Regulation of transendothelial migration of Tregs in irradiated HNSCCs by EphB4-ephrinB2 interaction

EphB4-ephrinB2 相互作用对受辐射 HNSCC 中 Tregs 跨内皮迁移的调节

• 批准号: 10643984
• 负责人: Laurel B Darragh
• 金额: $ 4.45万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10643984
• 关键词: AKT Signaling Pathway; Affect; Animal Model; Autoimmune; Binding; Biological Assay; Blood Vessels; CD4 Positive T Lymphocytes; CD8B1 gene; Cell Adhesion; Cell Communication; Cell physiology; Cells; Coculture Techniques; Combination immunotherapy; Cytometry; Cytoplasmic Tail; Data; Disease; Effector Cell; Embryonic Development; Endothelial Cells; Endothelium; EphB4 Receptor; Equilibrium; FOXP3 gene; Failure; Flow Cytometry; Genetic; Genetic Engineering; Goals; HPV-negative head and neck cancer; Head and Neck Cancer; Head and Neck Squamous Cell Carcinoma; Homing; Immune; Immune Evasion; Immune checkpoint inhibitor; Immunocompetent; Implant; Infiltration; L-Selectin; Ligands; LoxP-flanked allele; Malignant Neoplasms; Mediating; Methods; Modeling; Molecular; Molecular Target; Mus; Outcome; Participant; Pathway interactions; Patients; Peptides; Phenotype; Phosphorylation; Physiological; Play; Population; Pre-Clinical Model; Process; Proto-Oncogene Proteins c-akt; Publishing; Radiation therapy; Receptor Protein-Tyrosine Kinases; Regulation; Regulatory T-Lymphocyte; Research; Resistance; Resistance development; Role; STAT3 gene; Signal Transduction; Site; System; T cell differentiation; T-Cell Proliferation; T-Lymphocyte; Testing; Tumor Promotion; Up-Regulation; Vascular Endothelial Cell; Vascular Endothelium; angiogenesis; anti-PD-1; anti-tumor immune response; cell motility; cell type; effective therapy; effector T cell; high risk; immunological status; immunoregulation; in vivo; inhibitor; intravital microscopy; knock-down; migration; mouse model; novel; pharmacologic; preclinical study; radiation response; radioresistant; receptor; recruit; response; side effect; therapy resistant; trafficking; transcriptome sequencing; tumor; tumor growth; tumor microenvironment; tumor progression; tumor-immune system interactions

Abstract Resistance to radiation therapy (RT) remains a challenging problem for high-risk head and neck squamous cell carcinoma (HNSCC) patients. Anti-PD1 is now approved in the first- and second-line settings, indicating that the majority of these tumors are resistant to treatment. The field awaits the results of multiple trials testing whether combining immunotherapy and RT can help avoid resistance to RT, but many preclinical studies (including those from our lab) indicate that treatment resistance still develops despite such combinations. The preferential recruitment of unique immunosuppressive cell subtypes after RT to the tumor microenvironment (TME) in response to RT plays an important role in contributing to the promotion of tumor growth and progression. The tumor endothelium can act as a selective barrier that regulates the entry, stability, and activation status of immune cells, but the mechanistic underpinnings of this barrier activity remain poorly understood. The EphB4 receptor tyrosine kinase and its ligand ephrinB2 define novel molecular targets. Upon cell to cell contact, both EphB4 and ephrinB2 can signal via their cytoplasmic domains. Though they have been extensively studied in cell migration and angiogenesis in early embryonic development, little has been published on their role in modulating the cancer immune microenvironment. Our data show that ephrinB2 is expressed on the tumor vasculature and is upregulated by RT and that EphB4 is expressed on immunosuppressive regulatory T cells (Tregs). Inhibiting EphB4-ephrinB2 interaction with a peptide (TNYL-RAW) results in the selective, and exclusive, reduction in infiltration of Tregs while increasing activation of CD8+ and CD4+Foxp3- T cell populations. Tregs, we have demonstrated, play a key role in the development of resistance to RT, and their depletion re-establishes responsiveness to therapy in HNSCC murine models. We hypothesize that RT’s upregulation of ephrinB2 on tumor endothelial cells acts preferentially on EphB4 expressing Tregs, and blocking this interaction reduces their intratumoral infiltration and survival, which allows CD8+ and CD4+ effector cells to induce tumor regression. In Aim 1, we will analyze the mechanistic outcomes of Treg EphB4 and endothelial ephrinB2 interaction on Treg transendothelial trafficking, intratumoral homing, and survival using genetically engineered animal models with ephrinB2 deleted on endothelial cells or EphB4 deletion on Tregs after RT. In Aim 2, we will interrogate the cellular and molecular mechanisms triggered by the interaction between endothelial ephrinB2 and EphB4- expressing Tregs in endothelial-Treg co-culture assays after RT. Targeted inhibition of STAT3, AKT, Erk pathways will be done with pharmacological inhibitors based on our preliminary data. We expect that these studies will elucidate the molecular and cellular parameters of EphB4-ephrinB2 inhibitors and will provide the necessary information to develop a more effective therapy for RT resistant HNSCC.

抽象的 对于高危头颈鳞状细胞来说，放射治疗（RT）的抵抗仍然是一个具有挑战性的问题 癌症（HNSCC）患者。抗 PD1 现已批准用于一线和二线治疗，这表明 大多数这些肿瘤对治疗有抵抗力。该领域正在等待多项试验的结果来测试是否 结合免疫疗法和放疗可以帮助避免对放疗的耐药性，但许多临床前研究（包括那些 来自我们实验室的）表明，尽管有这种组合，治疗耐药性仍然会出现。优惠 RT后招募独特的免疫抑制细胞亚型至肿瘤微环境（TME） 对放疗的反应在促进肿瘤生长和进展方面发挥着重要作用。这 肿瘤内皮可以作为选择性屏障，调节肿瘤内皮细胞的进入、稳定性和激活状态。 免疫细胞，但这种屏障活性的机制基础仍然知之甚少。埃弗B4 受体酪氨酸激酶及其配体 ephrinB2 定义了新的分子靶点。当细胞与细胞接触时，两者 EphB4 和 ephrinB2 可以通过其细胞质结构域发出信号。尽管它们已被广泛研究 细胞迁移和血管生成在早期胚胎发育中的作用，但关于它们在早期胚胎发育中的作用的报道很少 调节癌症免疫微环境。我们的数据显示 ephrinB2 在肿瘤上表达 EphB4 在免疫抑制性调节 T 细胞上表达 （特雷格斯）。抑制 EphB4-ephrinB2 与肽 (TNYL-RAW) 的相互作用会导致选择性的、排他性的、 减少 Tregs 的浸润，同时增加 CD8+ 和 CD4+ Foxp3- T 细胞群的激活。特雷格， 我们已经证明，在 RT 耐药性的发展中发挥着关键作用，并且它们的消耗重新建立 HNSCC 小鼠模型对治疗的反应。我们假设 RT 上调 ephrinB2 肿瘤内皮细胞优先作用于表达 EphB4 的 Tregs，阻断这种相互作用会降低其 瘤内浸润和存活，使 CD8+ 和 CD4+ 效应细胞能够诱导肿瘤消退。在 目标 1，我们将分析 Treg EphB4 和内皮 ephrinB2 相互作用对 Treg 的机制结果 使用基因工程动物模型进行跨内皮运输、瘤内归巢和生存 RT 后，内皮细胞上的 ephrinB2 缺失或 Tregs 上的 EphB4 缺失。在目标 2 中，我们将询问 内皮细胞 ephrinB2 和 EphB4 相互作用触发的细胞和分子机制 RT 后在内皮-Treg 共培养测定中表达 Tregs。靶向抑制 STAT3、AKT、Erk 根据我们的初步数据，将使用药物抑制剂来完成通路。我们期望这些 研究将阐明 EphB4-ephrinB2 抑制剂的分子和细胞参数，并提供 开发更有效的治疗耐药性 HNSCC 的必要信息。