טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentWaked Samah
SubjectThe effect of Lenalidomide on Multiple Myeloma Associated
Macrophages
DepartmentDepartment of Medicine
Supervisors Professor Benjamin Brenner
Dr. Noam Benyamini
Dr. Tamar Katz


Abstract

Introduction: In multiple myeloma (MM), macrophages located in the bone marrow microenvironment play a crucial role in the fate of malignant cells. Under the influence of the surrounding milieu, monocytes can change their migratory capacity and differentiate into tumor-associated macrophages (TAMs). At the tumor site, TAMs contribute to tumor survival, proliferation, angiogenesis, immunosuppression, and drug resistance. Lenalidomide, an immunomodulatory drug, is used for the treatment of MM. Lenalidomide works through various mechanisms that involve direct cytotoxicity as well as the modulation of different components of the immune system e.g. T cells, NK cells. However, its effect on macrophages is not well known. The current study has aimed to explore the effect of Lenalomide on monocyte recruitment, macrophage polarization and pro-tumor functions.


 Methods: Monocytes isolated from peripheral blood mononuclear cells of healthy donors were allowed to migrate through transwell insert towards different conditioned mediums (CM). For macrophage generation, monocytes were cultured with M-CSF followed by incubation with IL-4 to obtain M2 macrophages. To generate TAMs, CM obtained from the BM of MM patients was used. The phenotype and functional properties of the generated macrophages were assessed.



Results: Monocyte migration towards CM obtained from MM cell lines (RPMI8226 or U266) or from BM mononuclear cells of MM patients was significantly higher compared to migration toward normal BM donors’ CM. Monocytes treated with Lenalidomide demonstrated significantly decreased migration toward CM of MM cell lines (RPMI8226 or U266) compared to untreated monocytes. The effect of Lenalidomide on monocyte migration toward patient-derived CM was diverse. While 4 samples demonstrated decreased migration compared to untreated cells, in 5 samples, it increased.


Both M2 macrophages and TAMs treated with Lenalidomide expressed higher levels of M2 markers CD206, CD163, and cytokine IL-10 compared to untreated macrophages. Functional assays showed that Lenalidomide increased endocytosis of both M2 macrophages and TAMs. Exposure to Lenalidomide led to suppression of T cell proliferation when T cells were co-cultured with either autologous M2 macrophages or TAMs. In addition, M2 macrophages treated with Lenalidomide demonstrated a reduction in IFN-γ secretion from T cells compared to untreated M2 macrophages. In an in vivo study, BM of mice treated with Len showed higher levels of CD206 mRNA transcripts compared with untreated mice.



Discussion: This study has suggested that Lenalidomide has a direct effect on monocyte/macrophage behavior in the microenvironment generated by MM cells. Lenalidomide is found to reduce monocyte migration, support polarization of macrophages towards the M2 phenotype and promote macrophage anti-inflammatory and immunosuppressive functions. These results suggest that Len not only has an immune-stimulation and anti-tumoral effects but may also have protumoral effects. Whether or not these effects may be related to lenalidomide resistance warrants further investigations. This may also be one of the reasons for the clinical advantage of combining Lenalidomide with other drugs. Hence, development of anti-macrophage therapeutics that target specific pathways associated with their development into M2-like macrophages might contribute to the armamentarium of agents for treating MM.