Abstract 4558: High-throughput, high content Imaging assay for assessing solid tumor phagocytosis in 3D spheroid model
Authors: Rudnik Michal , Grining Özlem Yavaş , Rotankova Nadezhda , Strebel Silvan , Chiovaro Francesca , Frey Olivier , Agarkova Irina , Moritz Wolfgang 
Published: April 2023

Abstract
Emerging studies have demonstrated that innate immune checkpoints, regulating Tumor-associated Macrophage (TAM) phagocytic activity, play a crucial role in immune escape and survival of cancer cells. CD47-SIRPα axis is the best studied example of the “don’t eat me” signal, frequently overexpressed on cancer cell. Targeting of CD47 protein has shown impressive results in clinical trials in hematologic malignancies but remained challenging for solid tumors therapy. Therefore, tools facilitating the development of novel innate immune checkpoint inhibitors are of great interest. To address that unmet need, we established a high-throughput, 384-well-format assay for efficacy assessment of compounds inducing macrophage phagocytosis. As a model of solid tumor, we employed 3D spheroids aggregated of human cancer cell lines (A375, HCT116, MCF7, SKOV3) expressing GFP or PDX material (LXFA1647 lung adenocarcinoma, MEXF2106 melanoma) and cancer associated fibroblasts, as well as Monocyte-Derived Macrophages (MDMs) which altogether were co-aggregated to form spheroids mimicking the tumor microenvironment and immune-competence. In parallel MDMs were co-cultured with matured spheroid to emulate tumor infiltration. Both models were cultured in automation and High Content imaging compatible Akura™ 384-well plate. To evaluate our system, we targeted CD47-SIRPα axis by treatment of 3D spheroid models with αCD47 antibody (H6D12) in combination with atezolizumab (αPD-L1 antibody). Tumor viability and growth were assessed by fluorescence measurements and ATP level. Phagocytosis of fluorescently labeled tumor cells was imaged by confocal microscopy and quantified by inhouse developed analysis pipeline. Immune cell activation was evaluated by a cytokine bead array. The treatment of cell line-based 3D models with αCD47 antibody resulted in an increased number of phagocytic macrophages followed by a decrease of fluorescence and size of tumor spheroids in a dose-dependent manner. Moreover, we observed an induced secretion of TNFα, IL-6, IL-10 and IL-RA after 48h. Similarly, in PDX-based models, we observed decreased spheroid size and ATP level at day 7. Notably, αCD47 antibody-induced level of cytokines was further increased in combination with atezolizumab. To conclude, we have developed a human disease relevant, high-throughput assay for the evaluation of novel therapeutic modalities targeting phagocytosis. Due to compatibility with microscopy and most of the biochemical assays, it represents a powerful tool for clinical candidate development.
Citation Format: Michal Rudnik, Özlem Yavaş Grining, Nadezhda Rotankova, Silvan Strebel, Francesca Chiovaro, Olivier Frey, Irina Agarkova, Wolfgang Moritz. High-throughput, high content Imaging assay for assessing solid tumor phagocytosis in 3D spheroid model. [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 4558.