Single‐Cell Transcriptomic Analysis Suggests That JUN May Regulate BAMBI to Promote Osteosarcoma Cell Migration and Invasion

Background Distant metastasis of osteosarcoma (OS) is one of the major factors contributing to poor patient prognosis, and its underlying mechanisms remain incompletely understood. In this study, we employed single‐cell RNA sequencing (scRNA‐seq) to assess intratumoral heterogeneity and identify immune diagnostic and therapeutic markers, providing novel insights to guide targeted therapy. Methods Key cell clusters were identified using InferCNV analysis and combined with differential gene expression analysis, pseudotime trajectory analysis, transcription factor regulatory network construction, and functional enrichment analysis; signaling pathways closely associated with OS metastasis were screened and subsequently validated through the GSE152048 dataset and in vitro functional experiments. Results In this study, a total of 10 cell clusters were identified. Immune cells exhibited higher infiltration levels in the metastatic group. Further dimensionality reduction and clustering of malignant osteoblastic cells revealed five subpopulations, among which the metastatic C1 and C3 subclusters exhibited more pronounced malignant features. The results showed that BAMBI was specifically downregulated during the transition from nonmetastatic to metastatic states. Further analysis indicated that JUN may maintain subcluster phenotypic stability by regulating BAMBI expression. Moreover, as a pseudoreceptor of the TGF‐ β signaling pathway, the downregulation of BAMBI can promote pathway activation, thereby enhancing the migration and invasion potential of OS cells. Furthermore, the results were validated in the GSE152048 dataset and cell experiments. Conclusion This study reveals that during the progression of OS from a nonmetastatic to a metastatic state, BAMBI expression is downregulated and may be regulated by JUN. The decreased expression of BAMBI leads to hyperactivation of the TGF‐ β signaling pathway, thereby promoting the migration and invasion of OS cells. These findings provide potential immune‐related and molecular targets that could inform single cell–guided strategies for targeted therapy and overcoming resistance.