Abstract 3541: A method to visualize and quantify effects of immunotherapies in a physiologically relevant 3D TME model
Authors: Daszkiewicz Lidia , Goverse Gera , Beztsinna Nataliia , de Man Saskia , Veenendaal Tomas , Klop Michelle , Okkes Daniel , Weterings Ashgard , Yan Kuan , Price Leo 
Published: June 2022

Abstract
INTRODUCTION: In vitro assays based on 3D co-cultures of cancer and immune cells present an opportunity to test diverse immunotherapies and understand their mechanisms of action in boosting anti-tumor immune responses in cancer patients. Incorporation of diverse cellular players, including cancer associated fibroblasts (CAFs) and suppressive myeloid cell populations, allows for faithful recapitulation of complex cellular interactions that occur in the tumor micro-environment (TME). The application of automation results in a high throughput platform which combines physiological relevance with assay robustness and reproducibility. Functional read-outs, such as migration of immune cells towards tumoroids, infiltration into the tumoroids and their killing are obtained by applying high content imaging, allowing for better understanding of the immune-modulatory profile of IO drugs. Here, we have applied image analysis to quantify the effects of different antibodies and small molecules targeting both the tumor and the T cells.
MATERIAL and METHODS: Tumoroids generated from breast and colon cancer organoids and cell lines were cultured in protein hydrogel. Partially HLA-matching T cells isolated from healthy PBMCs donors were added with or without activation to the 3D culture after incorporation of different suppressive populations, including CAFs and M2 macrophages. The capacity of effector T cells to infiltrate and subsequently kill tumor structures was visualized using high-content microscopy and quantified with morphometric image analysis software after reconstitution of 3D image stacks.
RESULTS and DISCUSSION: Automated 3D image and data analysis enabled discrimination of cellular interactions in a complex TME model. Depending on the activation status of T cells and the presence of suppressive cells in the environment, different levels of infiltration and tumor killing by effector T cells were measured. The suppression induced on the T cells by the environment was partially reversed by immune-modulators (e.g. CSF1R inhibitor) added to the co-culture, which was confirmed by increased IFNγ levels in culture supernatants. Two quantitative read outs that are strictly dependent on the 3D environment; T cell infiltration into tumor and decrease in tumor volume were combined to demonstrate immune suppression release induced by different immuno-modulators.
CONCLUSION: Our image-based platform described here allows for analysis of immunotherapy effects on cells that engage in a physiologically relevant spatial setting and in the presence of suppressive TME elements. Visualization and quantification of these complex cellular interactions within the TME empowers immunotherapy drug developers with a highly powerful tool to select the most promising candidates and better understand their mechanism of action, which ultimately will have an impact in the clinic.
Citation Format: Lidia Daszkiewicz, Gera Goverse, Nataliia Beztsinna, Saskia de Man, Tomas Veenendaal, Michelle Klop, Daniel Okkes, Ashgard Weterings, Kuan Yan, Leo Price. A method to visualize and quantify effects of immunotherapies in a physiologically relevant 3D TME model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3541.