Role of CD44v6 in AML and MM cell growth in vivo. (A) Leukemic blasts from AML patients (n = 25, see supplemental Table 1) and (B) malignant plasma cells from MM patients (n = 15, see supplemental Table 2) were analyzed by FACS. Leukemic blasts were grouped according to their French-American-British subtype (M0-M3, n = 6; M4-M5, n = 12; secondary AML, n = 7). Left: CD44v6 expression from representative cases. Right: CD44v6 RFI (see “Material and methods”) from each case. The dashed line represents the threshold arbitrarily defining CD44v6 positivity (RFI = 2). (C) Tumor cells were transduced with an LV encoding for a CD44v6-specific shRNA sequence (see supplemental Figure 1C) and tested for selective silencing by FACS after gating for the mOrange marker gene (see “Material and methods”). Upper panels: CD44v6 expression on wild-type THP-1 leukemia cells (wt) or on THP-1 cells transduced with the shRNA or with a control vector (CTR). Lower panels: CD44 expression in the same conditions. (D) THP-1 cells and (E) MM1.S myeloma cells were transduced with the shRNA or with the CTR vector and infused intravenously in NSG mice. A group of control mice were infused with wt tumor cells. After 4 weeks, mice were sacrificed and analyzed for engraftment in different organs. Left: percentages of CD33⁺/mOrange⁺ THP-1 cells in the liver or of CD38⁺/mOrange⁺ MM1.S cells in the BM of representative mice. Right: weights of THP1-infiltrated livers or percentages of BM-engrafted MM1.S cells from each mouse. The dashed band represents the normal range of mouse liver weight. Results from a 1-way ANOVA are shown when statistically significant (*P < .05; **P < .01). (F) Leukemic blasts from patient AML#4 (see supplemental Table 1) were transduced with the shRNA or with the CTR vector, infused intravenously in NSG mice, and followed in the peripheral circulation by FACS. Left: percentages of circulating mOrange⁺ leukemic blasts (mean ± SD from n = 5 mice per condition) at different time points. Right: percentages of mOrange⁺ leukemic blasts in the BM from each mouse at 8 weeks. Results from unpaired Student t test are shown for each time point (**P < .01; ***P < .001).