Establishment of a ZFP64::NCOA3 fusion-positive spindle cell/sclerosing rhabdomyosarcoma organoid model and evaluation of fusion-gene dependency.

Spindle cell/sclerosing rhabdomyosarcoma (SRMS) comprises a heterogeneous group of tumors characterized by distinct molecular alterations and clinical behaviors. Among them, ZFP64::NCOA3 fusion-positive SRMS represents an extremely rare subtype, and its biological characteristics remain poorly understood because of the lack of representative experimental models. In this study, we established a patient-derived organoid (PDO) line, designated OICI-RMS-1275, together with organoid-derived xenograft (ODX) models from a previously reported case of ZFP64::NCOA3 fusion-positive SRMS using a modified air-liquid interface organoid culture method. The established organoids exhibited stable long-term propagation and retained tumorigenic capacity following serial transplantation into NOD-scid IL2Rgnull mice. Histological and immunohistochemical analyses demonstrated preservation of the characteristic spindle cell morphology and diffuse expression of MyoD1 and PAX7 observed in the original tumor. Polymerase chain reaction and Sanger sequencing confirmed retention of the ZFP64::NCOA3 fusion transcript and fusion breakpoint in the organoids and ODXs. Lentiviral-mediated knockdown of the fusion gene significantly suppressed organoid proliferation, indicating dependency on ZFP64::NCOA3 for tumor growth. Transcriptomic analyses revealed preservation of major transcriptional features between the original tumor and ODXs, including myogenic lineage-associated gene expression programs. Fusion-gene suppression decreased expression of MYOD1 and DLK1 while increasing PPARG expression, suggesting involvement of the fusion in lineage-associated transcriptional regulation. Under adipogenic induction conditions, SRMS organoids exhibited lipid accumulation detected by Oil Red O staining regardless of fusion-gene suppression status, indicating preserved adipogenic differentiation potential. These findings establish the first PDO and ODX models of ZFP64::NCOA3 fusion-positive SRMS and demonstrate fusion-gene dependency in this rare molecular subtype. Our results further suggest that despite strong myogenic transcriptional programs, fusion-positive SRMS retains adipogenic differentiation capacity. These models provide a valuable platform for investigating disease biology and developing novel therapeutic strategies for rare fusion-positive rhabdomyosarcomas.