Although much is known about the gene mutations required to drive colorectal cancer (CRC) initiation, the tissue-specific selective microenvironments in which neoplasia arises remains less characterized. Here, we determined whether modulation of intestinal stem cell niche morphogens alone can exert a neoplasia-relevant selective pressure on normal colonic epithelium. Using adult stem cell-derived murine colonic epithelial organoids (colonoids), we employed a strategy of sustained withdrawal of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) inhibition to select for and expand survivors. EGFR-signaling-independent (iEGFR) colonoids emerged over rounds of selection and expansion. Colonoids derived from a mouse model of chronic mucosal injury showed an enhanced ability to adapt to EGFR inhibition. Whole-exome and transcriptomic analyses of iEGFR colonoids demonstrated acquisition of deleterious mutations and altered expression of genes implicated in EGF signaling, pyroptosis, and CRC. iEGFR colonoids acquired dysplasia-associated cytomorphologic changes, an increased proliferative rate, and the ability to survive independently of other required niche factors. These changes were accompanied by emergence of aneuploidy and chromosomal instability; further, the observed mitotic segregation errors were significantly associated with loss of interkinetic nuclear migration, a fundamental and dynamic process underlying intestinal epithelial homeostasis. This study provides key evidence that chromosomal instability and other phenotypes associated with neoplasia can be induced ex vivo via adaptation to EGF withdrawal in normal and stably euploid colonic epithelium, without introducing cancer-associated driver mutations. In addition, prior mucosal injury accelerates this evolutionary process.

尽管人们对驱动结直肠癌（CRC）发生所需的基因突变了解很多，但肿瘤发生的组织特异性选择性微环境仍然知之甚少。在这里，我们确定单独调节肠道干细胞生态位形态发生素是否可以对正常结肠上皮施加肿瘤相关的选择压力。使用成体干细胞衍生的小鼠结肠上皮类器官（colonoid），我们采用持续撤除表皮生长因子（EGF）和表皮生长因子受体（EGFR）抑制的策略来选择和扩大幸存者。经过几轮选择和扩增，出现了 EGFR 信号独立 (iEGFR) 类集落。源自慢性粘膜损伤小鼠模型的结肠样细胞显示出适应 EGFR 抑制的能力增强。iEGFR 类集落的全外显子组和转录组分析表明，与 EGF 信号传导、细胞焦亡和 CRC 相关的基因获得了有害突变和表达改变。iEGFR 类集获得了与发育不良相关的细胞形态学变化、增殖率增加以及独立于其他所需生态位因素生存的能力。这些变化伴随着非整倍体和染色体不稳定的出现；此外，观察到的有丝分裂分离错误与运动间核迁移的丧失显着相关，这是肠上皮稳态的基本动态过程。这项研究提供了关键证据，证明染色体不稳定性和其他与肿瘤相关的表型可以通过适应正常且稳定的整倍体结肠上皮中的 EGF 撤出而在体外诱导，而不会引入癌症相关的驱动突变。此外，先前的粘膜损伤加速了这一进化过程。