Circular RNAs (circRNAs) are recently identified as a naturally occurring family of widespread and diverse endogenous noncoding RNAs that may regulate gene expression in mammals [1-4]. They are unusually stable RNA molecules with cell type-or developmental stage-specific expression patterns [3, 5]. Exosomes are small membrane vesicles of endocytic origin secreted by most cell types. They contain a specific cargo of protein, mRNA and microRNA species, which can modulate recipient cell behaviors and may be used as biomarkers for diagnosis of human diseases [6]. Here, we show for the first time the presence of abundant circRNAs in exosomes. RNA-seq analyses revealed that circRNAs were enriched in exosomes compared to the producer cells. Furthermore, the sorting of circRNAs to exosomes may be regulated by changes of associated miRNA levels in producer cells, and may transfer biological activity to recipient cells. Additionally, more than 1 000 circRNAs were identified in human serum exosomes. circRNAs originated from human cancer xenografts could enter the circulation and be readily measured in the serum. Intriguingly, serum exosomal circRNAs were able to distinguish patients with colon cancer from healthy controls. Our results lay the foundation for development of circRNAs as a new class of exosome-based cancer biomarkers and suggest the potential biological function of exosomal circRNAs.

First we characterized circRNA transcripts by using RNA-seq analyses of ribosomal RNA-depleted total RNA from MHCC-LM3 liver cancer cells and cell-derived exosomes (Supplementary information, Figure S1). The prepared exosomes were examined by transmission electron microscopy (Supplementary information, Figure S2). A computational pipeline [3] based on anchor alignment of unmapped reads was used to identify circRNAs. Counts of reads mapping across an identified backsplice are normalized by read length and number of reads mapping (spliced reads per billion mapping (SRPBM)), which allows quantitative comparisons be-