In vitro treatment with TRAIL delays engraftment of pre-B ALL xenograft cells. Xenograft ALL-177 cells freshly isolated from the spleen of a donor mouse were treated in vitro with vehicle or TRAIL (1000 ng/mL) for 48 hours. Cells were serially diluted (range, 1 × 10⁶ to 3 × 10³) and injected into a total of 39 mice. (A-B) Half of the 39 mice (13 mice in the vehicle group, 10 mice in the TRAIL group, raw data are shown in supplemental Table 2) were killed 15 weeks after transplantation to evaluate dose-response. (A) Representative spleen sizes (left); immunohistochemistry of spleens stained for cells expressing human CD10 (middle; original magnification ×200); and bone marrow infiltration by human leukemia cells using flow cytometric analysis of human CD38 (right); numbers on top of the panels indicate amount of human leukemia cells injected per mouse. (B) Leukemic infiltration in bone marrow (BM) and spleen for all mice, as estimated by flow cytometric analysis of human CD38; each mouse is represented by a single dot. (C) From analysis of bone marrow and spleen, engraftment was considered positive if ≥ 1% human cells were detected in at least 1 organ (raw data listed in supplemental Table 2). LIC frequencies were calculated using Poisson statistics. Similar experiments were performed for ALL-54 and ALL-169 (supplemental Figure 3; raw data listed in supplemental Table 2). Decrease in LIC frequency by TRAIL treatment was calculated and indicated above bars. **P < .01 (Student t test). ***P < .001 (Student t test). Filled bars represent late mouse passage (passage 3 in ALL-54 and passage 4 in ALL-177); and bars with dots, early passage (cells that have been collected from a mouse injected with the primary sample). (D-E) To evaluate the kinetics of leukemic growth, the second half of the 39 animals transplanted with ALL-177 cells were observed over time (n = 8 mice per group; supplemental Table 4). Appearance of leukemic cells in peripheral blood (PB) was the readout, which was regularly determined by flow cytometry detecting human CD38. (D) Kinetics in 6 representative mice injected with 3 different cell numbers, for TRAIL and vehicle. (E) The relation between time to engraftment/time to death and numbers of leukemia cells injected per mouse; each mouse is represented by a single dot. Leukemic engraftment was defined as ≥ 1% leukemia cells in PB; overt leukemia/time to death was defined as ≥ 35% leukemia cells in PB. p.i. indicates postinjection. Regression curves and equations were calculated thereof and are indicated.