IMPACT OF SMALL EXTRACELLULAR VESICLES IN CHRONIC LYMPHOCYTIC LEUKAEMIA MICROENVIRONMENT

MORANDE, PABLO E; ETIENNE MOUSSAY; JANJI, BASSAM; GARGIULO, ERNESTO; JEROME PAGGETTI

Virtual (por la pandemia)

Congreso; EHA Congress 2020; 2020

European Hematology Association

Background Chronic Lymphocytic Leukaemia (CLL), the most common adult leukaemia of the western hemisphere, is characterized by the accumulation of abnormal B-lymphocytes in peripheral blood and lymphoid organs. CLL cells proliferation and survival is highly dependent on interactions with the microenvironment. Thus, to effectively target tumour progression, it is essential to understand the communication between CLL cells and surrounding tissues.Small extracellular vesicles (sEVs) are vesicles with size that ranges between 30 and 150nm. Small EVs are involved in cell-to-cell communication through the transfer of genetic material and proteins. Furthermore, sEVs possess direct functions carried out by sEV-ligands that are able to affect the biological functions of the targeted cells. The release of sEVs and their cargo depends on the cellular and physiological context. In cancer, tumour-derived sEVs are involved in the re-education of microenvironment cells promoting tumour proliferation, immune escape and metastasis.Aims The aim of the present project is to isolate and fully characterize the CLL microenvironment-derived sEVs (CLL-ME-sEVs) to investigate their role in CLL-tumour development and progression.Methods To obtain a biological representation of sEVs in the tumour microenvironment, we established a new protocol allowing us to isolate sEVs directly from the spleen of leukemic mice. Next, we characterized RNA (RNAseq and qPCR) and protein (mass spectrometry and WB) content of the CLL-ME-sEVs. To evaluate the impact of CLL-ME-sEVs in tumour immune escape we screened for a wide range of immune checkpoint ligands on their surface, as well as the corresponding receptors on T cells found in the tumour microenvironment. Finally, we evaluated functional activity of CLL-ME-sEVs by performing in vitro treatments on lymphocytes and by developing high throughput analysis of the targeted cells (e.g. transcriptomic)Results By crossing the Eµ-TCL1 transgenic strain with RAB27(ab) double knock-out (RAB27DKO) mice, we obtained a novel animal model in which murine-derived CLL cells cannot release sEVs (TCL1_Rab27DKO mice). We used this model to inject cells into recipient mice and found that CLL-derived sEVs are essential for disease development, since CLL development was abolished in their absence during adoptive transfer of TCL1_RAB27DKO cells into recipient mice, as compared to transfer of control Eµ-TCL1 cells. With the aim to analyse sEV composition, we isolated sEVs generated in vivo by CLL splenocytes of sick Eµ-TCL1 mice and their tumour microenvironment. CLL-ME-sEVs shown increased amount and complexity of RNA and protein content compared with CTRL-ME-sEVs, obtained from spleens of wild type animals. Furthermore, in our screening we detected the presence of multiple immune checkpoint ligands directly anchored on tumour-derived sEVs. Interestingly, we also found that the corresponding immune checkpoint receptors on CLL microenvironment T cells was highly expressed, which suggests a sEV-mediated immune escape strategy. Finally, functional results on T-regulatory (CD4+Foxp3+) and CD8+ cells incubated in presence of CLL-ME-sEVs shown upregulation of genes involved in immune suppression and cell exhaustion respectively.Conclusion Altogether, these results provide insights into the role of ME-sEVs in CLL development, and suggest a putative role of sEVs in immune modulation affecting leukemic progression.Session topic: 05. Chronic lymphocytic leukemia and related disorders - Biology & Translational Research