3D Culture Models of Partial-EMT and Its Regulation in Oral Cavity Squamous Cell Carcinoma

项目摘要 头颈部鳞状细胞癌（HNSCC）是癌症死亡的第六大原因。当前限制 在我们对HNSCC中瘤内细胞异质性（ITH）理解中， 新靶向治疗剂的分层和开发。我们的精液单细胞RNA测序（scRNA- seq）分析口腔鳞状细胞癌（OSCC）显示部分上皮间质细胞癌 过渡（p-EMT）计划，这是在HNSCC ITH的一个关键特征。p-EMT的特征在于表达一种 独特的间充质标志物集，没有上皮标志物或经典EMT表达的损失 转录因子，并与侵袭性和治疗失败。我们证明了 来自癌症相关成纤维细胞（CAF）的转化生长因子β 3（TGF-β3）通过癌症驱动p-EMT 细胞TGFβ诱导（TGFBI），独特亚群的主要标志物。作为TGFβ抑制剂的体外疗效 尽管靶向EMT尚未转化为临床，但显然需要更好的体内生物学模型。电流 p-EMT的建模受到单层培养的OSCC细胞系不能捕获ITH或p-EMT的阻碍。 相比之下，三维患者衍生的类器官（PDO）保持分化梯度，概括了 癌细胞异质性我们将使用OSCC PDO和CAFs来研究新的TGF-β3-TGFBI在OSCC中的作用。 轴驱动p-EMT和p-EMT作为对治疗反应的预测生物标志物的值。 首先，确定癌细胞TGFBI调节p-EMT以促进肿瘤生长的机制， 转移，我们将使用CRISPR干扰（CRISPRi）来敲低OSCC PDO中的TGFBI，并评估OSCC中的TGFBI。 对p-EMT的影响。为了模拟这种击倒的功能影响，我们将原位移植TGFBI- 调节的PDO进入免疫缺陷小鼠的颊粘膜，预期TGFBI敲低将 减少p-EMT、肿瘤形成和转移。第二，确定CAF影响的机制 通过TGF-β3-TGFBI轴表达OSCC p-EMT，我们将使用CRISPRi敲低OSCC CAF中的TGF-β3， 用OSCC PDO培养修饰的CAF。评估TGF-β3-TGFBI信号对异种移植瘤的影响 生长和转移，然后我们将原位共移植修饰的CAF和PDO到NSG小鼠中。第三、 为了确定p-EMT如何预测治疗反应，我们将治疗TGF β 1修饰的PDO， 将TGF-β3修饰的CAFs与一系列标准的OSCC化疗药物以及TGF-β 3抑制剂混合， TGFβ通路。为了支持使用p-EMT作为预测生物标志物，我们将处理我们的大型生物样本库， OSCC PDO与这一组相同的治疗，并确定p-EMT表达和 治疗反应我们预期TGF-β3-TGFBI轴诱导的p-EMT与 对标准和TGFβ靶向治疗的差异反应，支持其作为预测生物标志物的用途。 除了OSCC，我们的研究可能为针对p-EMT和其他罕见肿瘤的治疗提供了范式转变的基础。 亚群，以及驱动它们的旁分泌相互作用。

PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer death. Current limitations in our understanding of intratumoral cellular heterogeneity (ITH) in HNSCC cause major challenges in patient stratification and development of novel targeted therapeutics. Our seminal single cell RNA-sequencing (scRNA- seq) analysis of oral cavity squamous cell carcinoma (OSCC) revealed a partial epithelial-to-mesenchymal transition (p-EMT) program that is a key feature of ITH in HNSCC. p-EMT is characterized by expression of a unique set of mesenchymal markers, without loss of epithelial markers or expression of classical EMT transcription factors and is associated with invasiveness and treatment failure. We demonstrated that transforming growth factor beta-3 (TGF-β3) from cancer associated fibroblasts (CAF) drives p-EMT via cancer cell TGFβ-induced (TGFBI), a principal marker of the unique subpopulation. As in vitro efficacy of TGFβ inhibitors targeting EMT has not translated to the clinic, there is a clear need for better models of in vivo biology. Current modeling of p-EMT is hindered by the inability of OSCC cell lines in monolayer culture to capture ITH or p-EMT. In contrast, three-dimensional patient derived organoids (PDO) maintain differentiation gradients, recapitulating cancer cell heterogeneity. We will use OSCC PDOs and CAFs to investigate the role of the novel TGF-β3-TGFBI axis in driving p-EMT and the value of p-EMT as a predictive biomarker for response to therapy. First, to determine the mechanism by which cancer cell TGFBI regulates p-EMT to promote tumor growth and metastasis, we will use CRISPR interference (CRISPRi) to knock down TGFBI in OSCC PDOs and evaluate the impact on p-EMT. To model the functional impact of this knockdown, we will orthotopically transplant TGFBI- modulated PDOs into the buccal mucosa of immunodeficient mice, anticipating that TGFBI knockdown will reduce p-EMT, tumor formation, and metastasis. Second, to determine the mechanism by which CAF influence OSCC p-EMT via the TGF-β3-TGFBI axis, we will use CRISPRi to knock down TGF-β3 in OSCC CAFs and co- culture modified CAFs with OSCC PDOs. To assess the impact of TGF-β3-TGFBI signaling on xenograft tumor growth and metastasis, we will then orthotopically co-transplant modified CAFs and PDOs into NSG mice. Third, to determine how p-EMT predicts therapeutic response, we will treat TGFBI-modified PDOs, with or without admixed TGF-β3-modified CAFs, with a series of standard OSCC chemotherapies, as well as inhibitors of the TGFβ pathway. To support the use of p-EMT as a predictive biomarker, we will then treat our large biobank of OSCC PDOs with this same set of therapies and determine the relationship between p-EMT expression and therapeutic response. We anticipate that p-EMT, induced by the TGF-β3-TGFBI axis, will be associated with differential responses to standard and TGFβ-targeting therapies, supporting its use as a predictive biomarker. Beyond OSCC, our study may provide the basis for a paradigm shift in therapies targeting p-EMT and other rare subpopulations, as well as the paracrine interactions that drive them.