Using photopolymerizable collagen matrices to investigate how progressive stiffening alters lymphatic trafficking of cancer and immune cells in pancreatic ductal adenocarcinoma

项目摘要 需要更有效的体外模型来改善约90%的胰管炎患者的治疗 腺癌（PDAC）在诊断后五年内死亡。PDAC是致命的，难以治疗，由于高 转移率和免疫抑制微环境。疾病进展为淋巴 肿瘤周围的毛细血管将转移的癌细胞和抗肿瘤免疫细胞运输到淋巴 结PDAC还通过纤维化反应（结缔组织增生）进行进展，纤维化反应重塑肿瘤 细胞外基质（ECM），并随时间改变ECM硬度。然而，在如何实现这一目标方面仍然存在知识差距。 动态PDAC微环境调节与癌症和免疫细胞进入相关的淋巴相互作用， 大多数体外PDAC模型缺乏对刚度的时间控制以模拟进行性ECM 硬化我们的目的是使用PDAC特异性三维体外模型进行性硬化，以研究 ECM硬度的变化如何改变早期运输事件和免疫抑制。这项试点工作 有助于设计更复杂的PDAC进展预测模型的长期目标， 肿瘤和抗转移药物的发现和改善患者的预后。目标将检验中心假设 在肿瘤周边进行性ECM硬化增强了癌症和免疫细胞的运输， 在进入毛细淋巴管之前和之后能够迁移的机械敏感分子的调节。 我们提出了两个具体目标来实现我们的目标：（1）定义渐进硬化如何调节 淋巴管生成性、转移性、免疫抑制性、与趋化性/迁移、侵袭相关的表型，以及 成熟甲基丙烯酸化I型胶原将与透明质酸组合以代表PDAC ECM， 允许水凝胶在光暴露下膨胀到代表正常和肿瘤组织的水平。淋巴 内皮细胞、PDAC癌细胞和免疫细胞（树突状细胞、巨噬细胞）将单独组合 与水凝胶接触，并评估与转移、运输和免疫抑制相关的表型变化。 (2)确定机械敏感性如何调节淋巴管内皮细胞的屏障完整性和粘附性， 淋巴管内皮细胞、PDAC癌细胞和免疫细胞之间的运动性。单层淋巴管 内皮细胞将在经历逐渐硬化的水凝胶上生长。然后将单层 暴露于PDAC和免疫细胞，也经历了硬化，以评估功能结果： 粘附强度、竞争性粘附、跨单层迁移、内皮屏障完整性，以及 跨壁迁移这项工作是创新的，因为它使用组织工程来行使时间控制， ECM硬度和中心纤维化以及淋巴相互作用-当前体外研究中缺失的关键特征 PDAC模型-在我们对PDAC进展的理解中。这些试点研究将影响PDAC治疗 通过在研究潜在的治疗靶点方面取得重大进展， 淋巴转移和/或改变PDAC中免疫抑制和抗肿瘤免疫细胞的平衡。

PROJECT SUMMARY More effective in vitro models are needed to improve treatment for ~90% of patients with pancreatic ductal adenocarcinoma (PDAC) who die within five years of diagnosis. PDAC is deadly and difficult to treat due to high rates of metastasis and an immunosuppressive microenvironment. The disease progresses as lymphatic capillaries around the tumor transport metastasizing cancer cells and anti-tumor immune cells to the lymph nodes. PDAC also progresses, in part, through a fibrotic response (desmoplasia) that remodels the tumor extracellular matrix (ECM) and alters ECM stiffness over time. However, knowledge gaps remain on how the dynamic PDAC microenvironment regulates lymphatic interactions related to cancer and immune cell entry and trafficking, and most in vitro PDAC models lack temporal control over stiffness to mimic progressive ECM stiffening. Our objective is to use a PDAC-specific 3D in vitro model with progressive stiffening to investigate how changes in ECM stiffness alter early-stage trafficking events and immunosuppression. This pilot work contributes to the long-term goal of designing more complex, predictive models of PDAC progression for anti- tumor and anti-metastasis drug discovery and improved patient outcomes. Aims will test the central hypothesis that progressive ECM stiffening at the tumor periphery enhances cancer and immune cell trafficking via up- regulation of mechanosensitive molecules that enable migration before and after entering lymphatic capillaries. We propose two specific aims to accomplish our objective: (1) Define how progressive stiffening regulates lymphangiogenic, metastatic, immunosuppressive, phenotypes related to chemotaxis/migration, invasion, and maturation. Methacrylated type I collagen will be combined with hyaluronic acid to represent the PDAC ECM and allow the hydrogel to stiffen under light exposure to levels that represent normal and tumor tissues. Lymphatic endothelial cells, PDAC cancer cells, and immune cells (dendritic, macrophages) will be individually combined with hydrogels and assessed for phenotypic changes related to metastasis, trafficking, and immunosuppression. (2) Determine how mechanosensitivity regulates barrier integrity in lymphatic endothelial cells and adhesion and motility between lymphatic endothelial cells, PDAC cancer cells, and immune cells. Monolayers of lymphatic endothelial cells will be grown on hydrogels undergoing progressive stiffening. The monolayer will then be exposed to PDAC and immune cells that have also undergone stiffening to assess functional outcomes: adhesion strength, competitive adhesion, migration across the monolayer, endothelial barrier integrity, and transmural migration. This work is innovative in that it uses tissue engineering to exercise temporal control over ECM stiffness and center fibrosis and lymphatic interactions—key features that are missing from current in vitro PDAC models—in our understanding of PDAC progression. These pilot studies stand to impact PDAC treatment by making significant progress to investigate potential therapeutic targets of interactions that could that inhibit lymphatic metastasis and/or shift the balance of immunosuppressive and anti-tumor immune cells in PDAC.