Periodic Reporting for period 1 - EVIL (Reprogramming of the leukemic microenvironment by small extracellular vesicles: from characterization to therapeutic application)
Reporting period: 2021-12-01 to 2023-11-30
Chronic lymphocytic leukemia (CLL) is a hematological malignancy characterised by the accumulation of abnormal B lymphocytes in peripheral blood (PB) and lymphoid organs. It represents the most common form of leukemia in the Western hemisphere to affect the elderly, and remains incurable. In the last years, new molecules such as kinase inhibitors were introduced in the standard care of CLL, which partially replaced chemotherapy and antibody-based treatments. These new approaches showed an efficient control of the disease in most cases. However, some patients do not respond or become resistant to current treatments, therefore novel alternative therapies are being continuously developed. The heterogeneous clinical and biological features of CLL position this disease as an attractive, high incidence and easy-accessible sample model for the study of leukemia. The present project is focused on dissecting the cross-talk between small extracellular vesicles (sEV, i.e. exosomes), the CLL tumor cells and the microenvironment (ME), to unravel their role in this disease and explore their therapeutic potential.
Small EV are nano-sized vesicles (40-160nm in diameter) originating from the endosome, produced by all cell types and released to the extracellular space. They contain a lipid bilayer and various biomolecules whose composition depends on the parental cell and the physiological conditions in which they were produced. Increasing novel data position sEV as a key and complex cell communication system implicated in several physio-pathological processes. Particularly in cancer, sEV can positively influence disease progression by direct targeting of tumor cells and also by influencing the non-malignant cells that compose the tumor ME.
In line with the objectives stated in the 2020 Interim report of the Mission Board for Cancer of the European Commission (EC), this project faces the huge threat that CLL represents for Europe’s citizens and health systems and is positioned within the challenges from the beating cancer plan. We set as main objectives of this project to study the targeting and internalisation of sEV, to study the effects of sEV on immunosuppression and anti-tumor immunity, and to study the effects of sEV on metabolism.
Small EV are nano-sized vesicles (40-160nm in diameter) originating from the endosome, produced by all cell types and released to the extracellular space. They contain a lipid bilayer and various biomolecules whose composition depends on the parental cell and the physiological conditions in which they were produced. Increasing novel data position sEV as a key and complex cell communication system implicated in several physio-pathological processes. Particularly in cancer, sEV can positively influence disease progression by direct targeting of tumor cells and also by influencing the non-malignant cells that compose the tumor ME.
In line with the objectives stated in the 2020 Interim report of the Mission Board for Cancer of the European Commission (EC), this project faces the huge threat that CLL represents for Europe’s citizens and health systems and is positioned within the challenges from the beating cancer plan. We set as main objectives of this project to study the targeting and internalisation of sEV, to study the effects of sEV on immunosuppression and anti-tumor immunity, and to study the effects of sEV on metabolism.
By developing the present project we have successfully uncovered several key aspects regarding the pro-tumoral role that leukemic ME-sEV (LME-sEV) have in CLL in vivo, deciphering the mechanisms underlying their negative function in cancer. We described that LME-EVs carry immune-checkpoint proteins, such as PD-L1, GAL9, B7-H2, and VISTA. Single-sEV analysis combined with hierarchical stochastic neighbor embedding clustering confirmed that PD-L1 and GAL9 are often coexpressed on CD20+ MHC-II+ vesicles, suggesting LME-sEVs may act as functional units with immunosuppressive capabilities. Using fluorescence-labeled LME-sEVs, we detected that 5% to 10% of the T cells and 40% of B cells uptake sEVs in vivo, and that CD8+ T cells, but not Treg, CD4+ Tconv, and CD19+ B cells, have decreased expression of genes associated with immune response and amino acid transport and increased expression of genes involved in the inhibition of immunity and T-cell differentiation. We have further profiled gene expression and protein contents of sorted CD8+ T lymphocytes treated with LME-sEVs. The genes involved in CD8+ T-cell activation, survival, proliferation, and immune activation are significantly downregulated, whereas genes negatively associated with the above processes were increased in these cells. In addition, LME-sEV treatment inhibited oxidative phosphorylation and leads to an unfavorable metabolic profile in CD8 T cells.
On another hand, we have described the different metabolites that are found inside LME-sEV, proven the intrinsic capacity of sEV to internalise glucose and furthered into the pipelines for identifying the molecules involved in the uptake of sEV by targeted cells. Finally, we have accumulated experience in the development of biologically-engineered sEV for potential therapeutic applications.
On another hand, we have described the different metabolites that are found inside LME-sEV, proven the intrinsic capacity of sEV to internalise glucose and furthered into the pipelines for identifying the molecules involved in the uptake of sEV by targeted cells. Finally, we have accumulated experience in the development of biologically-engineered sEV for potential therapeutic applications.
There is a constant high demand on new therapeutic approaches for CLL, since it represents the most common form of leukemia in the Western hemisphere to affect the elderly and since it remains incurable. From the results obtained in this project and the publication/s out of it, we can conclude that LME-sEV have a deep negative impact in the immune control of leukemic cells in CLL, and thus represent targetable entities with great potential. We consider that all the experience accumulated by the host laboratory along the development of this project, mainly in the development of the BEsEV, will have in time a great impact on innovative therapeutic technologies.