Fig. 4.

GPNMB released by macrophages activates a stemness program in the MCA-1-mock cells. a Number of formed spheres in MCA-1-mock cells alone (black) or exposed to the CM from macrophages of DBA/2J/Gpnmb⁺ mice (CM-macro GPNMB, green) or DBA/2J mice (CM-macro control, blue) is shown. In addition, the number of spheres formed by MCA-1-mock cells exposed to murine recombinant GPNMB (gray) and by transduced MCA-1-GPNMB cells (turquoise) is shown. Results are expressed as the fold increase versus the number of initial cells seeded. Mean ± SD of three different experiments. b Flow cytometry analysis of stemness-related markers (CD199, CD117 and Sca1) in the MCA-1-Mock or MCA-1-GPNMB cells (black, red), in spheres derived from the MCA-1-GPNMB cells (turquoise) and in the MCA-1-mock cells treated with the GPNMB-containing CM from macrophages (green). (Mean ± SD six different experiments). c mRNA levels of stemness-related transcription factors in MCA-1-mock cells, MCA-1-GPNMB cells and their derived spheres. d Tumor growth of the MCA-1-GPNMB or MCA-1-mock cells injected i.m. (10⁵) into DBA/2J mice. e Number of spontaneous lung metastases; each dot represents an individual mouse. Results are from one representative experiment of two perfomed with similar results. f mRNA quantification of the indicated transcription factors in the tumors formed by the MCA-1-mock and MCA-1-GPNMB cells grown in DBA/2J mice (5 mice/group). Data are expressed as fold increase relative to the MCA-1-mock tumors. All samples were tested in triplicate. g Tumor growth of MCA-1-mock cells, MCA-1-GPNMB cells and their derived spheres injected i.m. (10²) into NSG mice. h Number of spontaneous lung metastasis. Each dot represents an individual mouse (pooled from two experiments). Statistical analysis: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (one-way ANOVA, b–d and f–g multiple t-test, e and h unpaired t-test with the Welch’s correction). Data are presented as mean ± SEM