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

摘要 对放射治疗的抵抗(RT)对于高危头颈部鳞状细胞来说仍然是一个具有挑战性的问题。 癌症(HNSCC)患者。反PD1现在在一线和二线设置中获得批准，这表明 这些肿瘤中的大多数对治疗都有抵抗力。该领域正在等待多项试验的结果，以测试 结合免疫疗法和RT可以帮助避免对RT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，尽管有这样的组合，治疗耐药性仍在发展。优惠待遇 放射治疗后特定免疫抑制细胞亚型在肿瘤微环境(TME)中的募集 对RT的反应在促进肿瘤的生长和进展中起着重要作用。这个 肿瘤内皮细胞可作为选择性屏障调节血管内皮细胞的进入、稳定和激活状态 免疫细胞，但这种屏障活动的机制基础仍然知之甚少。EphB4 受体酪氨酸激酶及其配体ewitinB2定义了新的分子靶点。当细胞与细胞接触时，两者 EphB4和ewitinB2可以通过它们的细胞质结构域发出信号。尽管它们已经被广泛研究在 细胞迁移和血管生成在早期胚胎发育中的作用，目前还鲜有报道。 调节癌症免疫微环境。我们的数据显示在肿瘤上表达了ePhirinB2 EphB4在免疫抑制调节性T细胞上表达 (Tregs)。抑制EphB4-eparinB2与多肽(TNYL-RAW)的相互作用导致选择性和排他性， 减少Tregs的渗透，同时增加CD8和CD4Foxp3-T细胞的激活。特雷格斯 我们已经证明，在抗逆转录病毒的发展中发挥了关键作用，并且它们的枯竭重新建立起来 HNSCC小鼠模型对治疗的反应性。我们假设RT上调ePhrinB2在 肿瘤内皮细胞优先作用于表达Tregs的EphB4，阻断这种相互作用会减少它们的 肿瘤内的浸润和存活，这允许CD8和CD4效应细胞诱导肿瘤消退。在……里面 目的1，我们将分析Treg EphB4和内皮eparinB2相互作用在Treg中的机制结果 使用基因工程动物模型的跨内皮细胞贩运、肿瘤内归巢和生存 RT后内皮细胞上的ewitinB2缺失或Tregs上的EphB4缺失。在目标2中，我们将审问 EphB4-EphB4与EphB4-EphB4相互作用触发的细胞和分子机制 RT后内皮细胞-Treg共培养检测Tregs的表达。靶向抑制STAT3、AKT、ERK 根据我们的初步数据，将使用药物抑制剂进行治疗。我们预计这些 研究将阐明EphB4-eparinB2抑制剂的分子和细胞参数，并将提供 为开发更有效的RT耐药HNSCC治疗方法提供必要的信息。

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.