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

结直肠癌(CRC)是全世界最常见的癌症之一。大多数与结直肠癌相关的死亡与转移有关。腹膜转移发生在APPR。25%的转移性结直肠癌患者预后较差。这种类型的转移扩散背后的分子机制尚不清楚。肿瘤的异质性被认为是肿瘤复发、转移和耐药的主要原因。虽然基于基因表达模式的分子CRC共识亚型已于2015年被描述，但肿瘤内异质性的问题至今仍未得到解决。只有对单细胞分辨率的转录学分析才有可能识别遗传异质性肿瘤中的侵袭性“驱动细胞”。有趣的是，我们可以证明在结直肠癌侵袭前沿，转录因子ATF2的表达具有显著的异质性。到目前为止，这可能阻止了对ATF2依赖功能的因果分析，并掩盖了它在肿瘤侵袭中的作用。我们发现ATF2可以作为肿瘤抑制因子，尤其是作为腹膜转移的抑制因子。使用CRISPR/Cas9产生的ATF2基因敲除(KO)细胞系，我们观察到在球体、小鼠和鸡的异种移植中，侵袭模式发生了变化。ATF2异质性肿瘤周围有一组边缘细胞，ATF2缺失，E-钙粘附素表达强，提示为所谓的领头细胞表型。从我们的前期工作中，我们假设ATF2在抑制转移级联中的第一步--去粘连方面具有新的功能。随着我们的项目，我们将继续我们的研究，并详细分析ATF2缺失对结直肠癌侵袭潜能的作用。我们将在最新的转基因3D肿瘤模型中模拟ATF2的肿瘤异质性，例如人和小鼠的器官。我们将用稳定转导的荧光标记追踪ATF2的丢失。我们提供了一种新的条件性ATF2 KO小鼠，用于ATF2丢失调节网络的机制研究。单细胞RNA分析将有助于确定ATF2信号通路的新治疗靶点，并将首次在鸡、有机化合物和小鼠模型上测试TROP2的抑制剂，以测试其在结直肠癌中的抗侵袭作用。

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.