TAA-derived CCL2 maintained MBSC stemness. (A) MBSCs treated with CCL2 or PBS for 72 hours and immunostained for Ki-67 and Sox2 (n = 3). (B) MBSCs cultured in the media mixed 1:1 with supernatant of FBS-free TAA culture or PBS and immunostained for Ki-67 (n = 3). (C) Up, expression of Sox2 in MBSCs expressing shCCR2#2 or scrambled shRNA in presence of CCL2 (n = 3). Down, expression of Sox2 in MBSCs co-cultured with CCL2-deficient or CCL2-normal TAA (n = 3). (D) Immunostaining for Sox2 and GFP in the co-culture of MBSCs and shCCL2-GFP expressing TAA after treatment with CCL2 (n = 4). (E) Sox2, Pax6, and Nestin mRNA levels in MBSCs cultured in the media mixed 1:1 with supernatant of FBS-free TAA culture or PBS (n = 3). (F) Immunostaining for Ki-67 and NeuN in disseminated MB slices cultured with or without PF6309 (n = 3). (G) Growth rate and tumor size of subcutaneous co-transplantation of MBSCs and TAA infected with shCCL2#3-GFP, shCCL2#5-GFP, or scrambled control (n = 6/group). (H) Immunostaining for Ki-67, NeuN, and GFP in subcutaneous xenografts (n = 6). Scale bars, 10 μm.