native spatial arrangement of cells and the extracellular matrix (ECM) remain poorly understood, as research has largely focused on genetics and regulatory networks rather than physical structure. This has limited our understanding of how physical structural organization connects to biofilm function. Here, we combine high-pressure-freezing cryo-SEM, depth-resolved confocal microscopy, and quantitative spatial analysis to examine 4-day-old mucoid and PAO1 biofilms in a near-native state. In contrast to the dense cellular aggregates often inferred from dehydrated samples, cryo-SEM revealed bacterial cells as individually embedded within a continuous extracellular matrix. Spatial statistics revealed a preferred intercellular spacing of approximately 1 m, broad spacing distributions, and only weak short-range clustering. Depth-resolved confocal analysis confirmed this sparse organization across larger biofilm volumes and revealed vertical stratification, with the highest bacterial volume fraction near the substrate and lower cell volume fraction toward the biofilm surface. Cryo-SEM further showed that mucoid biofilms contained aligned fibrillar structures within the biofilm interior, whereas PAO1 biofilms exhibited a denser, mesh-like matrix. These findings challenge prevailing views of biofilms as densely packed bacterial aggregates and establish a quantitative framework for understanding antimicrobial tolerance, cell-cell interactions, nutrient access, and biofilm mechanics in both clinically and environmentally relevant contexts.

microbiology↗

bioRxiv2025

Beyond the Matrix: Rethinking Antibiotic Tolerance in CF Biofilms Using 3D Models

Chronic lung infections in cystic fibrosis (CF) patients are associated with Pseudomonas aeruginosa biofilms exhibiting high antibiotic tolerance with no clear explanation. We investigate the role of the biofilm matrix in this antibiotic tolerance using 3D biofilm models based on acetylated alginate and DNA, mimicking mucoid biofilms. Printed from these bioinks seeded with P. aeruginosa (PAO1), these models support robust microcolony formation as observed in vivo and enable high-throughput assessment of antibiotic diffusion and efficacy. Surprisingly, antibiotic diffusion is not significantly impeded by acetylation or DNA incorporation. Despite this, bacterial tolerance increases tremendously upon encapsulation in alginate. Acetylation further enhances tolerance, particularly to tobramycin, ciprofloxacin, and colistin. The addition of DNA mitigates this effect in a drug-specific manner. While mucoid biofilms, in contrast to the biofilm models, show significant retardation of antibiotic penetration, they also get saturated with all tested antibiotics within 20 h. This demonstrates that direct interaction with alginate or DNA does not explain the slow diffusion of antibiotics in mucoid P. aeruginosa biofilms. Our findings challenge the view that diffusion limitation or antibiotics binding by biofilm exopolysaccharides dominate biofilm resilience and highlight the need to target matrix-induced bacterial adaptation in the development of antibiofilm therapies.

microbiology↗