Extracellular Vesicles from Chronic Lymphocytic Leukemia Cells Promote Leukemia Aggressiveness By Inducing the Differentiation of Monocytes into Nurse-like Cells Via an RNA-Dependent Mechanism

Background: Bidirectional interactions exist between Chronic Lymphocytic Leukemia (CLL) cells and the cells of their microenvironment (ME): CLL cells indeed received several stimuli such as BCR stimulation but they can also interact with the surrounding cells by the release of extracellular vesicles (EVs). EVs are small double membrane particles (from 100nm to 1µm) carrying proteins, DNA and several types of RNA such as microRNAs or Y RNAs. EV exchange is today considered as a new way of cellular communication.

Methods: The small RNA profile (microRNA and Y RNA) of CLL-EVs isolated by ultracentrifugation from primary and fresh leukemic cells cultured with/without BCR stimulation was performed by NGS (n=4) and validated by qPCR (n=25). CLL-EV integration was monitored using PKH67 dye. After treatment of purified monocytes with CLL-EVs (Mono+CLL-EVs), the morphology, the cytokine profile, specific microRNA and mRNA expression and their ability to support CLL cell survival were analyzed and compared to those of nurse-like cells (NLC), the tumor-associated macrophages of CLL.

Results: NGS highlighted 31 microRNAs differentially expressed between BCR stimulated/unstimulated conditions and qPCR confirmed the increase of 3 of them (n=25) in EVs: miR-146a-5p (fold: +15.1, p<0.0001), miR-132-3p (fold: +42.1, p<0.0001) and miR-155-5p (fold: +3.3, p=0.003). The levels of these microRNAs were also increased in the cells after BCR stimulation. After 24h of incubation of CLL PBMC with CLL-EVs, CLL-EVs were preferentially taken up by monocytes (P=0.0019, n=6). Furthermore, BCR CLL-EV induced modifications in monocytes suggesting NLC differentiation : after 24h of incubation with CLL-EVs, miR-155-5p, miR-146a-5p, miR-132-3p were increased in monocytes (fold: +4.8, +4.5; +5.6, respectively; P<0.05, n = 9) similarly to what happens in the differentiation of monocytes in NLC (fold: +5.6, +27.4, +12, respectively, P<0.01, n=10). No significant difference in terms of pri-microRNA expressions were observed between the different conditions, demonstrating that the increase in microRNA levels was due to microRNA transfer and to de novo transcription. After 5 days of incubation with CLL-EVs, we also observed the increase of the hy4 RNA in monocytes and after 12 days, this increase was statistically significant in monocytes treated with BCR CLL-EVs compared to control CLL EVs (+4.0 fold, n=10, P=0.0020). Although, Mono+CLL-EVs display a fibroblast morphology similar to M2 macrophages. In addition, gene expression and secretome modifications of monocytes were observed: the level of several cytokines (IL-10, IL-8, IL-6, BAFF, CCL2, SDF-1α) associated to NLC differentiation or previously described as pro-survival factors for CLL cells were increased after CLL-EV treatment. Functionally, monocytes treated with BCR CLL-EVs protect CLL cells from spontaneous apoptosis and induce their migration as well as the migration of other monocytes creating an amplification loop for the establishment of a protective microenvironment. Modifications induced by CLL-EVs are decreased/abolished when EVs are treated with a high dose of RNAs. Interestingly, we report, by transfection experiments, that hY4 is able to induce the expression of CCL24, a key gene in M2 macrophage differentiation while the investigated microRNAs were not.

Conclusions: we showed that BCR stimulation modifies the small RNA content of CLL-EVs and that the addition of leukemic EVs to monocytes leads to monocyte differentiation into NLCs establishing a protective microenvironment that supports leukemic cell survival. The transfer of hY4 RNA by CLL EVs is crucial to induce monocyte differentiation into NLC while microRNA transfer participates to NLC differentiation but is not essential.