(A) Effects of GDNF alone (right panel) or in association with a fixed concentration (10% vol/vol) of conditioned medium derived from stromal cells exposed to PI-PLC (PI-PLC/CM; left panel) on clonogenic growth of blast cells derived from 6 RET-expressing AML samples. T-cell–depleted leukemic blasts (1.0 × 105) were resuspended in 1 mL of IMDM containing 20% FCS and 0.8% methylcellulose and cultured in 100-μL aliquots in 96-well flat-bottomed microplates in the presence of increasing concentrations (0.5 to 10 ng/mL) of GDNF. Freshly prepared BMSC-derived PI-PLC/CM was added to semisolid medium (10% vol/vol) immediately before plating. After 7 days of incubation, aggregates with ≥40 cells were scored as colonies. Results are expressed as mean ± SEM of 6 to 8 replicates. (B) Fluorescence histograms showing the expression of membrane-associated GDNF/soluble GDNF receptors complexes by primary RET-expressing AML cells. Cells were exposed for 1 hour at 37°C to GDNF alone (100 μg/mL) or to a combination of GDNF and BMSC-derived PI-PLC/CM and then sequentially with chicken polyclonal antihuman GDNF (100 μg/mL) and rabbit fluorescein isothiocyanate-conjugated isotype-matched antichicken Igs. As controls, isotype-matched irrelevant chicken Igs were used.
