Abstract Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive childhood soft tissue sarcoma, driven by the expression of a fusion oncoprotein PAX3-FOXO1. It presents with high metastatic potential, with 80% of tumors already metastatic at diagnosis, and a less than 20% cure rate with current available treatment. Paracrine and distant signaling, including via small secreted extracellular microvesicles including exosomes (sEVs), play a critical role in priming the pre-metastatic niche and enhancing tumor metastasis, but their role in FP-RMS, and specifically relating to fusion oncoprotein signaling, has not been studied. In this study, we aim to investigate the role of sEVs in mediating the effects of PAX3-FOXO1 signaling on the tumor microenvironment at the pre-metastatic niche, hypothesized to promote FP-RMS invasion and metastasis. We use the mouse myoblasts C2C12 model, with expression of the fusion oncogene PAX3-FOXO1. sEVs are extracted by serial ultracentrifugation and verified by size analysis and western blotting. We utilize in vitro cell culture studies as well as immunocompetent mouse models for tail vein injections to study effect of sEVs on the lung tumor microenvironment. We show that sEVs play a key role in enhancing cell migration and invasion of recipient stromal cells by comparing their effects on fibroblasts using treatment with 1) conditioned medium; 2) sEVs extracted from conditioned medium; and 3) an sEV-depleted fraction of the conditioned medium, all derived from PAX3-FOXO1-expressing cells versus controls. Furthermore, through RNA sequencing, we analyze the transcriptome of recipient fibroblasts treated with sEVs derived from PAX3-FOXO1-expressing cells versus those derived from cells expressing empty-vector control and identify signaling pathways differentially influenced by oncogene-modulated sEVs. In vivo, we found that sEVs were predominantly taken up by alveolar macrophages in the lung, in addition to other cells. Immunofluorescence staining further revealed that the populations of macrophages and cancer-associated fibroblasts were increased in the lungs of mice receiving sEVs derived from PAX3-FOXO1-expressing cells, as compared to those receiving sEVs from control cells. Ongoing work is focused on dissecting the proteomic and molecular signaling affected by PAX3-FOXO1-modulated sEVs on both fibroblast and immune cell compartments. In conclusion, this study reports that PAX3-FOXO alters the microenvironment of the distant pre-metastatic niche through sEV-mediated distant signaling. Ongoing investigations will provide deeper insights into the mechanisms underlying tumor metastasis in FP-RMS, potentially identifying novel therapeutic molecular targets to address current treatment gaps. Citation Format: Yu-Lieh Lin, Monireh Asoudeh, Faiz Ahmad, Pratibha Mishra, Raya Saab. Effects of the PAX3-FOXO1 fusion oncogene on secreted microvesicle-mediated priming of the pre-metastatic niche in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6578.