Figure 4. TAMs promote the growth of CSCs through activation of bone stromal cells.

(A) Primary human monocytes were treated with IL-4, IL-13 and CM of 231BoM for 7 days and analyzed by FACS. The morphology of the cells was also shown. (B) CSCs of 231BoM or 231BoM-shHAS2 were co-injected with (right tibia) or without (left tibia) TAMs in the same animals. They were treated with or without 4-MU for 30 days. The normalized bioluminescent values were represented. P-value was calculated by Wilcoxon rank sum test (n=12–17). (C) CM from co-culture of TAMs and CSCs were subjected to Growth factor antibody array analysis. The position of PDGF-BB is indicated by a red box. (D) TAMs were co-cultured with CSCs from 231BoM or 231BoM-shHAS2 for 2 days. TAMs were then sorted by FACS, and the expression of PDGFB gene was quantified by qRT-PCR (n=3). (E) Immunofluorescent image of co-culture of TAMs with CSCs of 231BoM. (F) CSCs of 231BoM were treated with CM from various stromal cells that were pretreated with or without PDGF-BB (100 ng/ml) followed by measuring the growth of CSCs by the MTS assay (n=6). (G) CM from HS5, hFOB1.19 and BM-hMSC treated with 100 ng/ml of PDGF-BB were individually subjected to Cytokine antibody array analysis. (H) Growth rate of CSCs of 231BoM treated with FGF9 or FGF7 and a combination with FGFR inhibitor (PD173074) was measured by the MTS assay (n=3). (I) CSC population of 231BoM cells by the same treatment as described in (H) was measured by FACS (n=3). (J) Sphere formation in suspension culture of MCF7-BoM2d cells was measured as the average number of spheres per 500 cells and the results were plotted (n=6). (K) CSCs of 231BoM were co-injected with TAMs/sh-scramble (left tibia) or TAMs/shPDGFB (right tibia) in the same animals (n=5). (L) CSCs of 231BoM were co-injected with BM-hMSC/sh-scramble (left tibia) or BM-hMSC/shFGF7&shFGF9 (right tibia) in the same animals (n=5). The normalized bioluminescent values were represented.