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.

项目总结 头颈部鳞状细胞癌(HNSCC)是第六大癌症死亡原因。当前的限制 在我们对HNSCC瘤内细胞异质性(ITH)的理解中，给患者带来了重大挑战 新的靶向治疗药物的分层和发展。我们的开创性单细胞RNA测序(scRNA- 口腔鳞状细胞癌(OSCC)的序列分析显示部分上皮间质。 过渡(p-EMT)计划，这是HNSCC ITH的一个关键特征。P-EMT的特征是表达一种 一组独特的间质标志物，没有上皮标志物的丢失或经典EMT的表达 转录因子，并与侵袭性和治疗失败有关。我们证明了这一点 肿瘤相关成纤维细胞(CAF)转化生长因子β-3(转化生长因子-β3)通过肿瘤驱动p-EMT 细胞转化生长因子β诱导的(TGFBI)，是唯一亚群的主要标志。转化生长因子β抑制剂对AS的体外作用 靶向EMT尚未转化为临床，显然需要更好的体内生物学模型。当前 口腔鳞癌细胞系在单层培养中不能捕获ITH或p-EMT，阻碍了p-EMT的建模。 相比之下，三维患者衍生器官(PDO)保持分化梯度，概括 癌细胞异质性。我们将使用口腔鳞状细胞癌PDO和CAF来研究新型转化生长因子-β3-TGFBI的作用 AXIS在驱动p-EMT中的作用以及p-EMT作为预测治疗反应的生物标志物的价值。 首先，确定癌细胞TGFBI调节p-EMT促进肿瘤生长和 我们将使用CRISPR干扰(CRISPRi)来敲除口腔鳞癌PDO中的TGFBI，并评估 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- 将PDO调制到免疫缺陷小鼠的口腔粘膜，预期TGFBI基因敲除将 减少p-EMT、肿瘤形成和转移。第二，确定CAF影响的机制 通过转化生长因子-β3-β轴，我们将使用CRISPRI在口腔鳞癌CAF中击倒转化生长因子-GFP 3，并联合. 用口腔鳞状细胞癌PDOS培养修饰的CAF。转化生长因子-β-3-TGFBI信号转导对移植瘤的影响 然后，我们将把修饰的CAF和PDO原位联合移植到NSG小鼠体内。第三, 为了确定p-EMT如何预测治疗反应，我们将治疗TGFBI修饰的PDO，无论有没有 将转化生长因子-β3修饰的CAF与一系列标准的口腔鳞癌化疗药物以及 转化生长因子β途径。为了支持使用p-EMT作为预测生物标记物，我们将处理我们的大型生物库 并确定p-EMT表达与口腔鳞癌PDO之间的关系。 治疗反应。我们预计，由转化生长因子-β3-TGFBI轴诱导的p-EMT将与 对标准和转化生长因子β靶向治疗的不同反应，支持将其用作预测性生物标记物。 除了口腔鳞状细胞癌，我们的研究可能为针对p-EMT和其他罕见疾病的治疗模式转变提供基础 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.

• DOI: 10.3390/cancers15245810
• 发表时间: 2023-12-12
• 期刊: CANCERS
• 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.