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. 2014 Sep 16;5(23):12027–12042. doi: 10.18632/oncotarget.2482

Figure 3. TAMs stimulate melanosphere formation.

Figure 3

Data for all bar graphs are expressed as fold change in sphere-forming efficiency relative to untreated CD34− TICs. Bars represent mean ± SE, * P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001, N.S. not significant, 1-way ANOVA for B and D to F, 2-way ANOVA for C. (A) Flow cytometry sorting of immune cell populations. The Lin+ gate contain T, B and NK cells. Lin− cells are separated into CD11b+CD68− myeloid cells and CD11b+CD68+ TAMs. (B) Effect of immune cell populations on sphere formation: CD34− TICs were cultured with the immune cell populations sorted in A. (C) Effect of TAM inhibition on sphere formation: CD34− TICs were cultured with or without TAMs. Two CSF1R inhibitors Ki20227 (Ki; 100nM) and CD115-neutralizing antibody (α-CD115; 1μg/ml) were added for the duration of the culture. (D) Effect of BMDM on sphere formation: BMDM from tumor-bearing mice (RET+) and non-tumor bearing littermates (ret−) were polarized to M1 or M2 phenotype and co-cultured with CD34− TICs. (E) Effect of conditioned medium (CM) on sphere formation: CM generated from M1- or M2-polarized BMDM were added to CD34− TICs at 1/10th of stem cell media volume. (F) Effect of CSF1R inhibitors on the stimulatory effect of M2-BMDM CM (M2-CM): Ki20227 (100nM) and anti-CD115 (1μg/ml) were either added to the BMDM prior to CM generation (Ki20227 > CM, α-CD115 > CM) or added to the CM during sphere culture (CM+Ki20227, CM+α-CD115).