Understanding the Role of Extracellular Vesicles in Lenalidomide-Resistance Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy that develops by the accumulation of various genetic abnormalities. In recent years, the prognosis of MM has improved by the development of novel drugs including immunomodulatory drugs (IMiDs), proteasome inhibitors, and antibody drugs. However, MM cells acquired drug resistance by long-term exposure to these therapeutic drugs. MM is a multiclonal disease, and various clone subtypes develop within the bone marrow microenvironment. It has been suggested that drug resistant phenotype could transmit from resistant clones to sensitive ones, and also to immune cells, or mesenchymal stem cells, resulting in the change of bone marrow microenvironment suitable for MM cell survival. It has been shown that extracellular vesicles (EVs) are one of the means of information transmission. EVs are secreted from almost all cells, and the amount of EV secretion is particular high from cancer cells. It was already known that cancer-derived EVs transmitted information associated with cancer progressions such as angiogenesis, metastasis, and drug resistance to the surrounding cells. Thus, EVs were proposed to play an important role in acquisition of drug resistance even though the mechanisms have not been fully understood in MM. In order to understand the mechanism of drug resistance in MM mediated by EVs, lenalidomide resistant cell lines were established by long-term exposure of lenalidomide. The amount of EVs was measured by ExoScreen, which is ultra-sensitive detection method of EVs by measuring surface protein of EVs, such as, CD9 and CD63, and by the nanoparticle tracking analysis. We found that lenalidomide resistant cell lines in KMS21R, KMS27R and KMS34R cell lines secreted about twice more EVs than their parental cell lines, and the amount of EV secretion was correlated with the drug sensitivity of lenalidomide. Suppression of EV secretion by knockdown of TSG101, which is known for EV secretion-associated protein, did not affect lenalidomide resistance. We could suppress the EV secretion to two-thirds, however cell proliferation and caspase activity were not change. From these results, we postulated the two possibilities; 1) EV secretion pathway other than TSG101 is associated with drug resistance via EVs; 2) EV derived from lenalidomide resistant cells can affect the cells exist in bone marrow microenvironment. From these hypotheses, we have done the following experiments. Firstly, to identify the genes which involve