טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMilitsin Ruslana
SubjectTumor-derived microparticles from cells exposed to ionizing
radiation inhibit anti-cancer immune response
DepartmentDepartment of Medicine
Supervisor Professor Yuval Shaked


Abstract

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One of the routes cancers use to progress is by evasion of the immune system. Immune checkpoints are used by tumor cells to escape the immune system, among those is the programmed death 1 (PD-1) and its ligand PD-L1. Ionizing radiation (IR) anti-cancer therapy is usually effective by damaging the DNA leading to cell apoptosis, and by activating specific immune cells which in turn promote anti-tumor activity. However, it has also been shown that IR may promote tumor re-growth through macrophages or T regulatory cells, therefore suggesting that IR effective treatment is mainly regulated by its anti-tumor and pro-tumor activities on the immune system.

Extracellular vesicles (EVs) are vesicles shed from cell membrane outer layer upon stress, carrying biologic active cargo that affects target cells. EVs were found in normal and also in pathological conditions including cancer. Tumor extracellular vesicles (TEVs) were shown to confer resistance to chemotherapy partially by contributing to immune evasion. In this thesis, we hypothesized that TEVs may affect immune responses following radiotherapy, and therefore could explain their possible involvement in immune evasion and tumor re-growth.

Here we showed that TEVs derived from irradiated tumor cells suppress T cell immune activity and thus may support tumor progression. We found that IR doses of 2Gy and 6Gy did not affect the number TEVs shed from 4T1 and EMT6 breast cancer cell lines. However, we found that PD-L1 expression on TEVs was induced by IR especially in the 2Gy dose. Such results were obtained in both mice and breast cancer patients. Furthermore, to test the ability of TEVs to regulate T lymphocytes activity, splenocytes of non-tumor bearing mice were co-cultured in presence or absence of TEVs. T cell activity was significantly reduced following exposure to TEVs from 2Gy irradiated cells, as indicated by measuring CD8? cells by flow cytometry and by reduced secretion of granzyme B detected by ELISA.

We next defined the effect of TEVs on tumor growth and T cell activity in vivo. Mice bearing breast cancer that were injected with TEVs from irradiated cells resulted in larger tumor size when the mice were injected with TEVs from 2Gy radiation dose. Moreover, a reduction in T cell activity was observed in the tumors, spleens, and peripheral blood after 2Gy TEVs administration. Based on these results we also tested the combined therapy of IR and immunotherapy in mice bearing breast tumors. Tumor growth was delayed following single treatment with IR or anti-PD1. However, the combined therapy was not resulted in enhanced anti-tumor response. Indeed, elevated T-cell activity was observed in mice treated with anti-PD-1 monotherapy, but not in combination with IR, suggesting that other effects are involved in the regulation of the immune system following IR therapy.

Our results suggest that TEVs are involved in the regulation of T lymphocytes activity through the expression of PD-L1 and thus may support tumor immune escape. The combination of IR and immunotherapy, however, may need further investigation, uncovering additional mechanisms to explain the benefit of inhibiting the immune system following IR therapy.