Immunomodulatory effects of TAM in macrophages
TAM has a 100-fold lower binding affinity for ERα as compared with estrogens (Clarke et al., 2003) and may be administered in specific clinical conditions at dosages that lead to micromolar concentrations of TAM in plasma and breast target tissue. Based on the results of Figures 1 and 2, we explored whether TAM elicits ERα-independent, immunoregulatory effects similar to 4HT. 10 μM TAM induced a 7-fold induction of Vegfα expression (Figure 3A), while lower concentrations were ineffective. Moreover, TAM antagonized E2 action when tested in a 100-fold excess (0.001 μM E2 + 1 μM 4HT). Importantly, additive effects were observed when 10 μM TAM was used in combination with 0.1 and 10 μM E2. TAM activity is not mediated by ERα, as it is still observed in ERαKO macrophages, where the additive effects to E2 were also not observed (see Figure 3B). Cell viability assays did not provide evidence for cytotoxic effects of TAM at any concentration used (See Supplementary Figure 1). These data show that pharmacological concentrations of TAM exert immune-regulatory effects through off-target, ERα-independent mechanism of action.