Lineage tracing and clonal analysis of oral cancer initiating cells

描述(申请人提供)：口腔鳞状细胞癌(OSCCs)是头颈部鳞状细胞癌的绝大多数病例。目前口腔鳞状细胞癌的5年生存率仍保持在~50-60%，这主要是由于其耐药、复发、远处转移和诊断较晚所致。有人提出，在许多癌症类型中，包括口腔鳞状细胞癌，癌细胞被组织成异常的细胞系，包括癌症干细胞[CSCs，也称为癌症起始细胞(CICs)]及其更分化的子代癌细胞。最近在小鼠癌症模型中的几项研究使用谱系追踪来识别带有组织干细胞标记的癌细胞，并将其赋予驱动肿瘤扩张的长寿克隆。这种体内谱系追踪方法通常涉及三苯氧胺诱导Cre重组酶-雌激素受体融合蛋白(CreERT)的瞬时激活，这导致条件性Cre报告(例如，rosa26-Stop-YFP条件报告)的激活。CreERT的表达可以由细胞类型特异的启动子控制，表达CreERT的细胞及其子代细胞可以在Cre介导的重组(从而形成细胞谱系)的基础上被Cre报告基因(例如YFP)标记。尽管最近在小鼠模型中的谱系追踪研究提供了令人信服的证据支持完整肿瘤中CSC的存在，但它们几乎没有提供确保CSC自我更新所需的分子机制的线索，也没有提供关于不同的信号通路是否可能控制不同的CSC命运的线索。目前还不清楚CSCs是否形成一个同质群体，或者不同亚群的CSCs在化疗后是否会表现出不同的行为(例如，不同的生存)。在口腔鳞状细胞癌中，尽管人们认为口腔鳞状细胞癌与其转移、复发和治疗耐药有关，但口腔鳞状细胞癌干细胞的特征仍不明确。在这个R21项目中，提出了一种创新的方法，通过一种新的基于体内通路的谱系追踪策略来研究口腔鳞状细胞癌发展过程中的CSCs以及在已建立的致癌物诱导的小鼠OSCC模型中的治疗。这一方法涉及基于一组已知参与调节干细胞的通路的激活(例如，分别用于标记Wnt或Hedgehog信号反应细胞的Axin2-CreERT或Gli1-CreERT)，对癌细胞亚群进行基因标记(目标1)。通过定量比较不同途径标记的细胞群体之间的克隆结果，可以揭示哪些途径在CSCs中是活跃的，以及细胞命运选择如何受到途径活性的影响。在目标2中，这个小鼠模型中的口腔癌启动细胞/CSCs(即基于它们对干细胞相关途径的激活而定义的)如何在克隆水平上对治疗干预做出反应将进一步通过谱系追踪来确定。该项目的长期目标是使用这种基于通路的谱系追踪方法来确定哪些通路与口腔鳞癌干细胞的产生和再生(在治疗时)或与治疗耐药相关，并通过这一途径开发和测试针对口腔鳞癌干细胞的新疗法。

DESCRIPTION (provided by applicant): Oral Squamous Cell Carcinomas (OSCCs) represent the vast majority cases of squamous cell carcinomas of the head and neck. Currently the 5-year survival rate for OSCCs remains at ~50-60%, largely due to their therapy resistance, recurrence, distant metastasis, and late diagnosis. It has been proposed that in many cancer types, including OSCCs, cancer cells are organized into aberrant cell hierarchies, including cancer stem cells [CSCs, also known as cancer initiating cells (CICs)] and their more differentiated daughter cancer cells. Several recent studies in murine cancer models have used lineage tracing to identify cancer cells that bear markers of tissue stem cells, and which give ris to long-lived clones that drive tumor expansion. This in vivo lineage tracing approach typically involves tamoxifen-induced transient activation of a Cre recombinase-estrogen receptor fusion protein (CreERT), which leads to activation of a conditional Cre reporter (e.g., the Rosa26-Stop-YFP conditional reporter). CreERT expression can be controlled by a cell type-specific promoter and CreERT-expressing cells and their daughter cells can be genetically labeled by the Cre reporter (e.g., YFP) upon Cre-mediated recombination (thus forming a cell lineage). Although these recent lineage tracing studies in murine models have provided compelling evidence to support the existence of CSCs in intact tumors, they give few clues about the molecular mechanisms required to ensure CSC self- renewal, nor about whether different signaling pathways might control distinct CSC fates. It is also unclear whether CSCs form a homogeneous population or whether different subpopulations of CSCs might exhibit different behaviors (e.g., differential survival) upon chemotherapy. In OSCCs, although it is thought that CSCs are responsible for their metastasis, recurrence and therapy resistance, the identity of OSCC CSCs remains elusive. In this R21 project, an innovative approach is proposed to study CSCs during OSCC development and upon therapy in a well-established carcinogen-induced murine OSCC models by a novel pathway-based in vivo lineage tracing strategy. This approach involves genetically marking subsets of cancer cells based on activation of a panel of pathways known to be involved in regulating stem cells (e.g., Axin2-CreERT or Gli1-CreERT for labeling Wnt or Hedgehog signaling-responsive cells, respectively) (Aim 1). By quantitatively comparing clonal outcomes between different pathway-labeled cell populations, one can reveal which pathways are active in CSCs and how cell fate choices are affected by pathway activity. In Aim 2, how oral cancer-initiating cells/CSCs in this murine model (i.e., defined based on their activit for stem cell-related pathway) respond to therapeutic interventions at the clonal level will be further determined by lineage tracing. The long-term goal of this project is to use this pathway-based lineage tracing approach to identify which pathways correlate with generation and regeneration (upon therapy) of CSCs or with therapeutic resistance, and through this to develop and test novel therapy to target OSCC CSCs.