Moskalchuk, A. | bioRxiv Science

bioRxiv2025

3R-Compliant Murine Ex Vivo Platform for Scalable Brain Cancer Modeling and Drug Screening

Translating in vitro findings into in vivo applications remains a major challenge in brain cancer drug discovery, largely due to inadequate models for assessing neurotoxicity and tumor microenvironment (TME) dynamics. Here, we present a novel, highly modular murine ex vivo platform based on adult neural stem cells (aNSCs), designed for scalable, medium-throughput drug screening and mechanistic studies for brain cancer research. This system integrates progressive 2D and 3D models, including assembled models for tumor migration, cancer core models mimicking tumor localization within neural tissue and advanced cancer core models incorporating immune cells to recapitulate key TME interactions. With high reproducibility, defined cell composition and rapid formation within 72 hours, these assembloids provide a translationally relevant and ethical alternative to animal models. Aligned with 3R principles, this platform bridges the gap between in vitro and in vivo studies, offering a powerful tool for innovating brain cancer drug discovery.

cancer biology↗

bioRxiv2024

T4 phage RNA is NAD-capped and alters the NAD-capepitranscriptome of Escherichia coli during infection through a phage-encoded decapping enzyme

Nicotinamide adenine dinucleotide (NAD+) serves as a cap-like structure on cellular RNAs (NAD-RNAs) across all domains of life, including Escherichia coli. Beyond its role in metabolism, NAD+ also functions as a regulatory molecule in phage defense mechanisms. However, NAD-RNAs have not yet been identified during bacteriophage infections, and the mechanisms governing their synthesis and degradation in this context remain unknown. To address this gap, we used T4 phage infection of E. coli as a defined and well-characterized model system to study NAD-RNAs at the virus-host interface. Here, we report the first identification and characterization of NAD-RNAs during phage infection. Using time-resolved NAD captureSeq, we identified NAD-capped host and T4 phage transcripts and observed that the set of enriched NAD-capped RNAs varies across different infection phases. Importantly, NAD captureSeq identifies NAD-capped transcripts based on enrichment and does not directly report the fraction of molecules that are NAD-capped. Consequently, temporal changes in enrichment may reflect altered NAD-capping, altered transcript abundance, or a combination of both. We provide evidence that NAD-RNAs are generated by the host RNA polymerase by initiating transcription with NAD+ at canonical transcription start sites. By quantifying intracellular NAD+ and bulk NAD-capped RNA during infection, we observe parallel decreases in both parameters over the course of infection. Furthermore, we characterize NudE.1, a T4 phage-encoded Nudix hydrolase previously shown to have in vitro NAD-RNA decapping activity. Together, our work presents the first time-resolved analysis of an RNA modification in a host-phage system, defining the landscape, dynamics, and turnover of NAD-capped RNAs during infection and providing a framework for future studies addressing their regulatory functions in phage biology.

microbiology↗