TNT signaling between BCP-ALL cells and MSCs. (A) Representative confocal images (Z-stack) showing TNT networks (white arrowheads) between BCP-ALL cell line REH (DiI, yellow) and hTERT-immortalized MSCs (DiO, green) after coculture for 3 hours. Bidirectional exchange of lipophilic dye via TNTs was observed (green arrow for MSC to ALL, yellow arrow for ALL to MSC). Leukemic cells also formed TNT-like structures toward the fibronectin-coated substratum (orange arrowhead). (B) Graph showing quantification of dye transfer from DiI-stained NALM6 cells toward unstained hTERT-MSCs (cultured in 4:1 ratio) in time. Figure shows representative experiment (n = 3). (C) Graph showing quantification of dye transfer after 24 hours of coculture (cultured in 1:1 ratio). Left panel, Dye transfer from DiI-stained hTERT-MSCs toward unstained NALM6 cells. Right panel, The reciprocal experiment (also performed in a 1:1 ratio). White and gray histograms represent staining intensity at the start of each experiment. (D) Quantification of dye transfer in time of experiment as exemplified in panel C, performed with 2 different BCP-ALL cell lines (REH and NALM6). Dye transfer from MSCs toward ALL was compared with dye transfer from ALL toward MSCs (n = 4; 2-tailed t test, unpaired). (E) Graph showing quantification of dye transfer from DiI-stained NALM6 cells toward unstained hTERT-MSCs with (red histograms) or without (gray histograms) TNT inhibition. Cells were cocultured in 4:1 ratio for 6 hours. Three independent TNT inhibiting conditions were used: actin inhibition by cytochalasin D or latrunculin B, physical disruption by gentle shaking, or culture in a 3.0-μm transwell system. (F) Quantification of dye transfer experiment exemplified in panel E, performed with 2 different BCP-ALL cell lines (REH and NALM6) (n = 4; 1-tailed t test, unpaired). Data are means ± SEM; **P ≤ .01, ***P ≤ .001 (see also supplemental Figures 1 and 3, and supplemental Videos 1A-B and 2).