Abstract 7233: An in vivo co-injection model for studying macrophage polarization and therapeutic repolarization in tumor development

Abstract Macrophages play a critical role in tumor development by shaping the tumor microenvironment. In particular, the M2-polarized macrophage phenotype is associated with pro-tumoral functions such as promoting angiogenesis, immune suppression, and tumor progression. Consequently, numerous therapeutic approaches aim to repolarize M2 macrophages to mitigate their tumor-supportive behavior. While these effects are consistently and effectively demonstrated in vitro in our laboratory, translating these findings into in vivo models remains a significant challenge. Here, we present an in vivo co-injection model that allows the detailed study of macrophage-tumor cell interactions and the evaluation of potential therapeutic agents targeting macrophage polarization. First, we isolated monocytes from heathy donors and differentiated them in vitro into various phenotypes (M0, M1, and M2) according to our established protocols. We selected a tumor cell line that secretes high levels of macrophage survival promoting factors and has good in vivo growth characteristics. In this model we co-injected the tumor cells together with macrophages in NXG animals. Our results demonstrate that the co-injected M2 macrophages remains stable for over a week in the tumor microenvironment, closely mimicking the persistence observed in human tumors. Additionally, M0 macrophages undergo differentiation under the influence of the tumor microenvironment, reflecting the dynamic and plastic nature of macrophage polarization in vivo. Marker expression as well as functional properties, such as phagocytotic activity of pHrodo Zymosan particles ex vivo, was analyzed by flow cytometry. This model provides a biologically relevant system for studying macrophage-targeting therapies, complementing the robust and reproducible in vitro experiments routinely conducted in our laboratory. The presence of the M0 and M2 macrophages over a significant period of time enhance the model’s utility for evaluating substances designed to repolarize macrophages in vivo. In conclusion, this in vivo co-injection model represents a valuable tool for preclinical research into macrophage polarization and its impact on tumor progression. Its potential ability to replicate key aspects of macrophage-tumor cell interactions enables comprehensive investigations of therapeutic strategies targeting the tumor microenvironment. This approach has the potential to accelerate the development of novel treatments aimed at disrupting the pro-tumoral functions of M2 macrophages and enhancing anti-tumoral immune responses. Citation Format: Philipp Metzger, Valerie Oberhardt, Carla N. Castro, Holger Weber. An in vivo co-injection model for studying macrophage polarization and therapeutic repolarization in tumor development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7233.