Molecular profiling of glioblastoma-derived extracellular vesicles identifies small nucleolar RNAs as candidate liquid biomarkers for radiation- induced senescence.

Radiation-induced senescence (RIS) in glioblastoma (GBM) is an undesirable cell fate that inhibits tumor cell death and supports resistance and outgrowth. While senescence-targeting drugs are promising adjuvants, their clinical application will require proper patient selection based on post-treatment RIS burden. Current methods to evaluate senescence, however, are tissue-based, and given GBM's difficult anatomical location, post-treatment biopsies are impractical. Therefore, novel and less invasive biomarkers for TIS are urgently needed. To this end, we aimed to identify candidate extracellular vesicle (EV) liquid biomarkers for TIS by profiling senescence-associated cargo changes within GBM EVs. Using a panel of GBM patient-derived cell lines, we show that RIS is the primary functional state following radiation exposure and is associated with significant alterations in the cargo of senescent-derived EVs (senEVs). In particular, senEV transcriptomes have an increased abundance of senescence-associated RNA species and enrichment of senescence-associated gene sets. Most striking, however, was that senEVs are most differentiated by the significant enrichment of a panel of snoRNAs. This signature was conserved in 4/5 GBM models of RIS and was validated by qRT-PCR. Further analysis by mass spectrometry revealed that snoRNAs are likely co-packaged with their associating proteins, as senEVs had concurrent increases in these binding partners. Finally, in a preliminary patient cohort comparing plasma EVs obtained prior to surgery to those obtained after completion of their radiation therapy, we identified increased senescence-associated RNA such as CDKN2B and GLB1 and the snoRNA SNORA49 in post-radiation EVs. Altogether, this data suggests that senEV RNA species, and particularly snoRNAs, are a promising analyte for RIS-biomarker development. With further study, this work may open avenues for a companion diagnostic for senotherapeutics.