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

项目总结 需要更有效的体外模型来改进对~90%的胰管患者的治疗 腺癌(PDAC)，在确诊后五年内死亡。PDAC是致命的，很难治疗，因为 转移率和免疫抑制微环境。这种疾病以淋巴管的形式发展。 肿瘤周围的毛细血管将转移的癌细胞和抗肿瘤免疫细胞输送到淋巴中。 节点。PDAC的进展部分是通过重建肿瘤的纤维化反应(结缔组织增生)来实现的。 细胞外基质(ECM)，并随时间改变ECM的硬度。然而，知识差距仍然存在于如何 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 贩运，而且大多数体外PDAC模型缺乏对僵硬的时间控制来模拟渐进性ECM 变得僵硬。我们的目标是使用一种具有渐进性硬化的PDAC特异性3D体外模型来研究 细胞外基质硬度的变化如何改变早期的人口贩运事件和免疫抑制。这项试点工作 有助于设计更复杂、更具预测性的PDAC进展模型 肿瘤和抗转移药物的发现和患者预后的改善。AIMS将检验中心假说 肿瘤周围的渐进性ECM硬化通过Up-2促进肿瘤和免疫细胞的运输。 对机械敏感分子的调节，使其能够在进入毛细淋巴管之前和之后迁移。 我们提出了两个具体目标来实现我们的目标：(1)定义渐进性僵化如何调节 淋巴管生成、转移、免疫抑制、与趋化/迁移、侵袭和 成熟。甲基丙烯酸酯I型胶原将与透明质酸结合以代表PDAC ECM和 让水凝胶在光照下变硬，照射到代表正常组织和肿瘤组织的水平。淋巴 内皮细胞、PDAC癌细胞和免疫细胞(树突状细胞、巨噬细胞)将单独结合 使用水凝胶，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。淋巴单分子层 内皮细胞将在经历渐进性硬化的水凝胶上生长。然后，单层将是 暴露在PDAC和免疫细胞中，这些细胞也经历了僵硬，以评估功能结果： 黏附强度、竞争性黏附、跨单层的迁移、内皮屏障完整性以及 跨壁迁移。这项工作的创新之处在于，它使用组织工程学来对 细胞外基质僵硬、中心纤维化和淋巴相互作用--目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。这些先导性研究将对PDAC治疗产生影响 通过在研究可能抑制这种相互作用的潜在治疗靶点方面取得重大进展 淋巴道转移和/或改变PDAC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.