Figure 5

Figure 5

Angiogenic potential of malignant 3D multicellular spheroids. (A, B) RT-qPCR analysis of endothelial cell markers gene expression such as VEGF (A) and GATA2 (B) in BMT, AMT, and HMT spheroids on day 0, day 7, and day 10. (C, D) Flow cytometric analysis (C) and quantification (D) of VE-cadherin+ cells in BMT, AMT, and HMT spheroids on day 10. (E) RT-qPCR analysis of a-SMA, FSP, CD68, CD163, and CD206 mRNA levels in CD144+ cells isolated from BMT, AMT, and HMT spheroids on day 14. (F, G) RT-qPCR analysis of endothelial cell markers (VE-cadherin, VEGF, vWF, Endoglin, and CD31) (F) and angiogenic regulators (ZEB1, FLI1, GATA2, Etv2, Ets, and Tie2) gene expression (G) in THP-1 macrophages and CD68+ cells, respectively, isolated from BMT, AMT, and HMT spheroids on day 14h. (H). Representative images of BMT, AMT, and HMT spheroids on day 21 after placing the spheroid of day 14 on a collagen matrix and maintained in endothelial cell media for 7 days. Cell sprouting (red arrow) were detected in tumor spheroids. (I) RT-qPCR analysis of eNOS gene expression in CD68+ cells of these spheroids on day 21. GAPDH was used as loading control for RT-qPCR analysis. Fold change for CD144+ cells was calculated keeping the 2Dcells(control)at day 0as control and fold change for CD68+ cells was calculated keeping THP-1 monocytes (2D) as control. All experiments were performed in triplicates (n = 3). Data represented as mean ± S.D. 2D vs BMT over time *p< 0.05, **p< 0.01, ***p< 0.001; BMT vs AMT over time ^##p< 0.01, ^###p< 0.001; BMT vs HMT over time ^§p< 0.05, ^§§p< 0.01, ^§§§p< 0.001; ns, non-significant.