Tumor heterogeneity for ATF2 and its impact for invasive behavior of colorectal cancer

结直肠癌（CRC）是全球最常见的癌症之一。大多数CRC相关死亡与转移有关。腹膜转移发生在大约。25%的转移性结直肠癌，并与预后较差相关。这种类型的转移性扩散背后的分子机制是未知的。肿瘤异质性被认为是肿瘤复发、转移和治疗耐药的主要原因。尽管2015年已经描述了基于基因表达模式的分子CRC共识亚型，但迄今为止尚未解决肿瘤内异质性的问题。只有对单细胞分辨率的转录组学分析才有可能在遗传异质性肿瘤中识别侵袭性“驱动细胞”。有趣的是，我们可以证明在CRC肿瘤侵袭前沿，转录因子ATF 2的表达具有显著的异质性。到目前为止，这可能阻止了对ATF 2依赖性功能的因果分析，并掩盖了其在肿瘤侵袭中的作用。我们发现，ATF 2作为肿瘤抑制剂，特别是作为腹膜转移的抑制剂。使用CRISPR/Cas9产生的ATF 2敲除（KO）细胞系，我们观察到球状体、小鼠和鸡异种移植物中改变的侵袭模式。ATF 2异质性肿瘤被具有ATF 2缺失和强E-钙粘蛋白表达的边缘细胞群包围，这表明了所谓的前导细胞的表型。根据我们的前期工作，我们假设ATF 2在抑制去粘附（转移级联反应的第一步）方面具有新的功能。通过我们的项目，我们将继续我们的研究，并详细分析ATF 2丢失对CRC侵袭潜力的作用。我们将在最新的遗传修饰的3D肿瘤模型（如人类和小鼠类器官）中模拟ATF 2的肿瘤异质性。我们将用稳定转导的荧光标记物追踪ATF 2损失。我们有一个新的条件Atf 2 KO小鼠的机制研究的ATF 2损失调节网络。单细胞RNA分析将有助于确定ATF 2信号通路的新治疗靶点，并首次在鸡，类器官和小鼠模型中测试TROP 2（已确定的ATF 2靶点之一）的抑制剂，以确定其在CRC中的抗侵袭作用。

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide. The majority of CRC-related deaths are associated with metastasis. Peritoneal metastasis occurs in appr. 25% of metastatic CRC and is associated with worse prognosis. The molecular mechanisms behind this type of metastatic spread are unknown. Tumor heterogeneity is considered to be the major reason for tumor relapse, metastasis, and therapy resistance. Although molecular CRC consensus subtypes based on gene expression pattern have been already described in 2015, the problem of intratumoral heterogeneity was not solved so far. Only transcriptomic analyses on single-cell resolution have the potential to identify invasive „driver cells“ in genetically heterogeneous tumors. Interestingly, we could demonstrate at CRC tumor invasion front a remarkable heterogeneity for expression of the transcription factor ATF2. This might have so far prevented causal analyses for ATF2-dependent functions and have masked its role for tumor invasion. We showed that ATF2 acts as tumor suppressor, especially as inhibitor of peritoneal metastasis. Using CRISPR/Cas9 generated ATF2 knockout (KO) cell lines we observed an altered invasion pattern in spheroids, mouse, and chicken xenografts. The ATF2 heterogeneous tumor was surrounded by a marginal group of cells with ATF2 loss and strong E-Cadherin expression suggesting the phenotype of so-called leader cells. From our pre-work, we hypothesize a novel function for ATF2 in inhibition of de-adhesion, the first step in the metastatic cascade. With our project, we will continue our studies and analyze in detail the role of ATF2 loss for the invasive potential of CRC. We will simulate tumor heterogeneity for ATF2 in up-to-date genetically modified 3D tumor models such as human and mouse organoids. We will trace the ATF2 loss with stably transduced fluorescence markers. We have available a novel conditional Atf2 KO-mouse for mechanistic studies of the ATF2 loss-regulation network. Single-cell RNA analysis will help to identify new therapeutic targets of the ATF2 signaling pathway and for the first time inhibitors of TROP2, one of the identified ATF2 targets, will be tested in chicken, organoids, and mouse models for their anti-invasive effects in CRC.