Figure 7. CAFs induce GBM tumor growth intracranially in vivo.

(A) Kaplan-Meier curve showing intracranial implantation of 3.5 × 10⁴ GBM6 neurospheres with 5 × 10³ GBMpt3CAFs reduced survival compared with mice receiving 4.0 × 10⁴ GBM6 neurospheres, a threshold not associated with tumor formation in most mice (n = 10/group; P = 0.03). Compared with mice receiving 10⁵ GBM6 cells in neurospheres (higher number used to generate tumors), intracranial implantation of 3.5 × 10⁴ GBM6 neurospheres with 5 × 10³ CAFs upregulated cancer progression genes as determined by NanoString nCounter multiplex analysis using a PanCancer progression codeset, and as seen by (B) volcano plot showing significantly (P < 0.05) up- (to the right of rightmost vertical dashed line) and downregulated genes (to the left of leftmost vertical dashed line). (C) Heatmap showing significantly (P < 0.05) up- and downregulated genes constructed based on the log₂ (fold change) and significant P adjusted value. (D) Pathway analysis showing that CAFs upregulated HIF-1 signaling, EMT, and cell proliferation pathways in GBM6 tumors (P < 0.003). (E) Immunofluorescence images (×20 magnification) showing increased vasculature, labeled via rhodamine B-dextran perfusion in sections from mice with GBM6+CAFs, quantified by (F) total vessel area/hpf (P = 0.02); P = 0.0002) (3 mice/group; 8 fields/mouse; t test). (G) CAFs increased the percentage of macrophages that were CD206⁺ M2 protumoral macrophages in GBM6 neurosphere-derived tumors (P = 0.0096; t test). Scale bar: 20 μM. *P < 0.05; **P < 0.01; ***P < 0.001.