RESEARCH ARTICLE CLL Exosomes Modulate the Transcriptome and Behaviour of Recipient Stromal Cells and Are Selectively Enriched in miR-202-3p

Bi-directional communication with the microenvironment is essential for homing and sur-vival of cancer cells with implications for disease biology and behaviour. In chronic lympho-cytic leukemia (CLL), the role of the microenvironment on malignant cell behaviour is well described. However, how CLL cells engage and recruit nurturing cells is poorly character-ised. Here we demonstrate that CLL cells secrete exosomes that are nanovesicles originat-ing from the fusion of multivesicular bodies with the plasma membrane, to shuttle proteins, lipids, microRNAs (miR) and mRNAs to recipient cells. We characterise and confirm the size (50–100 nm) and identity of the CLL-derived exosomes by Electron microscopy (EM), Atomic force microscopy (AFM), flow cytometry and western blotting using both exosome-and CLL-specific markers. Incubation of CLL-exosomes, derived either from cell culture supernatants or from patient plasma, with human stromal cells shows that they are readily taken up into endosomes, and induce expression of genes such as c-fos and ATM as well as enhance proliferation of recipient HS-5 cells. Furthermore, we show that CLL exosomes encapsulate abundant small RNAs and are enriched in certain miRs and specifically hsa-miR-202-3p. We suggest that such specific packaging of miR-202-3p into exosomes results in enhanced expression of ‘suppressor of fused ’ (Sufu), a Hedgehog (Hh) signalling inter-mediate, in the parental CLL cells. Thus, our data show that CLL cells secrete exosomes that alter the transcriptome and behaviour of recipient cells. Such communication with microenvironment is likely to have an important role in CLL disease biology