Abstract 4614: Investigating lymphatic vessel remodeling and anti-tumor immunity in pancreatic cancer using tumor-on-chip and mouse models

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest tumors in humans. PDAC has an immunosuppressive tumor microenvironment, constituting for poor immunotherapy outcomes. Lymphatic vessels (LVs) transport leukocytes and antigens from tumor peripheries to draining lymph nodes (dLNs) for activation of anti-tumor immune responses. It is believed that impairment of the lymphatic function may result in delayed and ineffective immune activation against tumors. In this study, we created a three-dimensional (3D) tumor-on-chip model to examine phenotypical and functional changes in 3D engineered LVs, composed of human primary lymphatic endothelial cells (LECs) in co-culture with PDAC cells. Our in vitro studies revealed that PDAC cells induce lymphangiogenesis and tighten cell-cell junctions in LECs, forming “zipper-like” LEC junctions, compared to control LECs with “button-like” junctions. We introduced 70kDa dextran into the LV lumens and showed PDAC-induced zippering of junctions decreased vessel permeability. These results led us to hypothesize that altered LEC junctions may affect fluid drainage into the LVs in vivo. We inoculated PDAC cells into the mouse ear pinnae and performed whole-mount immunostaining and lymphatic drainage experiment by injecting Evans blue into the ears and measured the remained Evans blue in the ears. In the ear experiments, inoculated PDAC cells affected host LVs with originally button-like junctions to become zipper-like and impede lymphatic drainage. To identify a potential signaling pathway that may be responsible for LEC junction zippering, we used phospho-kinase arrays and discovered that PDAC cells induced LECs to increase phosphorylation of GSK3-β, JNK kinases, and enhanced the total protein level of β-catenin, implying that PDAC cells turned on the WNT signaling in LECs. In preliminary screening, we treated LECs co-cultured with PDAC cells with WNT-C59, an WNT inhibitor that blocks secretion of all WNT ligands, which resulted in decreased number of LV sprouting and less contractile actin-cytoskeleton in LECs. It is critical to know which molecules were secreted by PDAC cells and influenced the WNT signaling in LECs. With that in mind, we plan to perform mass spectrometry with PDAC-conditioned media to identify PDAC-specific secretome; and run RNA-seq with LECs isolated from mouse ears with or without PDAC to investigate molecular basis of the WNT activation in LECs and LV remodeling. Finally, we plan to examine the egress of leukocytes from primary PDAC tumor site, T cell activation in tumor-dLNs, and PDAC tumor growth with or without WNT inhibition in Kaeda mice. The identity and function of PDAC-egressed immune cells, and those which stayed within the primary tumor will enhance our understanding of the roles of LVs in orchestrating anti-tumor immunity in PDAC. Citation Format: Anna Maria Kolarzyk, Conor Loy, Renhao Lu, Iwijn De Vlaminck, Deborah Fowell, Esak Lee. Investigating lymphatic vessel remodeling and anti-tumor immunity in pancreatic cancer using tumor-on-chip and mouse models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4614.