Biology subjects

John, F.

Publications and source records attributed to John, F..

3 recordsLinked to original sources

bioRxiv2026

A targetable dependency on nonsense-mediated decay for proteostasis and immune control in small cell lung cancer

Small cell lung cancer (SCLC) is among the deadliest cancers with excessive somatic alterations, expectedly resulting in immunogenic epitopes. However, patient benefit from immunotherapy is limited. We found abundant frameshift mutations in SCLC, regarded as highly immunogenic, counterbalanced by a hyperactive nonsense-mediated decay (NMD) pathway, responsible for frameshift-mRNA degradation. NMD activity correlated with tumor mutational burden (TMB) across cancers, suggesting that SCLC depends on NMD for cellular homeostasis and immune evasion. NMD inhibition impaired SCLC proliferation and induced ER stress-dependent apoptosis in a TMB-dependent manner, thereby enabling pharmacological inhibition in vivo which effectively controlled tumor growth. By integrating genome and transcriptome sequencing with MHC-I immunopeptidomics and functional in vitro and in vivo assays, we identified that NMD inhibition boosted neoantigen expression and presentation by tumor cells and increased T cell recognition, thus enhancing overall tumor immunogenicity and further improving immunotherapy efficacy in vivo. Our work shows that SCLC - as a TMBhigh cancer - relies on NMD for survival and immune escape, uncovering a novel TMB-dependent tractable vulnerability for this devastating disease.

cancer biology↗

bioRxiv2026

CytoLight: A Rapid and Versatile Fluorescent-Based Labeling Method for Extracellular Vesicle Characterization and Tracking

Efficient, aggregation-free extracellular vesicles (EVs) labeling is essential for studying their dynamics in-vitro and in-vivo. However, traditional dyes introduce limitations including aggregation, membrane intercalation, fluorescence transfer and inconsistent performance across EV sources thus distorting quantification, altering surface properties and confounding uptake and biodistribution analyses. Here, we systematically evaluated CytoLight, a luminal dye traditionally used for live-cell imaging, as an alternative for EV quantification, characterization, uptake analysis and in-vivo tracking, benchmarking it against PKH26, CFSE and ExoBrite across multiple platforms. CytoLight generated stable, intravesicular fluorescence without aggregation or membrane alteration, eliminating artifacts characteristic of conventional dyes. Using fluorescence-NTA and single-EV flow cytometry, CytoLight showed more consistent labeling across EV types than CFSE or ExoBrite, while avoiding PKH-related micelle-driven artifacts and exhibited compatibility with CD81 dual-detection. In uptake assays, CytoLight produced EV-specific endocytosis-dependent internalization signals exceeding labeled-BPS/protein controls. In-vivo, CytoLight-labeled EVs enabled fluorescent biodistribution mapping showing conventional EV tropism patterns distinguishable from labeled-PBS controls. These findings establish CytoLight as an effective, aggregation-free EV-labeling strategy. Its stability, specificity, compatibility with single-EV platforms and reliable performance in both cellular uptake and biodistribution studies position CytoLight as a practical, scalable alternative to current dyes, providing a stronger foundation for standardized and reproducible EV research.

cell biology↗

bioRxiv2025

Engineered AAV9 as in vivo gene delivery platform for the selective transduction of TME cell subsets

Precise in vivo gene delivery to specific cell types remains a significant challenge in gene therapy, particularly for cancer immunotherapy applications. Here, we rationally engineered AAV9 to become a modular, receptor-targeted vector for selective in vivo gene delivery. We first identified the N272A and W503A mutations as effective in ablating the native tropism of AAV9. Subsequently, designed ankyrin repeat proteins (DARPins) were inserted into the GH2/3 capsid loop, redirecting vector specificity towards defined cellular receptors without compromising capsid integrity or yield. As a proof of concept, HER2-targeted DART-AAV9 vectors demonstrated highly selective transduction of HER2-positive tumor cells in vitro and in vivo, in both, subcutaneous and orthotopic glioblastoma models, with negligible transduction of off-target organs including liver, heart, and kidney. When equipped with immunomodulatory genes (anti-PD-1 or IL-2) HER2-DART-AAV9 mediated secretion of functional therapeutic proteins from transduced tumor cells. Additionally, our modular platform facilitated rapid generation of CD8-targeted DART-AAV9 vectors, enabling selective transduction of human CD8+ T cells. Importantly, the engineered vectors exhibited favorable resistance to neutralization by human serum and retained their specificity and potency in human blood, underscoring their potential for clinical translation. Together, these findings establish DART-AAV9 as a versatile, precise, and clinically promising gene delivery platform for cancer immunotherapy.

bioengineering↗