(H-316) In vitro characterization of macrophage phenotype in a spheroid tumor model of melanoma

Introduction: Macrophage-fibroblast interactions–mediated by cell-cell contact and secreted signals–are critical for maintaining tissue homeostasis. During tumor progression, cancer cells alter homeostatic networks between macrophages and fibroblasts, causing macrophages to transition from an anti-tumor, inflammatory phenotype to a pro-tumor, immunosuppressive phenotype. Understanding how macrophages evolve within the tumor will support development of targeted immunotherapies to reestablish an effective immune response. To enable studies of macrophage phenotypic evolution in the tumor microenvironment (TME), we engineered a 3D in vitro spheroid tumor model containing macrophages, fibroblasts, and cancer cells. Spheroid models have been shown to recapitulate the physical properties of tumors, including direct cell-cell interactions and spatial orientation of cells. Therefore, we hypothesized that tumor spheroids would be able to recapitulate tumor-associated macrophage (TAM) evolution in the TME.


Materials and Methods: We constructed a 3D in vitro spheroid tumor model of the melanoma TME using the immunogenic YUMMER1.7 murine melanoma tumor cell line, bone marrow-derived macrophages, and fibroblasts (3T3MEF). To form the tumor spheroids, cells were deposited onto an agarose-coated well and centrifuged to gather cells together (1). After 3 days in culture, the cells formed a spheroid. The spheroids were removed from the agarose-coated wells and embedded into a 1.5mg/ml collagen I gel droplet (Fig 1A). The spheroids were kept in culture for up to 6 days, and spheroid growth was tracked by fluorescent imaging (Fig 1B) and flow cytometry. We further performed phenotypic profiling of the macrophages with and without treatment and compared to in vivo murine melanoma tumors.


Results, Conclusions, and Discussions: We first evaluated spheroid growth and cell type proportions in the spheroid. By day 4, spheroids approximately doubled in size (Fig 1B), and proportions of cancer cells, fibroblasts, and macrophages were representative of in vivo melanoma tumors. We next evaluated the phenotypic evolution of the macrophages in the tumor spheroids by measuring expression of iNOS (associated with anti-tumor inflammation) and Arg1 (associated with pro-tumor immunosuppression) (Fig 1C). On day 0, few macrophages expressed Arg1 or iNOS; however, by day 6 there was a significant increase in Arg1+ macrophages, with a small population of iNOS⁺Arg1⁺ cells, reminiscent of in vivo melanoma YUMMER1.7 tumors. Agonistic CD40 (CD40ag), a macrophage-targeted immunotherapy, is being tested as a way to reprogram immunosuppressive macrophages in melanoma (2). Therefore, we next explored how CD40ag altered macrophage phenotype in the tumor spheroids (Fig 1D). Overall, we conclude that this in vitro model of the melanoma tumor enables investigation of TAM function during the early establishment of the immunosuppressive TME and during rescue of TAM anti-tumor function with macrophage-targeted treatment.


Acknowledgements (Optional): This project was funded by the NIH/NCI Cancer Systems Biology Consortium (U01CA238728) and by the Cancer Research Institute Technology Impact Award (CRI4253).


References (Optional): (1) Pizzurro, G. Cancers, 2021. (2) Weiss, S. Clinical Cancer Research, 2021.