Dynamic 3D interplay of primary human salivary cells and the basement membrane

描述(申请人提供)：头颈部癌症的治疗需要切除腺体组织并结合放射治疗，会对靶组织和周围组织造成重大损害。唾液腺是脆弱的，当受损时，功能丧失会导致唾液腺功能减退和口干，从而增加口腔健康风险和生活质量下降。目前，口干症还没有治愈方法，只有缓解与唾液功能丧失相关的不适的干预措施。一种新的治疗方法正在进行中，以解决这一未得到满足的需求，使用透明质酸(HA)水凝胶和切除的人唾液腺的原代细胞。可重复地将唾液腺泡组织成功能分泌单位是我们模型系统标准化的关键一步。我们的目标是模拟发育中的腮腺唾液腺组织的微环境，以最好地支持浆液性腺泡的组织。决定组织化腺泡的因素是基底膜(BM)沉积和管腔形成。利用实时成像光学和荧光显微镜，我们已经观察到在腺泡形成之前，HA水凝胶中的腺泡细胞的协调运动。在组织的早期阶段，腺泡细胞处于不断受到机械力影响的动态微环境中，我们假设机械力在3D中驱动着BM的沉积、管腔的形成和结构的完整性。在目标1中，我们打算确定在BM沉积和腺泡生长过程中参与细胞运动净协调的信号机制。在这种协调中提出的信号机制包括细胞-ECM界面的整合素信号，连接蛋白介导的细胞内和细胞间信号，以及Nesprin4核重新定位。在目标2中，我们将测量在3D中启动多细胞结构协调运动所需的牵引力。实时荧光成像和计算模型将被用来开发位移场和细胞牵引力图，并重建组织腺泡的细胞牵引力作为其大小和微环境的函数。在目标3中，我们将评估腺泡组织、管腔形成和结构完整性，以响应水凝胶的机械负荷。将评估不同大小和频率的载荷对腺泡组织和完整性的影响。这些特定目标的成功完成将(1)标准化用于工程唾液腺浆液性腺泡的模型系统以及在此优化HA水凝胶迭代的评估过程，(2)从根本上理解唾液腺泡的结构/功能关系，以及(3)提高该组织工程系统的翻译潜力。此外，私人助理吴丹妮尔博士将接受3D实验和计算培训，这对她作为组织工程独立研究员的未来至关重要，她将从多学科界面的操作中获得技能和视角，并将通过手稿和赠款撰写、指导和协作技能培训来推进她的长期职业目标。

DESCRIPTION (provided by applicant): Head and neck cancer treatments that require the resection of glandular tissue in combination with irradiation therapy cause significant damage to target and surrounding tissues. Salivary glands are vulnerable and when compromised, a loss of function causes hyposalivation and 'dry mouth' (xerostomia) that lead to an increase in oral health risk and a decline in quality of life. Currently, there is no cure for xerostomia, only interventions for alleviating the discomfort associated with loss of salivary function. A novel therapy is underway to address this unmet need using hyaluronic acid (HA) hydrogels and primary cells from resected human salivary gland. Repeatable organization of salivary acini into functional secretory units is a key step toward the standardization of our model system. Our goal is to mimic the microenvironment of parotid salivary gland tissue in development to best support the organization of serous acini. Determining factors for organized acini are basement membrane (BM) deposition and lumen formation. Using live imaging light and fluorescence microscopy, we have observed the coordinated motility of acinar cells in HA hydrogels, prior to acini organization. In early stage organization, acinar cells are in a dynamic microenvironment continuously influenced by mechanical forces, and we hypothesize that mechanical forces drive the BM deposition, lumen formation, and structural integrity of the acini in 3D. In Aim 1, we intend to identify the signaling mechanisms involved in the net coordination of cell motility durin BM deposition and growth of the acini. Signaling mechanisms proposed in this coordination include integrin signaling at the cell-ECM interface, connexin mediated intra- and intercellular signaling, and nesprin4 nuclear repositioning. In Aim 2, we will measure the traction forces required to initiate the coordinated movement of a multicellular structure in 3D. Live-fluorescence imaging and computational modeling will be used to develop displacement field and cellular traction maps, and reconstruct cellular traction forces of organizing acini as a function of their size and microenvironment. In Aim 3, we will evaluate acini organization, lumen formation, and structural integrity in response to mechanical loading of the hydrogel. Effects of varying magnitude and frequency loads on acini organization and integrity will be evaluated. Successful completion of these specific aims will (1) standardize the model system used to engineer serous acini of the salivary parotid gland as well as the evaluation process for optimizing iterations of the HA hydrogel herein, (2) yield fundamental understanding of salivary acini structure/function relations, and (3) advance the translational potential of this tissue engineered system. Additionally, the PI, Dr. Danielle Wu, will gain experimental and computational training in 3D, crucial for her future as an independent researcher in tissue engineering, will acquire skills and perspective from operating at a multidisciplinary interface, and will advance her long-term career goals with training in manuscript and grant writing, mentorship, and collaboration skills.