Exosomal genetic cargo shift dictated by RD3 deficiency steers progressive neuroblastoma evolution

Exosomes are central mediators of systemic cellular communication, directing tumor evolution through the dynamic exchange of genetic and molecular cargo. Neuroblastoma (NB), a lethal pediatric tumor of embryonal origin, where selection and enrichment of treatment-resistant cancer stem cell clones have decisive impact. Recently, we identified acquired RD3 deficiency prompts tumor cell plasticity and regulates tumor progression. Herein, we investigated whether RD3 controls delivery of exosomal genetic cargo. For this we utilized our custom archived Neural crest cell specific Cre conditional LoxP-directed RD3-Knockout (KO, Tg mice) driven human syntenic spontaneous NB. Serum exosomes were isolated, from RD3-KO mice and compared with their wild-type counterparts. Exosomal RNA was isolated using the Total Exosome RNA and Protein Isolation kit, characterized by nanoparticle tracking analysis, flow cytometry, transmission electron microscopy, and was then analyzed by paired-end Illumina RNA sequencing. Differential gene expression (DEG) was computed using log₂ fold change with log₂ FDR. DEG analysis identified an animal-independent signature of 53 upregulated and another 1,396 downregulated genes. Functional annotation in Ingenuity Pathway Analysis revealed enrichment in therapy-resistance pathways, including angiogenesis signaling by VEGF ( VEGFA, KDR, FLT1 ), NAD⁺ salvage pathway II mediated DNA damage response ( NMRK1, NT5C2, NT5C1A ), cell cycle control ( CDK6, CDC7, SIN3A ), pro-survival CXCR4 signaling ( HSP90AA1, RAB11FIP3 ), extracellular matrix remodeling ( COL1A1, ADAM12, HSPG2, CD31 ), and immune/inflammatory signaling (CD27, IL-1) pathways. Furthermore, oncogenic cell signaling pathways, including RhoGDI signaling ( CDC42, PAK1, ARHGEF2), regulating migration, invasion, and WNT/beta-catenin signaling ( SOX17, MAP3K7, WNT9A ), regulating stemness maintenance, were decisively reprogrammed. These results, for the first time, distinctly demonstrate that RD3 deficiency drives molecular rearrangements that promote therapy resistance, underscoring its central function in exosome-mediated cancer cell signaling that survives intensive multimodal clinical therapy. Crucially, our findings position RD3-regulated exosomal genetic cargo as a promising biomarker and therapeutic target for better clinical management of progressive NB. FUNDING: DOD-CA-210339; OCAST-HR19-04; NIH-P20GM103639