by Seiji Fukuda, Hal E. Broxmeyer, and Louis M. Pelus Flt3 ligand and the Flt3 receptor regulate hematopoietic cell migration by modulating the SDF-1α(CXCL12)/CXCR4 axis by Seiji Fukuda, Hal E. Broxmeyer, and Louis M. Pelus Blood Volume 105(8):3117-3126 April 15, 2005 ©2005 by American Society of Hematology

Migration of UCB CD34+ cells and Ba/F3 cells expressing human wild-type Flt3 (Ba/F3-Flt3 cells) to FL. (A) Freshly isolated CD34+ cells were suspended in 0.5% BSA/RPMI, and migration toward 10 ng/mL rhFL was quantitated by flow cytometry. Migration of UCB CD34+ cells and Ba/F3 cells expressing human wild-type Flt3 (Ba/F3-Flt3 cells) to FL. (A) Freshly isolated CD34+ cells were suspended in 0.5% BSA/RPMI, and migration toward 10 ng/mL rhFL was quantitated by flow cytometry. In replicate cultures, cells were incubated with or without 10 ng/mL FL in 0.5% BSA/RPMI, and cell viability was determined based on forward and side scatter. Percentage migration of CD34+ cells to a positive (0/+FL; ▪) and a zero (+FL/+FL; ▴) gradient along with background migration (0/0; ○) are shown and were calculated based upon migration of CD34+ cells divided by fold change in viable cells incubated with FL. Data are expressed as mean ± SEM of 3 experiments. *P < .05 compared with background migration in the absence of FL. (B) Ba/F3-Flt3 cells were washed and resuspended in RPMI with 0.5% BSA at 5 × 106/mL and 0.1 mL, added to the transwell with either escalating doses of rhFL in the lower chamber (0/+; •) or in both the upper and lower chambers (+/+; ○), and incubated for 4 hours at 37°C. Cells that completely migrated to the bottom chamber were enumerated, and percentage migration was calculated as described in “Materials and methods.” Data represent 1 of the 3 experiments with similar results. *P < .05 compared with background migration in the absence of FL. Error bars indicate mean ± SEM. (C) (Left) Percentage migration of control vector–transduced Ba/F3 cells (□) or Ba/F3-Flt3 cells (▪) in response to a positive gradient of increasing concentration of rhFL. (Right) Flt3 expression was analyzed by flow cytometry. *P < .05 compared with background migration in the absence of FL. Shaded curve indicates isotope staining; open curve, FIT3 staining. Error bars indicate mean ± SEM. (D) Ba/F3-Flt3 cells were extensively washed and incubated with 10 ng/mL rhFL or 100 ng/mL rhCXCL12 for up to 360 seconds, fixed, and stained as described in “Materials and methods.” The percentage increase in mean channel fluorescence of phalloidin-FITC compared with cells incubated without cytokines (•) is shown. □ indicates CXCL12; ▿, FL. Data represent 1 of the 2 experiments with equivalent results. *P < .05 compared with cells without cytokines. Error bars indicate mean ± SEM. Seiji Fukuda et al. Blood 2005;105:3117-3126 ©2005 by American Society of Hematology

Migration of hematopoietic cells in response to the combination of FL and CXCL12. Migration of hematopoietic cells in response to the combination of FL and CXCL12. (A) Migration of freshly isolated UCB CD34+ cells to a positive gradient of escalating doses of CXCL12 was analyzed in the absence (□) or presence of 10 ng/mL rhFL (▪) in the bottom chamber for 4 hours. Data are the mean ± SEM of 2 experiments. † denotes synergistic effect of FL plus CXCL12. (B) Migration of CXCR4+, Flt3+, and CD34+ cells to 10 ng/mL FL and/or 100 ng/mL CXCL12. Partially purified UCB CD34+ cells were stained with FITC anti-CD34, PE anti-Flt3, and Cy-chrome anti-CXCR4, and CD34+, CXCR4+, and Flt3+ cells (gate R1) were sorted by FACS. The isotype and CXCR4/Flt3 staining are shown. Cells were subjected to migration for 4 hours. Data represent the mean ± SEM of 2 experiments. † denotes synergistic effect of FL plus CXCL12. (C) Total CD34+ cells were subjected to migration to 10 ng/mL rhFL and/or 100 ng/mL rhCXCL12 for 4 hours. Intracellular Ki-67 expression in CD34+ cells was performed as described.40 Migrated cells were fixed with 1% paraformaldehyde overnight at 4°C and washed with PBS containing 0.25% Triton X-100 and 1% BSA. Cells were blocked with human IgG on ice for 10 minutes and then stained with FITC-conjugated isotype control or anti-Ki67 antibody in the same buffer on ice for 60 minutes. Cells that appeared below isotype staining were defined as Ki-67negative cells. The percentage of Ki-67negative cell migration (▪) was calculated as the number of migrated Ki-67negative cells divided by the number of Ki-67negative CD34+ input cells multiplied by 100. A similar calculation was performed for Ki-67positive cells (□). Data are the mean ± SEM from 4 experiments. *P < .05 compared with Ki-67positive cells by t test; † denotes synergistic effect of FL plus CXCL12. (D) Percentage migration of Ba/F3-Flt3 cells to FL, CXCL12, or the combination of FL and CXCL12. Exponentially growing Ba/F3-Flt3 cells were extensively washed and 5 × 105 cells subjected to in vitro transmigration assay to 10 ng/mL rh FL, 100 ng/mL rmCXCL12 or rhCXCL12, or FL plus CXCL12. Data are shown as mean ± SEM percentage migration for 3 experiments. *P < .05 compared with background migration; † indicates synergistic effect of FL plus CXCL12. (E) Percentage migration of Ba/F3-Flt3 cells toward FL (10 ng/mL) or CXCL12 (100 ng/mL) in the presence or absence of negative gradient of FL (10 ng/mL). Data are shown as mean ± SEM percentage migration for 3 experiments. *P < .05 compared with background migration; † indicates synergistic effect of FL plus CXCL12. (F) Migration of the human RS4;11 cells in response to 10 ng/mL FL and/or 100 ng/mL CXCL12. Cells were extensively washed and evaluated for migration for 4 hours in 0.5% BSA/RPMI. The data represent the mean ± SEM percentage migration for 1 of 2 experiments. *P < .05 compared with background migration; † indicates synergistic effect of FL plus CXCL12. Seiji Fukuda et al. Blood 2005;105:3117-3126 ©2005 by American Society of Hematology

Long-term migration of CD34+ cells to FL and/or CXCL12. Long-term migration of CD34+ cells to FL and/or CXCL12. (A) Freshly isolated UCB CD34+ cells were subjected to in vitro migration to a positive gradient of 10 ng/mL FL (▪), 100 ng/mL CXCL12 (⋄), or the combination of FL plus CXCL12 (•). Cell migration was quantitated at 4, 24, 48, and 72 hours. Replicate cultures were incubated with 10 ng/mL rhFL, 100 ng/mL CXCL12, or a combination of FL plus CXCL12 in the chemotaxis medium for 72 hours, and viable cell counts were determined by the forward and side scatter analysis. Percentage of migration was calculated based upon migration of CD34+ cells divided by fold change in viable cells. Data represent the mean ± SEM from 2 experiments. *P < .05 compared with background migration (0/0; ▵). (B) Migration of CD34+ cells in response to positive (0/FL+CXCL12; □) and zero (FL+CXCL12/FL+CXCL12; ▦) gradients of FL plus CXCL12 were determined for 48 hours. Data are the average ± SEM of 3 experiments. *P < .05 compared with background migration (0/0; ▪). Seiji Fukuda et al. Blood 2005;105:3117-3126 ©2005 by American Society of Hematology

Phosphorylation of MAPKp42/p44, Akt, CREB, and ATF-1 in Ba/F3-Flt3 cells stimulated with FL and/or CXCL12 and effects of selective pathway inhibitors on migration to FL and/or CXCL12. Phosphorylation of MAPKp42/p44, Akt, CREB, and ATF-1 in Ba/F3-Flt3 cells stimulated with FL and/or CXCL12 and effects of selective pathway inhibitors on migration to FL and/or CXCL12. (A) IL-3–starved cells were incubated with rhFL (10 ng/mL) and/or rhCXCL12 (100 ng/mL) for 5 minutes. Lysates were subjected to Western analysis for phosphorylation of MAPKp42/p44, Akt, CREB, and ATF-1. (B) Cells were preincubated with dimethyl sulfoxide (DMSO), 50 μM PD98059, 50 μM LY294002, or 10 μM H89 for 1 hour, washed twice, and subjected to migration to 10 ng/mL rhFL (□), 100 ng/mL rhCXCL12 (▪), or the combination of FL plus CXCL12 (▨) for 4 hours. ▦ indicates background migration (0/0). Cell viability was evaluated by forward and side scatter analysis following migration. Data represent mean ± SEM of percentage migration in 1 of 6 experiments with similar results. † indicates synergistic effect of FL plus CXCL12; P < .0001. (C) Ba/F3-Flt3 cells were preincubated with 10 μM AG1296 for one hour, washed, and evaluated for migration to 10 ng/mL FL (□), 100 ng/mL CXCL12 (▪), or a combination of FL plus CXCL12 (▨) as described in panel B. ▦ indicates background migration (0/0). Data represent the mean ± SEM relative percentage migration compared with cells pretreated with DMSO and migrating to FL plus CXCL12 (expressed as 100%) from 2 experiments of triplicate counts. † indicates synergistic migration to FL plus CXCL12; P < .0001. Seiji Fukuda et al. Blood 2005;105:3117-3126 ©2005 by American Society of Hematology

Effects of preculture of UCB CD34+ and Ba/F3-Flt3 cells with FL on migration to CXCL12, CXCR4 expression, and phosphorylation of intracellular signaling molecules. Effects of preculture of UCB CD34+ and Ba/F3-Flt3 cells with FL on migration to CXCL12, CXCR4 expression, and phosphorylation of intracellular signaling molecules. (A) Flow cytometric analysis of expression of cell surface CXCR4 on UCB CD34+ cells cultured with or without 100 ng/mL FL for 48 hours in 20% HI-FBS using antihuman CXCR4 antibody (clone 12G5). The filled and open histograms show isotype and CXCR4 staining, respectively. The top panel shows CXCR4 expression in cells incubated without FL, and the bottom panel shows cells pretreated with FL. The histogram is representative of 6 samples analyzed. (B) Percentage migration of UCB CD34+ cells cultured without (▪) or with 100 ng/mL FL (□) for 48 hours to CXCL12. After culture, cells were washed and subjected to migration assay with 100 ng/mL CXCL12. The panel represents 1 of the 3 experiments with similar results. *P < .001 compared with control cells without FL pretreatment. Data expressed are mean ± SEM. (C) Expression of cell surface CXCR4 on Ba/F3 cells or Ba/F3-Flt3 cells cultured with FL. Exponentially growing Ba/F3 cells or Ba/F3-Flt3 cells were cultured with (+) or without (–) 100 ng/mL rhFL in the presence of IL-3 for 24 hours. CXCR4 expression was analyzed by flow cytometry using antimouse CXCR4 antibody (clone 2B11). The filled and open histograms show isotype and CXCR4 staining, respectively. The data represent 1 of 3 experiments. (D) The graph shows mean ± SEM percent migration of Ba/F3-Flt3 cells to CXCL12 precultured without (▪) or with FL (□) for 24 hours from 3 experiments. *P < .001 compared with control cells without FL pretreatment. (E, top) Phosphorylation of MAPKp42/p44, Akt, CREB, and ATF-1 upon stimulation with 100 ng/mL CXCL12 in Ba/F3-Flt3 cells preincubated with (+) or without (–) FL. Following culture as described in panel C, cells were washed extensively and incubated with 100 ng/mL CXCL12 for up to 15 minutes. Protein phosphorylation relative to total protein was quantified by densitometry and shown in the bottom panel. ▵ indicates –FL; •, +FL. Seiji Fukuda et al. Blood 2005;105:3117-3126 ©2005 by American Society of Hematology

Migration, actin polymerization, and CXCR4 expression of Ba/F3 cells expressing wild-type or ITD-Flt3 mutations (W51, W73, and W78). Migration, actin polymerization, and CXCR4 expression of Ba/F3 cells expressing wild-type or ITD-Flt3 mutations (W51, W73, and W78). (A) Ba/F3 cells expressing wild-type Flt3 (▪) or ITD-Flt3 (W51, □; W73, ▨; and W78, ▦) were subjected to transwell migration in the presence or absence of a negative gradient of 100 ng/mL CXCL12 with no cytokine in the lower chamber. These mutations in the juxtamembrane domain of Flt3 cloned from the patients with AML induce IL-3–independent growth of Ba/F3 cells and the W51 and W78 ITD-Flt3 induce lethal myeloproliferative disease in a murine bone marrow transplantation assay.32 Data represent mean ± SEM percentage migration for 3 experiments. *P < .001 compared with wild-type Flt3, and ‡P < .001 compared with 0/0 gradient. (B) Baseline actin polymerization of Ba/F3 cells expressing wild-type or ITD-Flt3 expressed as mean fluorescence intensity of phalloidin-FITC. Symbols indicate same information as in panel A. Data are the mean ± SEM of triplicate measurements from 1 of 2 experiments with similar results. *P < .001 compared with wild-type Flt3. (C) Migration of Ba/F3-Flt3 (wild type or ITD-Flt3) to 100 ng/mL rhCXCL12 or 10 ng/mL FL plus 100 ng/mL CXCL12. Symbols indicate same information as in panel A. Data are expressed as mean ± SEM percentage migration for 3 experiments. *P < .0001 compared with wild-type Flt3. (D, left) Expression of cell surface CXCR4 on Ba/F3 cells expressing ITD-Flt3 compared with cells expressing wild-type Flt3 analyzed by FACS. Histogram represents 1 of the 3 experiments with similar results. The open histogram with bold line shows isotype staining of cells expressing wild-type Flt3. The isotype for other cells was equivalent to cells expressing wild-type Flt3 (not shown). The right panel represents mean ± SEM percentage reduction of CXCR4 in cells expressing ITD-Flt3 compared with cells expressing wild-type Flt3 from 3 experiments. Symbols indicate same information as in panel A. *P < .001 compared with wild-type Flt3. Seiji Fukuda et al. Blood 2005;105:3117-3126 ©2005 by American Society of Hematology

Cell cycle analysis of migrated wild-type or ITD-Flt3 Ba/F3 cells. Cell cycle analysis of migrated wild-type or ITD-Flt3 Ba/F3 cells. Cells that had migrated after 4 hours were collected, fixed overnight in 1% paraformaldehyde, and stained with 1 μg/mL propidium iodide (Sigma Aldrich) in 0.6% nonidet P-40 (NP-40)/PBS with 1 μg/mL RNase (Sigma Aldrich), as described.40 Data were analyzed using a FACScan and ModFIT and Cell Quest software (Becton Dickinson) and expressed as the mean ± SEM of spontaneously or CXCL12-induced migrating cells in S+G2/M phase from 3 experiments. ▪ indicates wild-type Flt3; □, W51; ▨, W73; and ▦, W78. *P < .05 compared with wild-type Flt3. Seiji Fukuda et al. Blood 2005;105:3117-3126 ©2005 by American Society of Hematology