ABSTRACT: To determine how macrophage states dictate cancer cell phenotype dynamics, we conducted a time-resolved, multiplexed scRNA-seq experiment of mixed cell spheroids composed of M-CSF macrophages and MC38 cancer cells. We collected the spheroids on days 1 and 5 after formation and processed approximately 9000 per condition. After demultiplexing and quality control, 178939 cells were retained and visualised by UMAP, recalculated separately for macrophages and tumor cells. We found that the macrophages occupy distinct transcriptional spaces on day 1, which reflect their prior cytokine imprinting. By day 5, macrophages converge onto a single “tumor-educated” cluster, indicating that paracrine cues from tumor cells override their original states and impose a common transcriptional programme. Macrophage centroid dispersion decreased, confirming transcriptional convergence. In contrast, transcriptional divergence of tumor cells increased between day 1 and 5. To define the macrophage state-dependent effects on tumor cells, we pre‑defined three transcriptomic modules—proliferation, epithelial‑to‑mesenchymal transition (EMT), and stress/hypoxia—based on our previous single‑cell atlas of MC38 spheroids (Schaer et al. 2023; Schaer et al. 2025). We then calculated a score for each module per tumor cell. On day 1, nearly all tumour cells scored as proliferative (high G2M_checkpoin score) regardless of macrophage polarization. By day 5, the score distributions diverged. Spheroids containing unpolarized and IFN‑γ macrophages remained proliferative, those with IL‑4‑ or TGF‑β macrophages lost their proliferation signature and shifted markedly toward EMT, while tumor cells in the absence of macrophages acquired a strong hypoxia signature. Spheroids with IL-10 polarized macrophages induced an intermediate pattern. Hence, anti‑inflammatory macrophages preferentially steer MC38 cells toward an EMT‑like, low‑proliferation, pro‑invasive state, whereas pro‑inflammatory IFN‑γ macrophages keep tumor cells locked in a proliferative programme. Simultaneously, the tumor microenvironment erases macrophage-of‑origin signatures, illustrating rapid, bidirectional cross-talk and plasticity within the 3D niche.
