解析 CAM 異種移植模型中 EMT 異質性的多模態分析平台：空間體學與計算驗證

Abstract

摘要 背景：SNAI1 是一種上皮-間質轉化（EMT）的主控轉錄因子（TF），具有雙重轉錄作用；其中，賴胺酸殘基 K146/K187 的乙醯化會使其功能從抑制因子轉變為激活因子，從而調整 EMT 在不同情境下的分子執行機轉。目前學界對於此切換下游機制理解仍相當有限。本研究以高惡性度漿液性卵巢癌（HGSOC）作為疾病模型，建立一個整合空間轉錄組學（ST）、計算分析與多重免疫螢光（mIF）之平台，以解析由 SNAI1 驅動的上皮-間質轉化異質性。 方法：將鉑類耐藥性高分化漿液性卵巢癌（HGSOC）細胞系 PEO4 經基因工程改造，使其分別表達 SNAI1-2R（乙醯化缺陷型）及空載向量（EV），再於雞胚絨毛尿囊膜（CAM）上建立腫瘤異種移植瘤。針對 PEO4-SNAI1-2R 與 PEO4-EV 異種移植瘤（每組 n=2），在 20 個具潛在影響區域內的 38 個 PanCK+/PanCK- 區段中，進行了 ST 分析（GeoMx DSP，癌症轉錄組圖譜；1.8k 個基因）。透過識別 PEO4-SNAI1-2R 與 PEO4-EV 之間的差異表達基因（DEGs），並將其輸入 MOSAIC（主力驗證已知上皮-間質轉化轉錄因子（EMT-TFs）直接靶標的整合式計算流程）進行分析。同時建立了一套微免疫螢光（mIF）工作流程，用以繪製 CAM 異種移植模型的細胞上皮-間質轉化狀態及腫瘤-宿主相互作用。 結果：透過空間定量分析共鑑定出 15 個差異表達基因（DEGs）。其中，有 4 個 DEGs 經由整合式計算分析流程 MOSAIC 評定為 SNAI1 的直接靶基因。研究發現，相較於 EV 腫瘤，ALCAM 是 SNAI1-2R 腫瘤中 PanCK+ 區段上調程度最高的基因（log2FC = 0.93，FDR < 0.001），且經 MOSAIC 評定為信度最高的 SNAI1 靶基因。隔室層級分析則進一步解釋 SNAI1-2R 異種移植瘤中的腫瘤內之異質性，而在 EV 異種移植瘤中則未有見此現象。此外，多重成像技術識別出上皮-間質轉化（EMT）的異質性，其中腫瘤區域同時共表達上皮（EpCAM）與間質（Vimentin）標記物，暗示由 SNAI1 驅動的混合型 EMT 狀態。 結論：綜上所述，本研究建立了一個概念驗證平台，將空間組學與計算證據相結合，以繪製高分化漿液性卵巢癌（HGSOC）中由 SNAI1 驅動的上皮-間質轉化（EMT）異質性圖譜。此框架為未來不同的 EMT 誘導模型及腫瘤背景提供了嶄新的方向。

Abstract Background: SNAI1 is a master epithelial-mesenchymal transition (EMT) transcription factor (TF) with dual transcriptional roles where the acetylation at lysine residue K146/ K187 switches its function from a repressor to an activator, contributing to the molecular execution of EMT in different contexts. The understanding of mechanistic programs downstream of this switch remains limited. Using high-grade serous ovarian carcinoma (HGSOC) as the disease model, this study establishes an integrated spatial transcriptomics (ST), computational and multiplex immunofluorescence (mIF) platform to resolve SNAI1-driven EMT heterogeneity. Methods: A platinum-resistant HGSOC cell line PEO4 was engineered to express SNAI1-2R (acetylation-deficient) and empty vector (EV). Their tumor xenografts were established in the chick chorioallantoic membrane (CAM). ST profiling (GeoMx DSP, Cancer Transcriptome Atlas; 1.8k genes) of the PEO4-SNAI1-2R and PEO4-EV xenografts (n=2 per group) was performed in 38 PanCK+/PanCK- segments across 20 regions of interest. Differentially expressed genes (DEGs) between PEO4-SNAI1-2R and PEO4-EV were identified and subjected to MOSAIC, an integrated computational pipeline for the verification of direct targets of known EMT-TFs. An mIF workflow was established to map the cellular EMT states and tumor-host interactions of the CAM xenografts. Results: A total of 15 DEGs were identified from spatial profiling. Among them, 4 DEGs were scored as direct SNAI1 targets by MOSAIC, an integrated computational pipeline. ALCAM was found to be the top upregulated gene in PanCK+ segments of SNAI1-2R versus EV tumors (log2FC = 0.93, FDR < 0.001) and scored as a SNAI1 target with highest confidence by MOSAIC. Compartment-level analysis further resolved intra-tumoral heterogeneity in SNAI1-2R but not EV xenografts. In addition, multiplex imaging identified EMT heterogeneity with tumor regions co-expressing epithelial (EpCAM) and mesenchymal (Vimentin) markers, suggestive of hybrid EMT states driven by SNAI1. Conclusion: Together, this study established a proof-of-concept platform linking spatial omics and computational evidence to map SNAI1-driven EMT heterogeneity in HGSOC. This framework provides the future roadmap to generalize across different EMT induction models and tumor contexts.