Modeling NRF2-Driven Anti-inflammatory, Tumor-promoting Macrophages In Vitro II

We employed 3D spheroid co-cultures to explore the paracrine and direct cell-contact effects of NRF2-driven macrophages on MC38 tumor growth. We seeded equal numbers of GFP+ MC38 cells and macrophages (WT or Keap1 KO) in ultra–low-adherence plates to form multicellular spheroids. Live-cell imaging showed that spheroids containing NRF2high macrophages reached significantly larger sizes over 120 hours compared to spheroids with WT macrophages. With multiplexed scRNA-seq of spheroids collected at 24 and 120 hours post-formation, we defined the directed effects of NRFhigh stress-TAMs on tumor cells. UMAP density projections visualize that the macrophage genotype did not affect tumor cell gene expression within the first 24 hours despite persistent NRF2-driven macrophage phenotypic diversity. However, by 120 hours, tumor cell transcriptomes diverged markedly, as visualized by an unequal shift in cell densities, which was much more pronounced in the spheroids containing Keap1 KO macrophages. Leiden clustering and subsequent cluster-based GSEA revealed that while most tumor cells co-cultured with wild-type macrophages remained in a proliferative state, those co-cultured with Keap1 KO macrophages transitioned into an epithelial-mesenchymal transition (EMT) state, indicative of high metastatic potential.