Endosomal mechanisms signaling oral cancer pain

项目摘要/摘要 GPCRs和RTK是跨膜受体，控制着许多生理和病理过程 疼痛过程包括癌症、疼痛和其他形式的慢性疼痛。这项提议的重点是信号 位于内体的GPCRs和RTK(eGPCRs和eRTK)可能起作用的机制 治疗癌症疼痛。潜在的假设是eGPCRs和eRTK在 人伤害性感受器和雪旺细胞的内涵体诱导持续激活和增敏 缓解疼痛。口腔癌微环境中的细胞(癌症细胞、免疫细胞和神经细胞)释放 激活GPCRs(例如，PAR2，CLR)和RTK(例如，TrkA， 伤害性感受器和雪旺细胞上的EGFR)。GPCRs和RTK经历依赖于笼蛋白的内吞作用和 组装信号复合体，控制离子通道的活性和表达，并持久 伤害性感受器和雪旺细胞的敏化，以驱动疼痛。一种反向翻译方法被用于 从3个特定的目标研究痛觉的内体信号。目标1确定eGPCR和 ERTK向疼痛发出信号。将评估口腔癌患者的疼痛(问卷调查、敏感性测试 口腔癌患者肿瘤的机械和化学刺激，以及并存情况的评估)。 手术切除的肿瘤、三叉神经和癌症上清液将被收集。特定于患者 雪旺细胞将从神经中分离和培养。将获得伤害性感受器和雪旺细胞 来自器官捐赠者。肿瘤上清液、蛋白酶、神经肽和生长的伤害性作用 调节伤害性感受器和雪旺细胞活性的因素将在小鼠身上进行研究。内吞抑制药 将被用于探索内吞作用和伤害性感受之间的联系。敏化和伤害性将是 与个体患者的疼痛表型相关。AIM 2定义eGPCR和eRTK疼痛信号 机械装置。肿瘤上清液以及eGPCR和eRTK配体的作用将被研究。细菌内吞作用 介导口腔癌疼痛的GPCRs和RTK将在人和小鼠的伤害性感受器和 通过共聚焦成像和生物物理分析检测雪旺细胞。生物传感器将被用来研究组件 伤害性感受器和雪旺细胞亚细胞室中的多蛋白信号复合体。 内体信号将与个体患者的疼痛表型相关。AIM 3开发 纳米粒(NP)对临床前疼痛模型中eGPCR和eRTK信号的拮抗作用 患者口腔癌疼痛。内体的酸性和光照将被用来触发NP的拆解和 拮抗剂的释放，并测试eGPCR和eRTK信号的贡献。它们在临床前疼痛中的应用 模型将内体信号与疼痛联系起来。加深对eGPCR和eRTK信号转导的理解 机制将为口腔癌疼痛的神经生物学提供洞察力。

PROJECT SUMMARY/ABSTRACT GPCRs and RTKs are transmembrane receptors that control a host of physiological and pathological processes including cancer pain and other forms of chronic pain. This proposal focuses on signaling mechanisms of GPCRs and RTKs located in endosomes (eGPCRs and eRTKs) that might be responsible for cancer pain. The underlying hypothesis is that eGPCRs and eRTKs generate long-lasting signals in endosomes of human nociceptors and Schwann cells that induce sustained activation and sensitization to mediate pain. Cells within the oral cancer microenvironment (cancer, immune, and neuronal cells), release proteases, neuropeptides and growth factors that activate GPCRs (e.g., PAR2, CLR) and RTKs (e.g., TrkA, EGFR) on nociceptors and Schwann cells. GPCRs and RTKs undergo clathrin-dependent endocytosis and assemble signaling complexes that control activity and expression of ion channels and long-lasting sensitization of nociceptors and Schwann cells to drive pain. A reverse translational approach is used to study endosomal signaling of pain in 3 Specific Aims. Aim 1 determines the contribution of eGPCR and eRTK signaling to pain. Oral cancer patient pain will be evaluated (questionnaires, sensitivity testing of patient oral cancer tumors with mechanical and chemical stimuli, and assessment of comorbidities). Surgically resected tumors, trigeminal nerve, and cancer supernatant will be collected. Patient-specific Schwann cells will be isolated and cultured from the nerve. Nociceptors and Schwann cells will be obtained from organ donors. Nociceptive effects of cancer supernatant, proteases, neuropeptides, and growth factors that mediate activity of nociceptors and Schwann cells will be studied in mice. Endocytosis inhibitors will be used to probe the link between endocytosis and nociception. Sensitization and nociception will be correlated with individual patient pain phenotypes. Aim 2 Defines eGPCR and eRTK pain signaling mechanisms. Effects of cancer supernatant, and eGPCR and eRTK ligands will be studied. Endocytosis of GPCRs and RTKs that mediate oral cancer pain will be studied in human and mouse nociceptors and Schwann cells by confocal imaging and biophysical assays. Biosensors will be used to study the assembly of multiprotein signaling complexes in subcellular compartments of nociceptors and Schwann cells. Endosomal signaling will be correlated with pain phenotypes in individual patients. Aim 3 develops nanoparticles (NP) to antagonize eGPCR and eRTK signaling in preclinical pain models that recapitulate patient oral cancer pain. Endosomal acidity and light will be exploited to trigger NP disassembly and antagonist release, and test contributions of eGPCR and eRTK signaling. Their use in preclinical pain models will link endosomal signaling to pain. A deeper understanding of eGPCR and eRTK signaling mechanisms will provide insight into the neurobiology of oral cancer pain.