Abstract Background Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma, categorized by two subtypes, fusion protein-positive (FPRMS) and negative (FNRMS), based on the existence of PAX3-FOXO1 fusion gene (P3F). Different features between the two subtypes could guide the use of distinct treatment approaches. We discovered that FNRMS exhibited enrichment of gene sets related to cell-extracellular matrix (ECM) interaction than FPRMS. ECM, providing physical support as a scaffold, regulates cancer development. We hypothesize that a distinct molecular feature, P3F, may cause the difference in cell-ECM interaction between FPRMS and FNRMS. Methods To confirm the higher cell-ECM interaction of FNRMS vs. FPRMS, we used confocal reflection microscopy (CRM), enabling us to monitor the fibrous structures of ECM. We generated the spheroids with FNRMS and FPRMS cell lines by hanging drop method and embedded them into collagen type I. Then, we assessed orientation and dynamic changes in the structure of collagen fibers surrounding spheroids. Also, we analyze the transcriptome of FPRMS cells with P3F knockdown to confirm if P3F regulates cell-ECM interaction negatively via RNA seq. Additionally, we examined ECM surrounding spheroids of P3F knockdown FPRMS cells if P3F modulates fibrous structures of ECM, indicating cell-ECM interaction. Results We showed that collagen fibers are oriented more perpendicular to the FNRMS spheroid surface, indicating the strong pulling and pushing effect of cells. However, fibers surrounding FPRMS spheroids showed parallel alignment to their surfaces, reflecting passively pushed by the spheroids. Additionally, fibers near FNRMS spheroids showed greater displacement than those near FNRMS spheroids, which suggests stronger cell-ECM interaction in FNRMS than in FPRMS. Furthermore, P3F knockdown FPRMS cells exhibited significant enrichment of gene sets related to cell-ECM interaction. Their CRM results showed that collagen fiber orientation was significantly perpendicular to the surfaces of spheroids of P3F knockdown FPRMS cells than those of control cells. P3F knockdown cell spheroids also displayed enhanced displacement of ECM fibers than control cells. These results suggest that P3F suppresses cell-ECM interaction, consistent with higher cell-ECM interaction of FNRMS than FPRMS. Conclusions The PAX3-FOXO1 gene may regulate cell-ECM interaction negatively, causing distinct features between FPRMS and FNRMS, which is crucial for various cellular functions and suggest different treatment between the two RMS subtypes. Citation Format: Chandra Kaladhar Vemula, Ivan Chavez, Anthony Chronopoulos, Jinseok Park. Effect of PAX3-FOXO1 fusion gene on cell-extracellular matrix interaction of Rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4269.