Abstract 5697: Image based quantification of myeloid cell repolarization and their interplay with the tumor microenvironment
Authors: Goverse Gera , Beztsinna Nataliia , Visser Benjamin , Spanjaard Emma , Yan Kuan , Price Leo , Daszkiewicz Lidia 
Published: September 2020

Abstract
BACKGROUND
The myeloid cell compartment plays an important role in anti-tumor immune responses and represents a heterogeneous population with both cancer-promoting and cancer-restraining actions. Unleashing the full potential of cancer immunotherapies requires an understanding of the cellular mechanisms that govern these opposite actions. We previously described a high-content screening platform to assess effects of immune modulators on the infiltration and killing of tumor organoids. Here, we incorporated the myeloid cell compartment to have a better representation of the human immune system in the tumor microenvironment (TME). In-house developed software was trained on a set of features that enabled discrimination between undifferentiated monocytes and polarized M1 and M2 macrophages and dendritic cells in 3D. This assay is well suitable to test cancer immunotherapies that target multiple cell types involved in anti-tumor immune responses and to understand in general the suppressive tumor environment.
MATERIAL and METHODS
Different myeloid populations were generated in 3D from monocytes derived from healthy donor PBMCs. Polarized M1 and M2 macrophages, DCs and undifferentiated monocytes were then co-cultured with either tumor conditioned media or spheroids derived from different cancer cell lines or colon rectal cancer organoids, growing in protein hydrogel. The cellular interactions were visualized using high-content microscopy and quantified with multiparametric morphometric analysis with OMinerTM software.
RESULTS
3D imaging and phenotypic analysis was used to classify different myeloid cell populations. This classification was confirmed functionally by profiling of released cytokines. In addition, our analysis revealed the different effects of tumor cells and the immunosuppressive microenvironment that they generated on the myeloid cells phenotypes. We also used this approach to demonstrate a repolarisation of M2 type macrophages into M1 type macrophages upon treatment with CSF1R inhibitor and a preventive effect of this compound on macrophage skewing induced by conditioned media from tumor cells of different origin.
CONCLUSIONS
The assay presented here enables visualization and quantification of effects of immunotherapies on myeloid cells using 3D phenotypic analysis. This co-culture system provides means to elucidate the bi-directional interplay between tumor and immune cells, allowing for analysis of functional reprograming of the suppressive population towards a M1 phenotype induced by drug candidates. This advanced platform for testing cancer immunotherapies also combines the complexity of the TME with the robustness of a high throughput screening platform.
Citation Format: Gera Goverse, Nataliia Beztsinna, Benjamin Visser, Emma Spanjaard, Kuan Yan, Leo Price, Lidia Daszkiewicz. Image based quantification of myeloid cell repolarization and their interplay with the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5697.