Blocking Axl activation inhibits FLT3-ITD⁺ AML in vivo. (A) SCID mice were injected subcutaneously with cells from the MV4;11 human FLT3-ITD⁺ AML cell line (5 × 10⁶ cells per mouse), and 10 days later treatment of the mice with PBS (n = 7), Ctrl-Fc (n = 7), or Axl-Fc (n = 10) was initiated as described in “Materials and methods,” followed by sacrifice and tumor measurement. Each filled circle indicates the weight of tumor mass from each mouse and the line shows the mean tumor weight of each experimental group. **P < .01, ***P < .001. (B) Irradiated SCID mice received an intravenous injection of primary human FLT3-ITD⁺ AML blasts (2 × 10⁷ cells per mouse). Four weeks later, thrice-weekly intraperitoneal administration of Ctrl-Fc (n = 4) or Axl-Fc (n = 5) was started. Eight weeks after injection of AML blasts, WBCs (left) and the percentage of human CD45⁺ cells (right) in peripheral blood were quantified. Each filled circle represents the data from each mouse and the line shows the mean of each experimental group. ***P < .001. (C) Irradiated SCID mice were intravenously injected with primary FLT3-ITD⁺ AML blasts from a patient (2 × 10⁷ cells per mouse; different from the patient sample used in panel B). Four weeks later, thrice-weekly administration of PBS (n = 5), PKC412 (n = 5), Axl-Fc (n = 5), or PKC412 plus Axl-Fc (n = 5) was started. Whereas PBS and Axl-Fc were administered with intraperitoneal injection, PKC412 was administered orally. Eight weeks after injection of AML blasts, the number of WBCs (top left) and the percentage of human CD45⁺ cells (top right) in peripheral blood were quantified. Each filled circle represents the data from each mouse and the line shows the mean of each experimental group. Kaplan-Meier plot of survival (bottom). Statistical significance was assessed using log-rank test (P = .02). Mice were sacrificed when they became moribund. The study ended at 90 days after the injection of primary patient FLT3-ITD⁺ AML cells into SCID mice. (D) At 90 days, all the mice still alive were killed and bone marrow was collected from each mouse and analyzed with fluorescence activated cell sorting to detect human CD45⁺ cells. Thin and thick lines indicate staining with isotype control antibody and anti–human CD45 antibody, respectively. The histograms shown are the representative of each experimental group.