Fig. 3

Ontogeny of GBM-educated myeloid cells. A Representative Iba1 staining in PDOXs depicting myeloid cells in invasive (PDOX P8), intermediate (PDOX P3), and angiogenic (PDOX P13) tumor growth, normal nude brain, and GL261 tumor. Tumor core and tumor border zones are highlighted. Arrows indicate examples of ramified (green), hyper-ramified (red), and amoeboid (black) myeloid cells. Scale bar: 50 µm. Sections were co-stained with hematoxylin. See more examples in Additional file 2: Fig S1B. B Representative Iba1 staining representing myeloid cells in PDOX P3 at different stages of tumor growth. Inserts represent sections of the entire mouse brains. Sections were co-stained with hematoxylin to visualize tumor cell density. C UMAP projection of reference-based mapping of myeloid cells from TME of GBM PDOXs and GL261 tumors and respective normal brains. Three myeloid cell entities were identified: microglia (Mg), peripheral monocyte-derived cells (Mo), and border-associated macrophages (BAMs). Inserts show expression of marker genes: pan-myeloid: Itgam (CD11b), Mg: P2ry12, Mo: Ly6c2, BAMs: Mrc1 (CD206). The color gradient represents expression levels. D Proportions of myeloid cell subpopulations in nude mouse normal brain (Nu-NB), PDOXs (9 models), C57BL6/N mouse normal brain (BL6-NB), GL261 tumors (3 collection time points: early, middle, late). Statistical difference between PDOXs (n = 9) and GL261 (n = 3) was evaluated with two-tailed Student’s t test with Bonferroni correction (***p < 0.001). E Flow cytometry-based quantification of CD45⁺CD11b⁺Ly6G⁻Ly6C⁻CD206⁻ Mg, CD45⁺CD11b⁺Ly6G⁻Ly6C⁺CD206⁻ Mo and CD45⁺CD11b⁺Ly6G⁻Ly6C⁻CD206⁺ BAMs in the Nu-NB and PDOX P3, P8, and P13 TME. For PDOXs P3 and P13 invasive zone and distant normal brain areas were also collected (n ≥ 3 mice/condition, mean ± SEM, one-way ANOVA with Tukey's HSD correction, ****p < 0.0001, *p < 0.05). See gating strategy in Additional file 2: Fig S4D