Inhibition of early steps of tumor cell bone colonization by circulating inactive ATX. (A) Detection of functionally active ATX and lysoPLD-deficient ATX-T209A expression in cell lysates and in conditioned media (CM) of CHO-dhfr⁺/ATX and CHO-dhfr⁺/ATX-T209A cells by immunoblotting using an anti-lysoPLD polyclonal antibody. (B) LysoPLD activity was quantified in cell culture CM in the presence or absence of BMP22 by fluorescence imaging on NightOwl using the ATX-Red compound (1 μM). Data are expressed as photon per second (Ph/s) over basal. Ctrl corresponds to parental CHO-dhfr⁺ cells. (C) BALB/c nude mice were treated with CM (300 μL/twice daily, i.p.) collected from transfected parental CHO-dhfr⁺ cells (Ctrl) or transfectants CHO-dhfr⁺/ATX (ATX) and CHO-dhfr⁺/ATX-T209A (ATX-T209A) cells 1 day before and the next 8 days after MDA-B02/luc cells injection (scale bar: 1 cm). Bone marrow cells were collected, cultured, and stained as described in Figure 5B (scale bar: 1 cm). (C) TCB colonies were counted. Results are expressed as mean of TCB (± SEM). Statistical analysis was performed using the nonparametric Mann-Whitney U test (***P < 0.05; vs vehicle-treated animals).