Capturing Cell Dynamics in Live Pancreatic Adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive and lethal cancers and is associated with a robust fibroinflammatory stromal response termed desmoplastic reaction. This stromal response causes the local microenvironment to significantly aid disease progression by providing drug free sanctuaries, immunosuppressive niches, and suppressing cytotoxic T lymphocyte infiltration and distribution, due in part to the increased intra-tumoral pressure and robust extracellular matrix proteins (ECM) density. In order for CD8+ cytotoxic T cells to infiltrate and eliminate cancer cells, they need to migrate efficiently through the dense tumor microenvironment (TME). Thus, altering external (ECM content/architecture) and internal (modulating microtubule (MT) dynamics in immune cell) factors has the potential to enhance efficient infiltration of native or engineered cytotoxic T lymphocytes so they effectively sample the tumor volume to combat disease. Therefore, to analyze the infiltration capabilities in a dense tumor environment, we optimized an approach to culture live tumor slices over 1-4 days in order to perform live cell imaging of carcinoma and immune cell dynamics in complex TMEs with nonlinear optical imaging platforms. From human peripheral blood or tumor-bearing mouse model of PDA, CD4+ or CD8+ cytotoxic T lymphocytes, respectively, were isolated, activated, labeled and later introduced to 3D collagen matrices and live murine PDA tumor slice explants, which has a complex multi-cellular environment and contains elements of the original TME and architecture. Furthermore, we used CRISPR technology to engineer T cells to lack GEF-H1 and alter MT→GEF-H1→RhoA pathway to determine its effect on cell motility. We employed two-photon excitation and second harmonic generation (SHG) imaging to visualize cell dynamics and ECM architecture, and quantify T cell migration behavior through 3D collagen matrices and the native PDA tumor architectures. To test approaches to re-engineer TMEs, we are specifically altering ECM composition and architecture in PDA and quantifying changes in T cell behavior. Thus, combined, these live measures and quantitative analysis will form the basis for our understanding of cell migration in the complex microenvironment and set the mark for our objective to modulate immunity in tumors.