Full-coverage landscape of extracellular RNAs, coding and non-coding, secreted by human glioma stem cells

Communication between glioblastoma brain tumor (GBM) and its microenvironment alters the parameters of tumor growth and host responses, and may be mediated in part by tumor-secreted RNA. The global repertoire of extracellular RNAs (exRNAs) released by GBM, however, has not been investigated. We have developed a protocol enabling quantitative, minimally biased analysis of vesicular and non-vesicular exRNA complexes, including microvesicles (MVs) and exosomes (collectively called extracelluar vesicles; EVs), as well as ribonucleoproteins (RNPs) and applied it to study exRNA in patient-derived glioma stem-like cultures (GSC). Despite the intertumoral heterogeneity, further exacerbated at the exRNA level, the extracellular complexes exhibit distinct RNA composition, with microvesicles most closely reflecting the cellular transcriptome, and exRNPs exhibiting the most discrete repertoire. Up to 90% of exRNA reads represent fragmented rRNA; the remaining content is enriched in small ncRNA species, such as miRNAs in exosomes, and precisely processed tRNA and Y RNA fragments in both EVs and exRNPs. EV-enclosed mRNAs are mostly fragmented, and UTRs are more abundant than ORF regions; nevertheless, some full-length transcripts are present. Overall, there is less than one copy of non-rRNA per EV. Our results suggest that massive EV/exRNA uptake would be required to ensure functional impact of transferred information to the normal recipient cells of the brain and predict the most impactful miRNAs in such conditions. This study also provides a catalog of diverse vesicular and non-vesicular exRNA species useful for biomarker discovery. Four low-passage patient-derived glioblastoma stem cell cultures were grown as neurospheres in serum-free media. Microvesicles (MVs), exosomes, and ribonucleoproteins (RNPs) were separated from conditioned media using sequential filtration, and RNA was isolated from each fraction. Cellular RNA was isolated from cultures in parallel. RNA was also isolated from MVs, exosomes, and RNPs from fresh media. Both long RNA library and small RNA libraries were prepared for each RNA sample, and sequenced on HiSeq2000. Cellular RNA of human or mouse primary normal brain cells (neurons, astrocytes, microglia, and endothelial cells) were also sequenced for small RNA libraries.