三阴性乳腺癌（TNBC）是最具侵袭性的人类癌症之一，并且预后不良。大约80％的TNBC病例属于分子基底样亚型，可以通过诱导分化来进行治疗。然而，诱导TNBC分化的策略尚未得到充分探索。我们使用自然化合物库构建了基于三维形态筛选模型来鉴定能够诱导TNBC细胞分化的潜在候选化合物。通过RT-qPCR、RNA-seq、流式细胞术、免疫荧光、SCENITH和无标签LC-MS/MS等鉴定了龙胆素的功效。通过药物亲和反应靶标稳定性（DARTS）测定法确定了龙胆素的直接靶标。还构建了异种移植小鼠模型来确认龙胆素的效果。我们发现，龙胆素，一种吲哚呋喃喹唑啉酮，可以在三维球体和小鼠体内模型中诱导基底型TNBC细胞向绒毛分化。机制上，龙胆素处理导致三维培养的TNBC细胞产生全局代谢应激和ROS水平升高。此外，NAC，一种ROS清除剂，妨碍了龙胆素诱导的TNBC细胞在三维培养中的分化。最后，我们确定富马酸水合酶（FH）是龙胆素的直接靶标。通过抑制FH和FH的敲除，在三维培养中一致诱导TNBC细胞分化。我们的结果为使用三维培养模型进行分化治疗药物发现提供了一种平台，并确定了龙胆素作为TNBC治疗的潜在化合物。© 2023年 BioMed Central Ltd., part of Springer Nature.

Triple-negative breast cancer (TNBC) is one of the most aggressive human cancers and has poor prognosis. Approximately 80% of TNBC cases belong to the molecular basal-like subtype, which can be exploited therapeutically by inducing differentiation. However, the strategies for inducing the differentiation of TNBC remain underexplored.A three-dimensional (3D) morphological screening model based on a natural compound library was used to identify possible candidate compounds that can induce TNBC cell differentiation. The efficacy of rutaecarpine was verified using assays: RT-qPCR, RNA-seq, flow cytometry, immunofluorescence, SCENITH and label-free LC-MS/MS. The direct targets of rutaecarpine were identified through drug affinity responsive target stability (DARTS) assay. A xenograft mice model was also constructed to confirm the effect of rutaecarpine in vivo.We identified that rutaecarpine, an indolopyridoquinazolinone, induces luminal differentiation of basal TNBC cells in both 3D spheroids and in vivo mice models. Mechanistically, rutaecarpine treatment leads to global metabolic stress and elevated ROS in 3D cultured TNBC cells. Moreover, NAC, a scavenger of ROS, impedes rutaecarpine-induced differentiation of TNBC cells in 3D culture. Finally, we identified fumarate hydratase (FH) as the direct interacting target of rutaecarpine. The inhibition of FH and the knockdown of FH consistently induced the differentiation of TNBC cells in 3D culture.Our results provide a platform for differentiation therapy drug discovery using 3D culture models and identify rutaecarpine as a potential compound for TNBC treatment.© 2023. BioMed Central Ltd., part of Springer Nature.