A co-culture system of macrophages with breast cancer tumoroids to study cell interactions and therapeutic responses

3D tumoroids have revolutionized in vitro/ex vivo cancer biology by recapitulating the complex diversity of tumors. While tumoroids provide new insights into cancer development and treatment response, several limitations remain. As the tumor microenvironment, especially the immune system, strongly influences tumor development, the absence of immune cells in tumoroids may lead to inappropriate conclusions. Macrophages, key players in tumor progression, are particularly challenging to integrate into the tumoroids. In this study, we established three optimized and standardized methods for co-culturing human macrophages with breast cancer tumoroids: a semi-liquid model and two matrix-embedded models tailored for specific applications. We then tracked interactions and macrophage infiltration in these systems using flow cytometry and light sheet microscopy and showed that macrophages influenced not only tumoroid molecular profiles but also chemotherapy response. This underscores the importance of increasing the complexity of 3D models to more accurately reflect in vivo conditions. [Display omitted] •Establishes three optimized breast cancer tumoroid-macrophage co-culture systems•Demonstrates physio-pathological relevance of the co-culture models•Co-culture impacts drug response profiles, emphasizing need to enhance model complexity•These 3D models can be tailored to address varied biological questions Cancer research has traditionally relied on 2D cell cultures and xenografts, failing to capture tumor complexity. Organoids, especially tumoroids derived from tumors, offer a promising alternative. However, current 3D models lack crucial immune cells, essential for tumor development and treatment response. Integrating immune cells into tumoroids is crucial to enhance their accuracy. We aimed to establish standardized methods for co-culturing human macrophages with breast cancer tumoroids, optimizing conditions to replicate macrophage-tumor interactions. Our study emphasizes the significance of macrophages in tumoroid drug responses, highlighting the necessity for more complex 3D models. 3D tumoroids replicate tumor diversity but lack immune cells. Raffo-Romero et al. devise three optimized co-culture methods for human macrophages and breast cancer tumoroids. By tracking the macrophage-tumoroid interactions, they show that macrophages influence tumor molecular profiles and chemotherapy response, emphasizing the need to enhance model complexity for accurate in vivo r