Identification and characterization of metastasis-initiating cells in esophageal squamous cell carcinoma in a pulmonary metastasis mouse model [scRNA-Seq]. Identification and characterization of metastasis-initiating cells in esophageal squamous cell carcinoma in a pulmonary metastasis mouse model [scRNA-Seq]

Background and aims: Cancer metastasis is the biggest obstacle to esophageal squamous cell carcinoma (ESCC) treatment. At present, understanding of its mechanism remains insufficient. Therefore, an in-depth exploration of the mechanisms of metastasis is crucial for early detection and intervention to reduce metastasis-related mortality. Methods: This study applied single-cell RNA sequencing analysis to investigate lung metastatic ESCC cells isolated from a pulmonary metastasis mouse model at multiple timepoints to characterize early metastatic microenvironment. Results: We identified a small population of parental ESCC KYSE30 cell line (Cluster S) resembled metastasis-initiating cells (MICs) because they could survive and colonize at lung metastatic sites. By comparing differential expression profiles between Cluster S and other subpopulations, we identified a panel of 7 metastasis-initiating signature genes (MIS), including CD44 and TACSTD2, to represent MICs of ESCC. Functional studies demonstrated that Cluster S cells (CD44high) exhibited significantly enhanced cell survival (resistances to oxidative stress and apoptosis), cell migration, invasion, stemness, and in vivo lung metastasis capabilities. Multiplex immunohistochemistry (mIHC) staining of 4 MISs (CD44, S100A14, RHOD, and TACSTD2) in ESCC cell lines and clinical samples found that differential MIS expression scores (dMISs) could predict lymph node metastasis, overall survival and risk of carcinothrombosis. GO and KEGG analyses revealed that CD44high cells were enriched in cell migration, organ development, stress responses, and neuron development, which might be related to the establishment of early metastatic microenvironment. Conclusion: This study identified CD44, S100A14, RHOD, and TACSTD2 as MISs to represent the MICs in ESCC populations and predict patient outcomes. Keywords: ESCC, metastasis-initiating cells, metastasis-initiating signatures, biomarker, scRNA-seq. Overall design: Human origin ESCC cell line labelled with luciferase and GFP (KYSE30-Luc-GFP) at in vitro state and retrieved from metastatic lungs of pulmonary metastasis mouse models at four timepoints (6 hours, 48 hours, 2 months, and 4 months) were collected via flow cytometry cell sorting (FACS), and undergone 10X Genomics single-cell transcriptome sequencing.

背景与目的：肿瘤转移是食管鳞状细胞癌（esophageal squamous cell carcinoma, ESCC）治疗面临的最大障碍。目前对其转移机制的认识仍存在不足，因此深入探究转移机制对于实现早期检测与干预、降低转移相关死亡率至关重要。
方法：本研究采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析方法，对多时间点下从肺转移小鼠模型中分离的肺转移性食管鳞状细胞癌细胞进行检测，以解析早期转移微环境的特征。
结果：本研究从亲本食管鳞状细胞癌KYSE30细胞系中鉴定出一个小细胞群（Cluster S），该细胞群类似转移起始细胞（metastasis-initiating cells, MICs），可在肺转移位点存活并定植。通过对比Cluster S与其他亚群的差异表达谱，我们筛选出包含CD44、TACSTD2在内的7个转移起始特征基因（metastasis-initiating signature genes, MIS），用于标识食管鳞状细胞癌的转移起始细胞。功能实验证实，Cluster S细胞（CD44高表达）的细胞存活能力（可抵抗氧化应激与细胞凋亡）、细胞迁移、侵袭、干性及体内肺转移能力均显著增强。对食管鳞状细胞癌细胞系及临床样本开展4个转移起始特征基因（CD44、S100A14、RHOD及TACSTD2）的多重免疫组化（multiplex immunohistochemistry, mIHC）染色后发现，差异MIS表达评分（differential MIS expression scores, dMISs）可用于预测淋巴结转移、总生存期及癌栓发生风险。GO与KEGG富集分析显示，CD44高表达细胞显著富集于细胞迁移、器官发育、应激反应及神经元发育通路，这可能与早期转移微环境的构建相关。
结论：本研究鉴定出CD44、S100A14、RHOD及TACSTD2作为转移起始特征基因，可用于标识食管鳞状细胞癌群体中的转移起始细胞，并预测患者预后。
关键词：食管鳞状细胞癌（ESCC）、转移起始细胞、转移起始特征基因、生物标志物、单细胞RNA测序（scRNA-seq）。
实验设计：本研究收集了体外培养的携带荧光素酶与绿色荧光蛋白标记的人源食管鳞状细胞癌KYSE30-Luc-GFP细胞系，并在6小时、48小时、2个月及4个月四个时间点从肺转移小鼠模型的转移肺组织中分离获取目标细胞，通过流式细胞分选（flow cytometry cell sorting, FACS）纯化后，开展10X Genomics单细胞转录组测序。