Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The alveolar subtype (ARMS) is clinically more aggressive, and characterized by an oncogenic fusion protein PAX3-FOXO1 that drives oncogenic cellular properties. Exosomes are small, secreted vesicles that affect paracrine signaling. We show that PAX3-FOXO1 transcript alters exosome content of C2C12 myoblasts, leading to pro-tumorigenic paracrine effects in recipient cells. Microarray analysis revealed alteration in miRNA content of exosomes, affecting cellular networks involved in cell metabolism, growth signaling, and cellular invasion. Overexpression and knockdown studies showed that miR-486-5p is an effector of PAX3-FOXO1, and mediates its paracrine effects in exosomes, including promoting recipient cell migration, invasion, and colony formation. Analysis of human RMS cells showed miR-486-5p is enriched in both cells and exosomes, and to a higher extent in ARMS subtypes. Analysis of human serum samples showed that miR-486-5p is enriched in exosomes of patients with RMS, and follow-up after chemotherapy showed decrease to control values. Our findings identify a novel role of both PAX3-FOXO1 and its downstream effector miR-486-5p in exosome-mediated oncogenic paracrine effects of RMS, and suggest its possible use as a biomarker.

Exosomes are important intercellular communication vehicles, secreted into body fluids by multiple cell types, including tumor cells. They have been demonstrated to contribute to the metastatic progression of tumor cells through paracrine signaling. Tumor exosomes contain intact and functional proteins, mRNA and miRNA that may alter the cellular environment to favor tumor growth. We evaluated the protein cargo of exosomes derived from the childhood tumor rhabdomyosarcoma (RMS) and the molecular pathways they are implicated in to decipher their role in the progression of this aggressive disease. We conducted a mass spectrometry analysis of exosome content isolated from five RMS cell lines: three of embryonal RMS (ERMS) and two of alveolar RMS (ARMS) histology and verified results by multiple reaction monitoring and western blot analyses. Results revealed 161 common proteins in ERMS-derived exosomes and 122 common proteins in ARMS-derived exosomes, of which 81 proteins were common to both subtypes. Using both PANTHER gene classification and Pathway Studio software, we assessed the perturbed biological processes and altered pathways in which the exosomal proteins are involved. The 81 commonly expressed proteins included those involved in “cell-signaling,” “cell-movement,” and “cancer.” Pathways engaging the identified proteins revealed 37 common pathways including “integrin signaling pathway,” “inflammation mediated by chemokine and cytokine signaling pathway,” and “angiogenesis.” Finally, a comparison of exosomal proteins of RMS cells with publicly available datasets from other cancer cells revealed that 36 proteins are specific and endogenous to the RMS-exosomes. Taken together, our results reveal that RMS-derived exosomes carry a protein cargo that contributes to conserved cellular signaling networks across multiple cell lines, and we also identify RMS exosome-specific proteins that should be further evaluated as possible novel biomarkers for this tumor.