Uterine Leiomyosarcoma Cell-Derived Extracellular Vesicles Induce the Formation of Cancer-Associated Fibroblasts

Objective: Uterine leiomyosarcoma (ULMS) is a rare malignant tumor, which is aggressive, and has a poor prognosis even during its early stages. Extracellular vesicles (EVs) carry cargo, such as microRNAs (miRNAs), which are involved in intercellular communication in the tumor microenvironment and other processes. Because there are no studies on EV-related miRNAs in ULMS, we identified EV-related miRNAs in ULMS and examined their function. Methods: Small EVs (sEVs) and medium/large EVs (m/lEVs) were extracted from ULMS cells by ultracentrifugation and their basic characteristics were evaluated. Then, small RNA sequencing was done to obtain EV-related miRNA profiles. Next, miRNA expression levels in sera and tissues of ULMS patients were compared with those of myoma patients. Results: miR-654-3p and miR-369-3p were indicated to be highly expressed in both sera and tissues of ULMS patients. These two miRNAs are also highly expressed in ULMS cell lines and ULMS-derived EVs. Some cancerassociated fibroblast (CAF) markers were increased when fibroblasts were treated with ULMS-derived EVs. Furthermore, fibroblasts took up EVs derived from ULMS as determined by confocal laser microscopy. In addition, the transfection of the two candidate miRNAs into fibroblasts significantly increased some CAF markers, particularly ACTA2. Conclusion: miR-654-3p and miR-369-3p are highly expressed in ULMS-derived EVs, indicating that these EVrelated miRNAs induce the formation of cancer-associated fibroblasts.