DataSheet_1_Proteomics and liquid biopsy characterization of human EMT-related metastasis in colorectal cancer.docx

Tumor cells undergo epithelial-mesenchymal transition (EMT), however, there is a room of disagreement in role of EMT heterogeneity to colorectal cancer metastasis (mCRC) evolution. To uncover new EMT-related metastasis proteins and pathways, we addressed the EMT status in colorectal cancer liver metastasis patient-derived CTCs to identify proteins that promote their distant metastasis. And then, we performed a comparative proteomic analysis in matched pairs of primary tumor tissues, adjacent mucosa tissues and liver metastatic tissues. By integrative analysis we show that, unstable Epithelial/Mesenchymal (E/M)-type CTCs had the strongest liver metastases formation ability and the proportion of E/M-type CTCs correlated with distant metastases. Using an optimized proteomic workflow including data independent acquisition (DIA) and parallel reaction monitoring (PRM), we identified novel EMT-related protein cluster (GNG2, COL6A1, COL6A2, DCN, COL6A3, LAMB2, TNXB, CAVIN1) and well-described (ERBB2) core protein level changes in EMT-related metastasis progression, and the proteomic data indicate ERBB2, COL6A1 and CAVIN1 are promising EMT-related metastatic biomarker candidates. This study contributes to our understanding of the role that EMT plays in CRC metastasis and identifies heterogeneous EMT phenotypes as a key piece for tumor progression and prognosis. We further propose that therapies targeting this aggressive subset (E/M-type) of CTCs and related protein may be worthy of exploration as potential suppressors of metastatic evolution.

肿瘤细胞经历上皮-间质转化（EMT）过程，然而，关于EMT异质性在结直肠癌转移（mCRC）演进中的角色，存在一定争议。为了揭示与EMT相关的新的转移蛋白和通路，本研究针对结直肠癌肝转移患者来源的循环肿瘤细胞（CTCs）中的EMT状态进行研究，以鉴定促进其远处转移的蛋白。随后，我们对匹配的原发肿瘤组织、邻近黏膜组织及肝脏转移组织进行了比较蛋白质组学分析。通过整合分析，我们揭示不稳定的上皮/间质（E/M）型CTCs具有最强的肝脏转移形成能力，且E/M型CTCs的比例与远处转移相关。利用优化后的蛋白质组学工作流程，包括数据非依赖性采集（DIA）和平行反应监测（PRM），我们鉴定了新的EMT相关蛋白簇（GNG2、COL6A1、COL6A2、DCN、COL6A3、LAMB2、TNXB、CAVIN1）以及已描述的（ERBB2）核心蛋白在EMT相关转移进展中的水平变化，蛋白质组学数据表明ERBB2、COL6A1和CAVIN1是具有潜力的EMT相关转移生物标志物候选者。本研究有助于我们理解EMT在CRC转移中的作用，并确定异质性EMT表型是肿瘤进展和预后的关键因素。我们进一步提出，针对这种具有侵袭性的CTCs亚群（E/M型）及其相关蛋白的治疗方案可能值得探索，作为抑制转移演进潜力的潜在策略。