原发肿瘤的空间基因表达谱与来自侵袭前沿的侵袭性CTC释放相关，导致OSCC的早期复发

Background Oral squamous cell carcinoma (OSCC) exhibits high incidences of relapse and treatment failure. To understand the molecular mechanisms driving these poor outcomes, we explored the spatial and temporal heterogeneity of primary tumors and their circulating tumor cells (CTCs) using whole transcriptome analysis. Methods Bulk RNAseq was performed on three tumor regions (T1-invasive front, T2, T3) and the tumor-free margin (TFM) from early (EarlyR) and no recurrence till 2 years (NoR) patients (N=5 per group). Ultra-low cell RNAseq of CTCs was performed at diagnosis and at post-therapy/recurrence. Differential gene expression analysis and pathway enrichment using fgsea and clusterProfiler, with the Hallmark and Reactome databases, were performed to identify biologically relevant genes/pathways. Results Spatially distant tumor regions of EarlyR patients were highly diversified from their adjacent free margin in comparison to the NoR group, as indicated by a significantly high number of DEGs - T1 vs TFM (EarlyR-2969, NoR-23), T2 vs TFM (EarlyR-4220 vs NoR-50), and T3 vs TFM (EarlyR-3883 vs NoR-1239). We ruled out that these increases in DEGs were not due to differences in TFMs of the EarlyR and NoR groups (DEGs-17). The pathway analysis revealed enrichment of metastasis-related pathways, including EMT, TNFA signalling via NFκB, and ECM remodelling, in the T1-invasive front of the EarlyR tumor, while the other regions(T2/T3) showed enrichment of proliferation-related pathways, indicating that the invasive front -T1 might be the source of release of aggressive CTCs. We identified SERPINE1, BMP2, CXCL10, CXCL11, ICAM1, IFIH1, IFIT2, IL15, IL15RA, IRF1, JUNB, TGFB1, and TNC as hub genes across multiple pathways enriched in T1, which may contribute to the aggressiveness of this tumor region. Among these genes, IFIT2, IRF1, and JUNB were also upregulated in baseline CTCs of the EarlyR group, indicating the importance of tracking these genes as markers of aggressive CTCs. The PCA analysis of CTC data revealed that, although baseline CTCs are transcriptomically similar between the EarlyR and NoR groups, the post-therapy CTCs are more evolved in the EarlyR group than in the NoR group. We also identified a key molecular marker, FNBP1L, overexpressed in the tumor invasive front (T1) of EarlyR and in their CTCs at pretherapy and posttherapy. However, FNBP1L was downregulated in other regions of the primary tumor (T2/T3), suggesting that aggressive CTCs originate primarily from the invasive front, and FNBP1L (implicated in cytoskeletal reorganisation) might be a key player driving early recurrence and metastasis. Conclusion OSCC tumors from EarlyR patients undergo extensive spatial and temporal transcriptomic evolution, and the invasive front is plausibly involved in the release of aggressive CTCs marked by expression of IFIT2, IRF1, JUNB, and FNBP1L.