Abstract B044: Exosomes under RD3 command: A new frontier in neuroblastoma biology

Abstract Genetic and molecular cargo delivered by exosomes play a pivotal role in cellular communication at the systemic level and define tumor evolution. Children presented with Neuroblastoma (NB), the deadly, developmental tumor, experience hematogenous metastasis with frequent relapses in rapidly decreasing time. Hematogenous and/or bone marrow (BM) metastasis is attributed to NB cell to BM-mesenchymal stem cell communication. Recently, we identified the tumor evolution stabilization function of Retinal degeneration protein 3 (RD3) in NB. Here, we investigated the function of RD3 in designing the NB cell exosomal protein cargo. NB (CHLA-42) cells derived from the BM of a patient with hematogenous metastasis that expresses constitutive levels of RD3 were reverse engineered to stably silence RD3 (RD3 - clone). Exosomes from parental and RD3- clones were isolated from serum-free cultures (Invitrogen), characterized (Nano Tracking Analysis, NS300), and subjected to protein isolation (Total exosome protein isolation kit, Invitrogen). Exosomal protein cargo were compared utilizing global proteomic profiling using Data Independent Acquisition-based mass spectrometry. Proteomic profiling archived a total of 4073 proteins with RD3-dependent expressional fluctuations in 3334 proteins. Interestingly, RD3 presence in NB cells uniquely deployed 138 proteins while RD3 loss led to the deployment of a different 601 proteins. Molecular function mapping of the RD3 presence dependent deployment of 138 proteins indicated tumor suppression and apoptosis (e.g., APAF1, TFGBR3, SMAD4); genomic stability; metabolic reprogramming (e.g., NT5DCs, HPX, PYGL); ECM remodeling; immune modulation and immune cell adhesion (e.g., CD109, ICAM2, PLXDC2); regulation of mitochondrial and DNA repair function (e.g., COX11, TOMM20, NME4); angiogenesis; tumor Invasion and metastasis; migration; tumor-stoma interaction (e.g., MMP1, ADAMTSs, SERPINE1, SPON1); transcription; signaling and differentiation regulation; EMT and proliferation; (e.g.,CAMK4,ANXA1, ZNFs). For the first time, these results demonstrate that cellular RD3 levels determine the protein cargo in the exosomes. More importantly, RD3 expressing cells deploy unique set of proteins that are known to regulate tumor evolution. Together, this study unveils that BM-metastatic NB cells deploy exosomal protein cargo and further imply that RD3 determines the NB cell systemic communication and NB evolution. FUNDING: This work was funded by Department of Defense, DoD CA-210339; Oklahoma Center for the Advancement of Science and Technology, OCAST-HR19-045; and the National Institutes of Health P20GM103639. Citation Format: Afsana Parveen Jahir Hussain, Poorvi Subramanian, Sivaroopan Aravindan, Natarajan Aravindan. Exosomes under RD3 command: A new frontier in neuroblastoma biology [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr B044.