To identify genes important for colorectal cancer (CRC) development and metastasis, we established a new metastatic mouse organoid model using Sleeping Beauty (SB) transposon mutagenesis. Intestinal organoids derived from mice carrying actively mobilizing SB transposons, an activating KrasG12D, and an inactivating ApcΔ716 allele, were transplanted to immunodeficient mice. While 66.7% of mice developed primary tumors, 7.6% also developed metastatic tumors. Analysis of SB insertion sites in tumors identified numerous candidate cancer genes (CCGs) identified previously in intestinal SB screens performed in vivo, in addition to new CCGs, such as Slit2 and Atxn1. Metastatic tumors from the same mouse were clonally related to each other and to primary tumors, as evidenced by the transposon insertion site. To provide functional validation, we knocked out Slit2, Atxn1, and Cdkn2a in mouse tumor organoids and transplanted to mice. Tumor development was promoted when these gene were knocked out, demonstrating that these are potent tumor suppressors. Cdkn2a knockout cells also metastasized to the liver in 100% of the mice, demonstrating that Cdkn2a loss confers metastatic ability. Our organoid model thus provides a new approach that can be used to understand the evolutionary forces driving CRC metastasis and a rich resource to uncover CCGs promoting CRC.

为了鉴定对结直肠癌（CRC）发展和转移重要的基因，我们利用睡美人（SB）转座子诱变建立了一种新的转移性小鼠类器官模型。来自携带主动动员 SB 转座子、激活的KrasG12D和失活的ApcΔ716等位基因的小鼠的肠类器官被移植到免疫缺陷小鼠体内。66.7% 的小鼠出现原发性肿瘤，7.6% 的小鼠也出现转移性肿瘤。通过对肿瘤中 SB 插入位点的分析，除了Slit2和Atxn1等新的 CCG 之外，还发现了先前在体内进行的肠道 SB 筛选中发现的许多候选癌症基因 (CCG) 。正如转座子插入位点所证明的那样，来自同一小鼠的转移性肿瘤彼此之间以及与原发性肿瘤具有克隆相关性。为了提供功能验证，我们敲除小鼠肿瘤类器官中的Slit2、Atxn1和Cdkn2a并移植到小鼠体内。当这些基因被敲除时，肿瘤的发展得到促进，表明它们是有效的肿瘤抑制因子。Cdkn2a敲除细胞也在 100% 的小鼠中转移到肝脏，这表明Cdkn2a缺失赋予了转移能力。因此，我们的类器官模型提供了一种新方法，可用于了解驱动 CRC 转移的进化力量，并为发现促进 CRC 的 CCG 提供了丰富的资源。