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

口腔鳞状细胞癌部分EMT的3D培养模型及其调控

• 批准号: 10723327
• 负责人: Anuraag Suhrid Parikh
• 金额: $ 16.55万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723327
• 关键词: 3-Dimensional; Adherent Culture; Automobile Driving; Biological Models; Biology; Blocking Antibodies; CRISPR interference; Cancer Etiology; Carboplatin; Cell Line; Cells; Cessation of life; Cetuximab; Cisplatin; Clinic; Coculture Techniques; Coupled; Data; Development; Disease; Doxycycline; Epithelium; Fibroblasts; Fluorouracil; Foundations; Future; Genes; Grant; Head and Neck Cancer; Head and Neck Squamous Cell Carcinoma; Heterogeneity; Immunodeficient Mouse; Immunohistochemistry; In Vitro; Malignant Neoplasms; Mediator; Mesenchymal; Modeling; Mus; Neoplasm Metastasis; Oral mucous membrane structure; Organoids; Paclitaxel; Pathway interactions; Patients; Prediction of Response to Therapy; Predictive Value; Protein Isoforms; Recurrence; Regulation; Research Personnel; Resistance; Role; Seminal; Series; Signal Induction; Signal Transduction; Skull Base Chordoma; TGFBR1 gene; Testing; Transforming Growth Factor beta; Transplantation; Treatment Failure; Tumor Promotion; Work; antitumor effect; biobank; cancer cell; chemotherapy; clinical predictors; epithelial to mesenchymal transition; in vivo; in vivo Model; inhibitor; knock-down; malignant mouth neoplasm; mouth squamous cell carcinoma; new therapeutic target; novel; paracrine; patient response; patient stratification; predicting response; predictive marker; prevent; programs; response; response biomarker; single-cell RNA sequencing; targeted agent; targeted treatment; transcription factor; transforming growth factor beta3; treatment response; tumor; tumor growth; tumor heterogeneity; tumor xenograft

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.

项目概要 头颈鳞状细胞癌（HNSCC）是癌症死亡的第六大原因。目前的局限性。 我们对 HNSCC 瘤内细胞异质性 (ITH) 的理解给患者带来了重大挑战 我们的开创性单细胞 RNA 测序 (scRNA-) 的分层和开发。 seq）口腔鳞状细胞癌（OSCC）分析显示部分上皮间质 过渡（p-EMT）程序是 HNSCC 中 ITH 的一个关键特征，其特征是 a 的表达。 一组独特的间充质标记物，不会丢失上皮标记物或经典 EMT 的表达 我们证明了转录因子与侵袭性和治疗失败有关。 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），作为TGFβ抑制剂的体外功效的独特亚群的主要标志。 针对 EMT 尚未转化为临床，目前显然需要更好的体内生物学模型。 p-EMT 的建模受到单层培养中 OSCC 细胞系无法捕获 ITH 或 p-EMT 的阻碍。 相比之下，三维患者衍生类器官（PDO）维持分化梯度，概括了 我们将使用 OSCC PDO 和 CAF 来研究新型 TGF-β3-TGFBI 的作用。 驱动 p-EMT 的轴以及 p-EMT 作为治疗反应的预测生物标志物的价值。 首先，确定癌细胞TGFBI调节p-EMT促进肿瘤生长和增殖的机制。 转移，我们将使用 CRISPR 干扰 (CRISPRi) 敲低 OSCC PDO 中的 TGFBI 并评估 对 p-EMT 的影响 为了模拟这种敲低的功能影响，我们将原位移植 TGFBI-。 将 PDO 调节到免疫缺陷小鼠的颊粘膜中，预计 TGFBI 敲低将 减少 p-EMT、肿瘤形成和转移 其次，确定 CAF 影响的机制。 OSCC p-EMT 通过 TGF-β3-TGFBI 轴，我们将使用 CRISPRi 敲低 OSCC CAF 中的 TGF-β3，并共同- 培养修饰的 CAF 与 OSCC PDO 评估 TGF-β3-TGFBI 信号对异种移植肿瘤的影响。 生长和转移，然后我们将修饰的 CAF 和 PDO 原位共移植到 NSG 小鼠中。 为了确定 p-EMT 如何预测治疗反应，我们将使用或不使用 TGFBI 修饰的 PDO 进行治疗 混合 TGF-β3 修饰的 CAF，与一系列标准 OSCC 化疗药物以及 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库 采用同一组疗法的 OSCC PDO 并确定 p-EMT 表达与 我们预计由 TGF-β3-TGFBI 轴诱导的 p-EMT 将与治疗反应相关。 对标准疗法和 TGFβ 靶向疗法的不同反应，支持其作为预测生物标志物的用途。 除了 OSCC 之外，我们的研究可能为针对 p-EMT 和其他罕见疾病的治疗范式转变提供基础 亚群，以及驱动它们的旁分泌相互作用。

项目成果

Experimental Modeling of Host-Bacterial Interactions in Head and Neck Squamous Cell Carcinoma.

头颈鳞状细胞癌宿主-细菌相互作用的实验模型。

• 发表时间: 2023-12-12
• 影响因子: 5.2
• 作者: Okolo, Ogoegbunam;Honzel, Emily;Britton, William R;Yu, Victoria X;Flashner, Samuel;Martin, Cecilia;Nakagawa, Hiroshi;Parikh, Anuraag S
• 通讯作者: Parikh, Anuraag S