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

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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.