American Society of Hematology

Figure 1

$Figure 1. CML stem cells retain transplantation and oncogenic capacity following Bcr-Abl reversion. (A) Irradiated CD45.2+ mice (8 Gy) transplanted using unfractionated (uf) CD45.1+ BM cells isolated from SCLtTA/Bcr-Abl dtg or wt controls (n = 12/12). (i) Bcr-Abl expression in recipient mice was controlled by absence (expression) or presence (no expression) of tetracycline in drinking water. Bcr-Abl expression was induced after transplantation for 25 days and the phenotype analyzed in dtg and controls (n = 3/3) by FACS of BM and spleen cells, spleen weight and histology, ratio of donor:host LSK cells, and Bcr-Abl expression. (ii) Bcr-Abl expression was reverted for 48 days and a cohort of remaining mice (n = 4/4) analyzed. (iii) BM cells from reverted dtg and controls were retransplanted using CD45.1+ FACS-sorted cells (n = 5/5). Bcr-Abl expression was reinduced and the phenotype was assessed 69 days after retransplantation. (Bi-ii) PB was analyzed in CD45.1+ retransplanted and reinduced mice 34 days after secondary transplantation. FACS of (C) BM and (D) spleen. Mean percentages of donor cells (CD45.1+), recipient cells (CD45.2+), granulocytes (Gr1+/CD11b+), immature granulocytes (Gr1low/CD11b+), donor granulocytes (Gr1+/CD45.1+), immature donor granulocytes (Gr1low/CD45.1+), B cells (B220+), T cells (CD3+), or erythroid cells (Ter119+). Spleen weights on (E) autopsy, (F) spleen histology, (G) donor:host ratio within BM LSK, and (H) Bcr-Abl expression in secondary recipients. (I) BM and LSK cells from 4-week induced dtg and control mice were cultured with either 50nM dasatinib, 100nM dasatinib, or DMSO for 48 hours. The level of p-CrkL was measured by Western blotting. (J) Apoptosis in total BM, Gr1, and LSK cells was assessed by annexin V staining in cells from 4-week induced dtg (n = 7) and control (n = 5) mice that were treated for 48 hours using 100nM dasatnib or DMSO. Histologic analyses of spleen were performed using NACE stain and are shown at magnifications of (insets) 10× and 20×. Shown is 1 representative spleen histology from each group. LF indicates lymphoid follicle. Data represented as mean ± SEM; *P < .05.$

CML stem cells retain transplantation and oncogenic capacity following Bcr-Abl reversion. (A) Irradiated CD45.2⁺ mice (8 Gy) transplanted using unfractionated (uf) CD45.1⁺ BM cells isolated from SCLtTA/Bcr-Abl dtg or wt controls (n = 12/12). (i) Bcr-Abl expression in recipient mice was controlled by absence (expression) or presence (no expression) of tetracycline in drinking water. Bcr-Abl expression was induced after transplantation for 25 days and the phenotype analyzed in dtg and controls (n = 3/3) by FACS of BM and spleen cells, spleen weight and histology, ratio of donor:host LSK cells, and Bcr-Abl expression. (ii) Bcr-Abl expression was reverted for 48 days and a cohort of remaining mice (n = 4/4) analyzed. (iii) BM cells from reverted dtg and controls were retransplanted using CD45.1⁺ FACS-sorted cells (n = 5/5). Bcr-Abl expression was reinduced and the phenotype was assessed 69 days after retransplantation. (Bi-ii) PB was analyzed in CD45.1⁺ retransplanted and reinduced mice 34 days after secondary transplantation. FACS of (C) BM and (D) spleen. Mean percentages of donor cells (CD45.1⁺), recipient cells (CD45.2⁺), granulocytes (Gr1⁺/CD11b⁺), immature granulocytes (Gr1^low/CD11b⁺), donor granulocytes (Gr1⁺/CD45.1⁺), immature donor granulocytes (Gr1^low/CD45.1⁺), B cells (B220⁺), T cells (CD3⁺), or erythroid cells (Ter119⁺). Spleen weights on (E) autopsy, (F) spleen histology, (G) donor:host ratio within BM LSK, and (H) Bcr-Abl expression in secondary recipients. (I) BM and LSK cells from 4-week induced dtg and control mice were cultured with either 50nM dasatinib, 100nM dasatinib, or DMSO for 48 hours. The level of p-CrkL was measured by Western blotting. (J) Apoptosis in total BM, Gr1, and LSK cells was assessed by annexin V staining in cells from 4-week induced dtg (n = 7) and control (n = 5) mice that were treated for 48 hours using 100nM dasatnib or DMSO. Histologic analyses of spleen were performed using NACE stain and are shown at magnifications of (insets) 10× and 20×. Shown is 1 representative spleen histology from each group. LF indicates lymphoid follicle. Data represented as mean ± SEM; *P < .05.

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