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

描述（由申请人提供）：头颈癌治疗需要切除腺组织并结合放射治疗，对靶组织和周围组织造成重大损伤。唾液腺是脆弱的，当受到损害时，功能丧失会导致唾液分泌不足和“口干”（口干症），从而导致口腔健康风险增加和生活质量下降。目前，没有治愈口干症，只有干预措施减轻与唾液功能丧失相关的不适。一种新的治疗方法正在进行中，使用透明质酸（HA）水凝胶和切除的人唾液腺原代细胞来解决这一未满足的需求。唾液腺泡可重复组织成功能分泌单位是我们模型系统标准化的关键一步。我们的目标是模拟腮腺唾液腺组织在发育中的微环境，以最好地支持浆液腺泡的组织。有组织腺泡的决定因素是基底膜（BM）沉积和管腔形成。利用实时成像光和荧光显微镜，我们观察了HA水凝胶中腺泡细胞在腺泡组织之前的协调运动。在组织的早期阶段，腺泡细胞处于动态的微环境中，不断受到机械力的影响，我们假设机械力在3D上驱动了BM的沉积、管腔的形成和腺泡的结构完整性。在目的1中，我们打算确定在脑基质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传导机制包括细胞- ecm界面的整合素信号传导、连接蛋白介导的细胞内和细胞间信号传导以及nesprin4核重定位。在目标2中，我们将测量在3D中启动多细胞结构的协调运动所需的牵引力。活体荧光成像和计算模型将用于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Aim 3中，我们将评估水凝胶机械载荷下的腺泡组织、管腔形成和结构完整性。不同大小和频率的负荷对腺泡细胞组织和完整性的影响将被评估。这些特定目标的成功完成将(1)规范用于唾液腮腺浆液腺泡工程的模型系统，以及本文中HA水凝胶优化迭代的评估过程，(2)对唾液腺泡结构/功能关系的基本理解，以及(3)提高该组织工程系统的转化潜力。此外，PI Danielle Wu博士将获得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，她将获得在多学科界面操作的技能和观点，并将通过手稿和拨款写作、指导和协作技能方面的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.