Fig 2: Positive expression of JunB in MM cells correlates with vessel density in an innovative 3D MM model and in patient-derived bone marrow biopsies.A–C Positive expression of JunB in MM cells correlates with bone marrow microvessel density in a 3D MM model. A Generation of a 3D MM microenvironment. KM-105 stroma cells were pre-seeded overnight onto poly-e-caprolactone scaffolds (PCLS). TetR-shJunB/MM.1S cells were then added in the presence or absence of doxycycline, transferred into the bioreactor together with fluorescent-tagged-HUVE cells (CellTracker™ Deep Red dye) co-cultured for up to 72 h in RPMI-1640 media with 2% FBS. B Representative Z-stack confocal images of GFP+ TetR-shJunB/MM.1S cells (green) together with stroma cells, alone, or together with fluorescence-tagged HUVE cells (red). Scale bars = 100 µm. C Quantification of DAPI-positive, GFP+ TetR-shJunB/MM.1S and fluorescence-tagged HUVE cells in Z-stack confocal images of the 3D cultures. Image processing and analysis was performed with FiJi ImageJ. All cells (DAPI stain, blue), TetR-shJunB/MM.1S cells (green), HUVECs (red); *p < 0.005. D-E Positive expression of JunB in MM cells correlates with vessel density in patient-derived bone marrow biopsies. D Hematoxylin and eosin (HE) (left panels), JunB (middle vertical panels), and CD31 (right panels) immunohistochemical staining of BM sections from three representative MM patients with low (negative) JunB expression and MVD (upper panel), intermediate JunB expression and MVD (middle panels), and high JunB expression and MVD (lower panels), respectively (40× magnification). Scale bars = 50 µm. The quantification was performed as described in “Materials and Methods”; results for each BM sample are listed in Supplementary Table 3. E The percentage of JunB-positive cells is shown as mean ± SD and is associated with increased BM MVD. p = 0.0036 using one-way ANOVA analysis followed by Tukey’s multiple comparisons test.