Organotypic 3d Models To Characterize The Molecular Requirements For Nk And T-Cell Infiltration

Multiple studies have demonstrated that the presence of tumor infiltrating lymphocytes is strongly correlated with increased survival. However, the level of infiltration within a specific tumor entity and between various tumor types varies widely. Increasing the degree of lymphocyte infiltration in a tumor represents an innovative drug concept. Organotypic 3D models consisting of tumor spheroids, stromal fibroblasts and Natural Killer (NK) or T-cells could represent valuable models to appropriately model infiltration and establish novel therapeutic concepts in the area of immune-oncology. As a first step in the development of a model for lymphocyte infiltration, the cytotoxicity and infiltration of human NK cells in 3D tumor spheroids were analyzed. Different NK cell lines and primary NK cells had a toxic effect on 3D tumor spheroids upon co-culture. Cryosections of co-cultured tumor spheroids showed infiltrated NK cells inside tumor spheroids. Infiltrated NK cells recovered from tumor spheroids were characterized by flow cytometry. Results showed that a higher infiltration correlates with cytotoxicity on tumor spheroids and transcriptomes of infiltrated vs. non infiltrated NK cells differ substantially. IL-12 stimulation increased the killing effect of NK cells on tumor spheroids, inducing higher expression of Perforin, IFNγ and GranzymeB levels. 3D tumor spheroids were embedded in a Matrigel/Collagen mixture with fibroblasts and the effect of compounds on NK cell infiltration was monitored. It was demonstrated that not only IL-12 increased NK cell infiltration, but also several compounds. Moreover, encapsulated 3D models with different cell types (tumor cells, fibroblasts, NK or T-cells) were established at a bioreactor scale to mimic the in vivo situation of complex tumor tissues. We could demonstrate NK and T-cell infiltration into these encapsulated 3D tumor spheroids. These encapsulated model systems permit long-term cultures to study the effect of compounds and/or NK or T-cells on later stages of tumorigenesis. In conclusion, this study demonstrates that organotypic 3D models are a valuable tool for the analysis of the molecular mechanisms that regulate lymphocyte infiltration into tumors. Citation Format: Wolfgang Sommergruber, Annika Osswald, Viola Hedrich, Marta Majewska, Helmut Dolznig. Organotypic 3D models to characterize the molecular requirements for NK and T-cell infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1655. doi:10.1158/1538-7445.AM2017-1655