Gastric cancer (GC) is histologically classified into intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC), and the latter is poorly differentiated and highly metastatic. In this study, using quantitative real-time polymerase chain reaction, we described a complete protocol for in vivo CRISPR–Cas9-based knockout screening of essential genes for DGC metastasis. We functionally screened 30 candidate genes using our mouse DGC models lacking Smad4, p53, and E-cadherin. Pooled knockout mouse DGC cells were transplanted into a spleen of syngeneic immunocompetent mice to study clonal advantages in context of a complex process of liver metastasis. Tmsb4x (thymosin beta-4 X-linked), Hmox1, Ifitm3, Ldhb, and Itgb7 were identified as strong candidate genes that promote metastasis. In particular, Tmsb4x enhanced DGC metastasis and stomach organoid-generated tumor growth in in vivo transplantation models. Tmsb4x promoted tumor clonogenicity and anoikis resistance. In situ hybridization analysis showed that Tmsb4x is highly expressed in E-cadherin-negative mouse DGC models compared with mouse IGC and intestinal cancer models. E-cadherin deficiency also increased Tmsb4x expression in stomach organoids via Wnt signaling activation. Collectively, these results demonstrate that Tmsb4x promotes DGC metastasis. In addition, this experimental system will aid in the identification of novel target genes responsible for DGC metastasis.

中文翻译：

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使用定量 PCR 进行体内 CRISPR-Cas9 基因敲除筛选鉴定胸腺素 β-4 X-连锁促进弥漫型胃癌转移

胃癌（GC）在组织学上分为肠型胃癌（IGC）和弥漫型胃癌（DGC），后者分化差、转移性高。在这项研究中，我们使用定量实时聚合酶链反应，描述了基于 CRISPR-Cas9 的体内 DGC 转移必需基因敲除筛选的完整方案。我们使用缺乏 Smad4、p53 和 E-钙粘蛋白的小鼠 DGC 模型功能性筛选了 30 个候选基因。汇集的敲除小鼠 DGC 细胞被移植到同基因免疫活性小鼠的脾脏中，以研究复杂肝转移过程中的克隆优势。Tmsb4x（胸腺素 beta-4 X-linked）、Hmox1、Ifitm3、Ldhb和Itgb7被鉴定为促进转移的强候选基因。特别是，Tmsb4x 在体内移植模型中增强了 DGC 转移和胃类器官产生的肿瘤生长。Tmsb4x 促进肿瘤克隆形成和失巢凋亡抗性。原位杂交分析表明，与小鼠 IGC 和肠癌模型相比，Tmsb4x 在 E-cadherin 阴性小鼠 DGC 模型中高表达。E-钙粘蛋白缺乏还通过 Wnt 信号激活增加了胃类器官中 Tmsb4x 的表达。总的来说，这些结果表明 Tmsb4x 促进 DGC 转移。此外，该实验系统将有助于识别负责 DGC 转移的新靶基因。