[Impact of small extracellular vesicles on anti-tumor immunity in chronic lymphocytic leukemia in vivo]. (Doctoral thesis)

Small extracellular vesicles (sEV) are nano-sized particles known for their implications in cell-to-cell communication. In cancer, tumor-derived sEV are responsible for the re-education of surrounding healthy cells, promoting tumor proliferation, immune escape and metastasis. In our study, we focused for the first time on the functional characterization of leukemia microenvironment-derived sEV (LME-sEV) by isolating small vesicles directly from the spleen of pre-clinical leukemic mice (Eµ-TCL1). First, we deeply characterized LME-sEV by proteomics analysis and by single-particle flow and imaging cytometry, and identified immune checkpoint molecules. With the aim to define sEV involvement in leukemogenesis, we crossed the Eμ-TCL1 and RAB27(ab) double knock-out (RAB27DKO) transgenic strains, obtaining a novel animal model (TCL1-RAB27DKO) in which sEV release from murine-derived CLL cells is impaired. Then, our results show that LME-sEV are essential for chronic lymphocytic leukemia (CLL) development in vivo, and their absence strongly impacts tumor development and progression. Given the highly immunosuppressive microenvironment in CLL, we focused our attention on LME-sEV impact on lymphocytes. Our data showed that LME-sEV are able to actively decrease CD8+ T cell anti-tumor activity both in vivo and in vitro by impacting transcriptomic and proteomic profile and transferring miRNA. These lead to a diminished synthesis of pro-inflammatory cytokines, to the expression of exhaustion markers and to a decrease cytotoxic activity. Altogether, our data demonstrate for the first time that LME-sEV play a key pro-tumoral role in vivo and suggests that LME-sEV negatively modulate the anti-tumor immune response.