(A) Spheroid GFP fluorescence (magenta) and annexin V (cyan). (B) Integrated fluorescence across the spheroid area. Data are mean ± 95% CI of 42 replicates. (C) Spheroids were grown in microwell plates for scRNA-Seq experiments and GFP fluorescence across the spheroid area was quantified for ≥3,000 spheroids per condition and used for cell density correction in F (gray, MC38; red, MC38+heme-TAMs) (ANOVA with Tukey-Kramer post-test corrected with P 0.001 for all comparisons, except MC38 day 8 vs. MC38 day 10, P = 0.99). (D) Multiplexed scRNA-Seq of MC38 tumor cell spheroids (only MC38) or mixed-cell-type spheroids (MC38 + heme-TAM) on days 4, 8, and 10 after formation. After macrophage exclusion, cell densities were scaled by the mean spheroid size before projecting on the UMAP. (E) Leiden clustering defined 3 clusters per experiment, a dominant functional annotation for each cluster was determined by GSEA. Dot plots depict the fraction of tumor cells within each functional state per time point. (F) GFP-MC38 spheroids (top) and mixed GFP-MC38+heme-TAM-tomato spheroids (bottom) were transferred from microwell plates to a flat glass-bottom plate on day 4 after spheroid formation and embedded into an extracellular matrix. Cell invasion was imaged 24 hours later. (G) Four days after formation, spheroids were embedded into an extracellular matrix (t = 0 hours), and cell invasion was measured by live-cell imaging every 4 hours. Top: Representative inverted bright-field images. Scale bar: 0.5 mm. Bottom: The invading cell front was automatically segmented and quantified over time. Data are mean ± 95% CI of 21 replicates analyzed within 1 representative experiment. (H) Spheroids were collected on day 5 after formation and injected i.v. into C57BL/6J mice. Lungs were collected 24 days after injection. t test. Scale bar: 5 mm.