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CHEMOKINES
Unique SDF-1–induced activation of human precursor-B ALL cells as a result
of altered CXCR4 expression and signaling
Asaf Spiegel, Orit Kollet, Amnon Peled, Loya Abel, Arnon Nagler, Bella Bielorai, Gideon Rechavi, Josef Vormoor, and Tsvee Lapidot
The mechanisms governing migration and
extramedullary dissemination of leukemic cells remain obscure. In this study
the migration and in vivo homing to the
bone marrow of nonobese diabetic severe combined immunodeficient (NOD/
SCID) mice injected with human precursor-B acute lymphoblastic leukemia (ALL)
cells in comparison to normal CD34ⴙ
progenitors (both cord blood and mobilized peripheral blood) was investigated.
Although migration and homing of both
cell populations was dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4
interactions, major differences in recep-
tor expression as well as the migratory
capacity toward various concentrations
of SDF-1 were found. Furthermore, unlike
normal CD34ⴙ progenitors, in vivo homing of the leukemic cells was superior
when recipient NOD/SCID mice were not
irradiated prior to transplantation. In addition, we report differences in the adhesion molecules activated following SDF-1
stimulation, documenting a major role for
very late antigen 4 (VLA-4), but not VLA-5
and lymphocyte function-associated antigen-1 (LFA-1), in homing of precursor-B
ALL cells. Interestingly, Toxin-B and pertussis toxin inhibited the homing of the
leukemic cells but not that of normal
CD34ⴙ progenitors or normal CD10ⴙ/
CD19ⴙ precursor-B cells, revealing differences in CXCR4 signaling pathways that
are based on changes that acquired by the
leukemic cells. Altogether, our data provide
new insights into different SDF-1–induced
signaling, activation, and consequent motility between normal CD34ⴙ and precursor-B
ALL progenitors, which may lead to improved clinical protocols. (Blood. 2004;103:
2900-2907)
© 2004 by The American Society of Hematology
Introduction
Among the various leukemias, B-cell precursor acute lymphoblastic leukemia (ALL) represents the most common childhood leukemia. Approximately 70% of children are cured1; however, the need
exists to improve the outcome in nonresponders, high-risk patients,
and patients who relapse. Novel treatment strategies based on a
better understanding of the biology of this type of leukemia, in
particular the mechanisms that regulate migration and dissemination of the malignant clone, may assist in achieving this goal.
Immune deficient severe combined immunodeficient (SCID)
and nonobese diabetic (NOD)/SCID mice have been used as a
functional, preclinical model for in vivo engraftment and dissemination of human pre-B ALL cells.2,3 By using this model it was shown
that the engraftment kinetics of human pre-B ALL blasts correlates with
the prognosis of the disease in the original patients.4-6
Stromal-derived factor-1 (SDF-1; also named CXCL12), the
ligand of the CXCR4 receptor, is constitutively produced by many
cell types, including immature osteoblasts and endothelial cells
within the bone marrow (BM) as well as by epithelial cells in many
organs, including the central nervous system.7-9 Human and murine
SDF-1 differ in only one amino acid and are cross-reactive.10
SDF-1 is the most powerful chemoattractant for undifferentiated
human CD34⫹ hematopoietic progenitors11,12 and is the only
chemokine known to induce high levels of directional migration of
both human CD34⫹/CD38⫺ and murine Sca-1⫹/ckit⫹/Lin⫺ stem
cells.13,14 We have previously shown the essential role of SDF-1/
CXCR4 interactions in both homing to the murine BM and
high-level multilineage repopulation by human CD34⫹/CD38⫺/low
SCID repopulating cells (SRCs) in NOD/SCID mice that received
transplants.8,13,15
SDF-1, originally cloned from a stromal cell line as a pre-B cell
growth factor, is essential for normal B-cell development. Mice
that lack SDF-1 or CXCR4 exhibit many lethal defects, including
impaired B-cell lymphopoiesis and lack of BM seeding by
hematopoietic progenitors.16,17 Mutations in human CXCR4 lead to
WHIM (warts, hypogammaglobulinemia, immunodeficiency, and
myelokathexis) syndrome, a combined immunodeficiency disease
that is characterized by neutropenia as well as deficient B- and
T-cell abundance and function.18 SDF-1 is also involved in
proliferation and survival of various cells, including normal human
CD34⫹ cells and pre-B ALL cells.19,20
SDF-1 regulates many interactions between primitive human
CD34⫹ cells and the BM microenvironment. In particular, this
ligand activates cell adhesion and transendothelial migration which
is mediated by the major integrins very late antigen 4 (VLA-4),
VLA-5, and lymphocyte function-associated antigen-1 (LFA1).21,22 Transmigration of human leukemic cells through BM
fibroblasts is also mediated by SDF-1/CXCR4 interactions, and the
malignant cells use the ␤1 integrins VLA-4 and VLA-5.23 It has
From the Department of Immunology, The Weizmann Institute of Science,
Rehovot, Israel; Gene Therapy Institute, Hadassah University Hospital,
Jerusalem, Israel; Departments of Bone Marrow Transplantation and Pediatric
Hemato-Oncology, Sheba Medical Center, Ramat-Gan, Israel; Sackler School
of Medicine, Tel Aviv University, Tel-Aviv, Israel; and Department of Pediatric
Hematology and Oncology, University Children’s Hospital, Muenster, Germany.
Supported in part by grants from the Minerva Foundation, Israel Cancer
Research Fund (ICRF), and the Israel Science Foundation.
Submitted June 12, 2003; accepted November 5, 2003. Prepublished online as
Blood First Edition Paper, November 20, 2003; DOI 10.1182/blood-2003-06-1891.
2900
Reprints: Tsvee Lapidot, Department of Immunology, The Weizmann Institute
of Science, Rehovot 76100, Israel; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology
BLOOD, 15 APRIL 2004 䡠 VOLUME 103, NUMBER 8
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BLOOD, 15 APRIL 2004 䡠 VOLUME 103, NUMBER 8
CXCR4 EXPRESSION AND ACTIVATION IN PRE-B ALL CELLS
2901
been shown that CXCR4 desensitization by pretreatment of human
ALL cells with high levels of SDF-1 in vitro prior to their
transplantation decreases their homing and engraftment levels in
NOD/SCID mice that receive transplants.24
Chemokines can induce distinct signaling pathways that mediate cell growth, transcriptional activation, as well as cell motility. In
vitro SDF-1–induced chemotaxis is inhibited by pertussis toxin
(PTX), demonstrating that the 7 transmembrane receptor CXCR4 is
coupled to G␣i proteins.11 Intracellular events induced by SDF-1
include elevation of cytoplasmic Ca2⫹ levels, activation of phosphoinositide 3-kinase (PI-3 kinase), and phosphorylation of mitogenactivated protein kinase kinase/extracellular signal regulated kinase (MEK/ERK) in several cell types.25-27
Actin cytoskeleton organization, a vital part of cell migration,
has been shown to be regulated by the Rho proteins Rho, Rac, and
Cdc42. These proteins belong to the Ras superfamily of small
guanosine triphosphate (GTP) binding proteins and function as key
regulators of many essential cellular processes, including actin
cytoskeleton organization, gene transcription, and cell adhesion.28
Cdc42 has been shown to be involved in SDF-1–induced T-cell
chemotaxis.29
In the present study, we investigated the role of SDF-1/CXCR4
interactions in the migration (both in vitro and in vivo) of human
precursor-B ALL cells, as well as the integrins and signaling
pathways activated on SDF-1 stimulation. The signaling pathways
involved in the migration of the precursor-B ALL cells in comparison to CD34⫹ cells were examined, revealing both similarities as
well as differences between normal and leukemic progenitor cells
in their CXCR4 expression, activation, and SDF-1–mediated
directional migration.
density MNCs were collected after standard separation on Ficoll-Paque
(Amersham Pharmacia Biotech) and washed in PBS. Granulocyte colonystimulating factor (G-CSF)–mobilized peripheral blood (MPB) cells were
obtained from healthy donors for clinical transplantation. CD34⫹ cells were
purified by using the magnetic activated cell sorting (MACS) cell isolation
kit and AutoMACS magnetic cell sorter (Miltenyi Biotec, Bergisch
Gladbach, Germany) according to the manufacturer’s instructions, and they
were incubated overnight in medium supplemented with 50 ng/mL stem
cell factor.
Materials and methods
Flow cytometry analysis
Cells
All human cells were obtained after informed consent and were used in
accordance with the procedures approved by the human experimentation
and ethics committees of the Weizmann Institute. Precursor-B ALL cell
lines Nalm-6 (kindly provided by Prof H. Ben-Bassat, Hadassah Medical
School, Jerusalem, Israel), A1, B1, G2, and BRE (kindly provided by Prof
M. Freedman, The Hospital for Sick Children, Toronto, ON, Canada) were
derived from freshly obtained peripheral blood composed primarily of blast
cells from children with poor prognosis B-lineage ALL.30 Cells were grown
in Iscove modified Dulbecco medium (IMDM) (Biological Industries, Beit
Haemek, Israel) supplemented with 10% fetal calf serum (Biological
Industries), L-glutamine (Biological Industries), and penicillin and streptomycin antibiotics (GibcoBRL, Grand Island, NY). Leukemic cells from
patients who had been newly diagnosed (Table 1) were diluted in
phosphate-buffered saline (PBS) and centrifuged on Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden). Washed mononuclear cells
(MNCs) were then cryopreserved in 10% dimethylsulfoxide, 40% fetal calf
serum, and IMDM prior to use. Human cord blood (CB) samples from
full-term deliveries were diluted 1:1 in PBS without Mg2⫹/Ca2⫹. Low-
Mice
NOD/SCID mice (NOD/LtSz Prkdcscid)31 were bred and maintained under
defined flora conditions in individually ventilated (high-efficiency particlearresting filtered air) sterile microisolator cages (Techniplast, Varese, Italy)
at the Weizmann Institute. All the experiments were approved by the animal
care committee of the Weizmann Institute. In some experiments the
8-week-old mice were irradiated with a sublethal dose of 375 cGy from a
cobalt source prior to transplantation. Leukemic cells (5-20 ⫻ 106 cells) or
CD34⫹ cells (5 ⫻ 105 cells) were injected into the tail vein of irradiated (24
hours after irradiation) or nonirradiated mice. For in vivo blocking
experiments, the cells were first preincubated for 30 minutes with 3 ␮g/106
cells of a blocking mouse antihuman VLA-4 (MCA697), anti–VLA-5
(MCA1187), anti–LFA-1 (MCA1149) (Serotec, Oxford, United Kingdom),
or anti-CXCR4 monoclonal antibody (mAb; clone 12G5; Pharmingen, San
Diego, CA) without washing. In other homing experiments, cells were
pretreated with SDF-1␣ (1 ␮g/mL, 20 hours, 37°C; PeproTech, Rocky Hill,
NJ), 100 ng/mL Clostridium difficile toxin B-10463 (Tox-B; 100 ng/mL, 20
hours, 37°C; kindly provided by Dr Aktories, Universitat Freiburg,
Freiburg, Germany), PTX (100 ng/mL, 2 hours, 37°C), or chelerythrine
chloride (CC; 10 ␮M, 30-60 minutes; Calbiochem, San Diego, CA) prior to
injection. Cells were recovered from the BM of the mice 16 hours after
transplantation, MNCs were counted, and the presence of human cells was
detected by flow cytometry.
Flow cytometry analysis was done as previously described.32 BM cells from
mice that received transplants were flushed and resuspended in fluorescence
activated cell sorting (FACS) buffer (PBS with 0.1% bovine serum albumin
[BSA], and 0.02% sodium azide). Cells were stained with human-specific
direct-labeled antibodies, 10 ␮L/mL purified antimouse CD16/CD32 Fc
receptor (PharMingen), and 1% human plasma and incubated for 30
minutes at 4°C. Human cells and murine BM cells from mice not receiving
transplants were used as a positive and negative control, respectively. The
presence of human precursor-B ALL cells was identified by staining with
anti-CD45–FITC (Immuno Quality Products, Groningen, The Netherlands), anti-CD10–FITC, or anti-CD19–FITC (Becton Dickinson, San Jose,
CA) antibodies. Human CD34⫹ cells were detected in the BM by using
human-specific anti-CD34–FITC (Becton Dickinson) and anti-CD38–
phycoerythrin (PE; Beckman Coulter, Brea, CA) antibodies. The level of
CXCR4 expression on cells was detected with PE-conjugated anti-CXCR4
antibodies (PharMingen). After staining, cells were washed in FACS buffer
and analyzed by flow cytometry (FACSCalibur and CellQuest software;
Becton Dickinson). When cells were stained for integrin expression,
nonconjugated antibodies against VLA-4, VLA-5, and LFA-1 (Serotec)
were used, and they were detected by using secondary FITC-conjugated
Table 1. Clinical information regarding cells from patients with newly diagnosed precursor-B ALL
Patient no.
Source
% Blasts
1
PB
95
c-ALL
BCR/ABL negative, MLL/AF4 negative, TEL/AML1 negative
2
PB
93
Pro-BALL
t(4;11), BCR/ABL negative, MLL/AF4 positive, TEL/AML1 negative, MLL/ENL negative
3
BM
92.5
c-ALL
BCR/ABL negative, MLL/AF4 negative, TEL/AML1 negative
4
PB
95
c-ALL
BCR/ABL negative, MLL/AF4 negative, TEL/AML1 negative
5
BM
100
6
PB
70
PB indicates peripheral blood.
Immunophenotype
Molecular data
c-ALL
t(12;21)
Pro-BALL
t(4;11)
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BLOOD, 15 APRIL 2004 䡠 VOLUME 103, NUMBER 8
SPIEGEL et al
F(ab⬘)2 fragment goat antimouse immunoglobulin G (IgG) (Jackson, West
Grove, PA). In experiments testing homing and migration of normal
precursor-B cells, CB MNCs were triple-stained with CD45-allophycocyanin (APC), CD10-FITC, and CD19-PE (Becton Dickinson). Human
leukocytes were gated according to their expression of the pan-leukocyte
marker CD45, and among this population the number of CD10⫹/CD19⫹
precursor-B cells was determined.
Chemotaxis assays
Chemotaxis experiments were assayed by using transwells (6.5-mm
diameter, 5-␮m pore) (Corning, Corning, NY) as previously described.22
Human cells (1-2 ⫻ 105) suspended in 100 ␮L medium were added to the
upper chamber, and 600 ␮L medium with or without SDF-1 (10-1000
ng/mL) (PeproTech) was placed in the bottom chamber. After 4 hours at
37°C, migrating (bottom chamber) cells were counted for 30 seconds with
the use of a FACSCalibur (Becton Dickinson). In some experiments, 1 to
2 ⫻ 106 cells/mL was preincubated (30 minutes at 4°C) in 100 ␮L medium
containing either 5 ␮g control isotype-matching mAb (Becton Dickinson)
or murine mAb specific to human CXCR4 (12G5 clone; Pharmingen). In
other experiments, 1 to 2 ⫻ 106 cells/mL was preincubated in medium
containing PTX (100 ng/mL, 2 hours, 37°C), CC (2-10 ␮M, 30-60 minutes,
37°C), or the CXCR4 antagonists T22 and T140 (kindly provided by Prof
N. Fujii, Kyoto University, Kyoto, Japan) (1 and 10 ␮M, respectively, 1
hour, 37°C), with appropriate controls. When the effect of SDF-1 desensitization or Tox-B was tested, cells were preincubated for 20 hours with 1
␮g/mL SDF-1 or 100 ng/mL Tox-B prior to migration.
Intracellular CXCR4 staining
Intracellular CXCR4 staining was done as previously described.33 In brief,
CXCR4 expressed on the cell surface was blocked with nonconjugated
antihuman CXCR4 mAb (clone 12G5, 10 ␮g/mL, 1 hour, 4°C). Cells were
fixed with paraformaldehyde (4%, 20 minutes at room temperature; BDH,
Poole, England) and then permeabilized with Triton X-100 (0.5%, 10
minutes at room temperature; Sigma, St Louis, MO). Anti-CXCR4–PE
mAb was used to label the cells for flow cytometry for 30 minutes, 4°C. The
cells were washed with PBS after each step.
Gelatin zymography
Normal CB CD34⫹ cells or precursor-B ALL cell lines (B1, G2, Nalm-6)
were incubated in serum-free RPMI (2 ⫻ 106 cells/mL) with or without
SDF-1 (1, 10, or 500 ng/mL) at 37°C for 40 hours as described elsewhere.34
Cell-conditioned media was then collected for zymographic analysis of
matrix metalloproteinase 2 (MMP-2) and MMP-9 activity. Media samples
were mixed with nonreducing sample buffer and loaded on 10% sodium
dodecyl sulfate (SDS)–polyacrylamide gel copolymerized with 1 mg/mL
gelatin derived from porcine skin (Sigma). After electrophoresis, gels were
washed clear of SDS for 30 minutes in 2.5% Triton X-100, washed 3 times
with H2O, and incubated at 37°C for 16 hours in developing buffer (50 mM
Tris (tris(hydroxymethyl)aminomethane) pH ⫽ 8, 5 mM CaCl2, 200 mM
NaCl, and 0.02% Brij). The gels were then stained with 0.25% Coomassie
Brilliant Blue and destained (5% acetic acid, 10% MeOH) until clear bands
appeared, indicating the presence of MMP-2 and MMP-9. Conditioned
media from HT-1080 cells secreting both MMP-2 and MMP-9 served
as control.
Statistics
Results of experimental points are reported as mean ⫾ SE. Statistical
significance was determined by Student t test for differences in means.
Results
Migration and homing of precursor-B ALL cells are dependent
on CXCR4/SDF-1 interactions
The crucial role of SDF-1 and CXCR4 in the homing and
engraftment of human CD34⫹ progenitor cells, including CD38⫺/low
SRCs has been shown in our lab.8,13,15 We set forth to determine
whether a similar mechanism is involved in the migration and
homing of precursor-B ALL cells. First, the capacity of precursor-B
ALL cells to respond to SDF-1 in vitro was examined. All
precursor-B ALL cell line cells tested expressed CXCR4 and
migrated through transwell filters toward SDF-1 placed in the
bottom chamber. Incubation with high concentrations (1 ␮g/mL) of
SDF-1 leading to receptor desensitization inhibited migration by
80%, and the neutralizing monoclonal anti-CXCR4 12G5 antibody
decreased in vitro migration by 40%. Complete inhibition of in
vitro migration was achieved when G2 or Nalm-6 cells were
preincubated with the CXCR4 peptide antagonists T22 and T140
(Figure 1A). As can be seen in Figure 1B, there is a strong
correlation between CXCR4 expression level and the ability of the
cells to migrate. Whereas untreated G2 cells express high levels of
CXCR4 on their membrane, SDF-1 desensitization caused a
Figure 1. Migration and homing of precursor-B ALL
cells depend on SDF-1/CXCR4 interactions. (A) Results show average percentage ⫾ SE of in vitro migration
of untreated (control), pretreated (␣-CXCR4, T22, or
T140) or SDF-1–desensitized (20 hours in IMDM containing 1 ␮g/mL SDF-1) Nalm-6 and G2 cells to 125 ng/mL
SDF-1 (at least 3 experiments for each cell line). (B) Cell
surface CXCR4 expression levels of unlabeled (a), T22
pretreated (b), SDF-1–desensitized (c), or untreated (d)
G2 cells were ascertained. (C) Cells (5-20 ⫻ 106) from
patients with newly diagnosed precursor-B ALL (patient
nos. 1-5) or cell lines were injected into NOD/SCID mice
either untreated, after blocking with anti-CXCR4–neutralizing antibodies (Nalm-6, G2, A1, BRE), or after 20 hours
of incubation with 1 ␮g/mL SDF-1 (Nalm-6 and G2).
Results show percentage of homing of human cells to the
BM 16 hours after transplantation relative to control
untreated cells (⫽ 100%). Three or more mice were used
for each cell line in each treatment. Cells from noninjected mice were used as a negative control. (D) A
representative experiment showing homing of G2 cells.
The number represents the number of human CD45⫹
cells per 106 acquired cells. *P ⬍ .05 compared with
control.
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BLOOD, 15 APRIL 2004 䡠 VOLUME 103, NUMBER 8
significant decrease in CXCR4 levels. Pretreatment with the T22
peptide inhibitor caused an even stronger internalization from the
membrane, which may explain the total inhibition of directional
migration.
We found that similar to migration, homing of precursor-B ALL
cells (both cell line and newly diagnosed patient cells) to the BM of
NOD/SCID mice was also dependent on SDF-1/CXCR4 interactions. Homing of precursor-B ALL cell line cells to the BM was
significantly reduced by 60% after SDF-1 desensitization and an
even greater reduction (90%) was observed following pretreatment
of the cells with neutralizing anti-CXCR4 antibodies (Figure
1C-D). Similarly, cells from patients with newly diagnosed precursor-B ALL that were incubated with neutralizing anti-CXCR4
antibodies prior to injection showed complete inhibition in homing
to the BM of nonirradiated NOD/SCID mice (Figure 1C). All
together these results demonstrate that CXCR4 signaling plays a
crucial role in SDF-1–mediated directional migration and homing
of precursor-B ALL cells.
Precursor-B ALL and normal CD34ⴙ cells exhibit different
expressions of CXCR4, SDF-1–induced migration,
and need for irradiation of recipient mice
Although sublethal irradiation prior to cell injection is essential for
optimal homing and engraftment of normal human CD34⫹ cells to
hematopoietic organs of NOD/SCID mice,8,35 it was previously
shown that sublethal irradiation is not a prerequisite for precursor-B ALL engraftment of NOD/SCID mice.5 Engraftment is
composed of several distinct stages, first homing to the BM
microenvironment, followed by retention and repopulation. We
investigated whether irradiation is important for homing, the first
crucial step of engraftment, by comparing the homing ability of
precursor-B ALL cells into the BM of irradiated versus nonirradiated mice. Irradiation was not needed for successful homing of
precursor-B ALL cell line cells. Moreover, a 15-fold increase in the
total number of cells that homed to the BM was detected when
NOD/SCID recipient mice were not preconditioned by total body
irradiation (TBI) (Figure 2A, log scale).
These results might be explained by increased sensitivity to
SDF-1; therefore in vitro migration of precursor-B ALL cell line
cells toward different concentrations of SDF-1 was tested. We
found that, although migration capacity of precursor-B ALL cells
CXCR4 EXPRESSION AND ACTIVATION IN PRE-B ALL CELLS
2903
reached high and maximal levels already at low levels of SDF-1
(10 and 50 ng/mL, respectively), a significant decrease was
detected in the migration toward very high levels of SDF-1 (1
␮g/mL). In contrast, migration of normal CD34⫹ cells (both CB
and MPB) reached peak levels of migration only at higher
concentrations of SDF-1 (125 ng/mL), and no significant decrease was detected when SDF-1 levels were further increased
(Figure 2B).
To understand these differences in the response to SDF-1, the
level of expression of intracellular and cell surface CXCR4 were
checked. Precursor-B ALL cells expressed high levels of cell
surface CXCR4, yet only low levels of intracellular CXCR4 were
detected. In contrast, in normal CB and MPB CD34⫹ cells most of
the CXCR4 molecules were localized within the cells, and only a
small part of the receptors were expressed on the membrane surface
(Figure 2C). These differences in the expression levels of intracellular and cell surface CXCR4 suggest different CXCR4 regulation
in normal and malignant human progenitor cells and may explain
different cell behavior in response to SDF-1.
Different patterns of MMP secretion by the precursor-B ALL
and CB CD34ⴙ cells
Matrix metalloproteinases (MMPs) are proteolytic enzymes secreted by normal and malignant cells and have been shown to play
a crucial role in cell motility and invasion through the extracellular
matrix (ECM).36 SDF-1 induces expression and secretion of
MMP-2 and MMP-9 by normal CD34⫹ cells, which, in turn, can
inactivate this chemokine.34,37 To determine the role of MMPs in
the motility of the normal and leukemic progenitors, the levels of
MMP-2 and MMP-9 secretion were assayed by zymography. CB
CD34⫹ cells and precursor-B ALL cells exhibited a different
pattern of MMP secretion. Whereas normal CB CD34⫹ cells
secreted MMP-9 but only low levels of MMP-2, precursor-B ALL
cell line cells (B1, G2, and Nalm-6) secreted mainly MMP-2 and
only low or undetectable levels of MMP-9. Furthermore, stimulation of the leukemic cells with SDF-1 at different concentrations
had no effect on MMP secretion. This finding is in contrast to
CD34⫹ cells in which MMP secretion is enhanced in response to
high levels of SDF-134 (Figure 3). The different patterns of MMP
secretion exhibited by the 2 cell populations may explain the
differences in their ability to migrate and home into the BM.
Figure 2. Differences in CXCR4 expression, SDF-1–induced migration, and irradiation requirement between precursor-B ALL and CD34ⴙ cells. (A) Precursor-B ALL
cell line cells (5-20 ⫻ 106) were injected into NOD/SCID mice, either untreated (f) or 48 hours after total body irradiation (375 cGy; u). The total number (average ⫾ SE of at
least 3 experiments) of human cells that homed to the murine BM 16 hours after transplantation is shown. This was calculated according to the number of human cells per 106
MNCs acquired by flow cytometry multiplied by the total number of MNCs in the BM. *P ⬍ .05 compared with homing into nonirradiated mice. (B) Fold increase (compared with
migration to 125 ng/mL SDF-1) of spontaneous or SDF-1–induced migration (at the indicated concentrations) of precursor-B ALL (䡺; G2, Nalm-6, BRE cells; average ⫾ SE of
at least 3 experiments performed in duplicates for each cell line) and normal CB (f) or MPB (u) CD34⫹ cells. *P ⬍ .05 relative to migration toward 125 ng/mL SDF-1. (C)
Immunofluorescence detection of intracellular (gray line), cell surface (black line), or isotype control (dotted line) expression of CXCR4 levels of normal CD34⫹ cells, G2 cells,
or precursor-B ALL cells from patients with newly diagnosed disease (patient nos. 1, 3, 4, and 5). Representative experiments of at least 3 independent experiments for each
group are shown.
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SPIEGEL et al
BLOOD, 15 APRIL 2004 䡠 VOLUME 103, NUMBER 8
Involvement of key signaling molecules in SDF-1–induced
cell motility
Figure 3. Different pattern of MMP secretion by precursor-B ALL and CB CD34ⴙ
cells. CB CD34⫹ and precursor-B ALL cells were cultured for 40 hours with or without
SDF-1. The presence of MMP-2 and MMP-9 in the conditioned media was detected
by gelatin zymography. Conditioned media from HT-1080 cells secreting both MMP-2
and MMP-9 served as control. A representative of 3 independent experiments
conducted is shown.
Role of adhesion molecules in homing of precursor-B ALL
and normal CB CD34ⴙ cells
Bradstock et al23 have demonstrated that ALL blasts migrate into
layers of BM fibroblasts in vitro by using the ␤1 integrins VLA-4
and VLA-5. We have shown that homing of normal human CB
CD34⫹ cells into the BM of NOD/SCID mice is equally dependent
on VLA-4, VLA-5, and LFA-1.15 We set out to test the direct effects
of ␤1 and ␤2 integrins on the homing of precursor-B ALL cells to
the BM of NOD/SCID mice. Precursor-B ALL cells were treated
with neutralizing antibodies against each of the integrins separately
before transplantation. Homing of precursor-B ALL cell line cells,
as well as cells from patients with newly diagnosed precursor-B
ALL into the BM was significantly reduced by blocking VLA-4
(80% and 85%, respectively) compared with nontreated cells.
Pretreatment of cells with anti–VLA-5 or anti–LFA-1 antibodies
did not inhibit homing of precursor-B ALL cell line cells, and a
40% and 45% (respectively) decrease in homing was detected
when patient cells were injected. Altogether, homing of cells after
pretreatment with VLA-4–neutralizing antibodies was significantly
lower than that of VLA-5 and LFA-1 pretreated cells (Figure 4).
These results demonstrate that, although all 3 integrins play an
equally important role in the homing of normal CD34⫹ cells,
activation of VLA-4 is the most prominent in homing of precursor-B ALL cells.
Figure 4. VLA-4 has the most prominent role in mediating homing of precursor-B ALL cells. Precursor-B ALL cells were pretreated with specific neutralizing
antibodies for the specified integrins prior to injection into NOD/SCID mice. Results
show percentage (compared with control ⫽ 100%) ⫾ SE of human cells recovered
from the BM of injected mice and are the average of 3 cell lines (A-1, G2, BRE) or 5
patient cells (patient nos. 1-5). Each group contains 6 or more mice. *P ⬍ .05
compared with both control as well as ␣-VLA-5 and ␣-LFA-1 pretreatment.
The chemokine receptor CXCR4 is a G-protein–coupled receptor.
PTX, an inhibitor of signal transduction mediated by the G␣i
subunit, almost completely abrogates in vitro migration of normal
human CD34⫹ cells.11,15 Similarly, we found that in vitro migration
of precursor-B ALL cells toward a gradient of SDF-1 was also
inhibited by PTX. In addition, homing of precursor-B ALL cells
was inhibited by 70% after pretreatment with PTX; however, only a
small and not significant decrease was detected in homing of MPB
CD34⫹ cells, and a slight increase was detected in homing of CB
CD34⫹ cells following pretreatment with PTX15 (Figure 5A-B).
The distinct response to PTX between the normal and leukemic
cells is not due to differences in receptor internalization, because
SDF-1–induced CXCR4 internalization was not affected by PTX
pretreatment in both the normal and the malignant cells (Figure
5C-D). In addition, PTX inhibited the SDF-1–induced calcium
mobilization in both normal and malignant cells in a similar
manner (data not shown). This finding is in accordance with a
previous report by Amara et al38 that showed G␣i-independent
CXCR4 internalization in T cells.
Rho proteins are key regulators of signal transduction and
mediate actin cytoskeleton rearrangement and cell motility.28 We
tested whether these proteins have a role in SDF-1–induced
hematopoietic cell migration. Pretreatment of precursor-B ALL
cells with Toxin B, a single-chained 270-kDa molecule that
specifically inactivates the Rho proteins RhoA, Cdc42, and Rac,
resulted in a significant reduction in migration and homing of the
Figure 5. SDF-1/CXCR4 interactions activate different signaling pathways in
migration and homing of precursor-B ALL and CD34ⴙ cells. Migration and
homing of cells following pretreatment with pertussis toxin (PTX; 100 ng/mL, 2 hours,
37°C), Toxin B (ToxB; 100 ng/mL, 20 hours, 37°C), or chelerythrine chloride (CC; 10
␮M, 30-60 minutes, 37°C) were tested. (A) Results show average percentage ⫾ SE
of in vitro migration of pretreated human precursor-B ALL, CB CD34⫹, and MPB
CD34⫹ cells compared with control untreated cells (⫽ 100%). Results of migration of
precursor-B ALL cells represent average of G2, BRE, and Nalm-6 cells (at least 3
experiments for each cell line). (B) Results show percentage of homing of pretreated
human precursor-B ALL, CB, and MPB CD34⫹ cells to the BM 16 hours after
transplantation compared with control untreated cells (⫽ 100%). Results represent
average ⫾ SE of cell lines G2, B-1, and A-1, as well as cells from patient nos. 1, 2, 3,
4, and 6. Cell viability was greater than 90% before injection. *P ⬍ .05 in comparison
of precursor-B ALL with normal CD34⫹ cells for each treatment. G2 cells (C) or CB
CD34⫹ cells (D) were incubated with or without PTX (100 ng/mL) for 2 hours and then
stimulated with increasing concentrations of SDF-1. Surface expression of CXCR4
was determined by flow cytometry. Results show average of 3 independent
experiments compared with control cells not stimulated with SDF-1 (⫽ 100%).
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BLOOD, 15 APRIL 2004 䡠 VOLUME 103, NUMBER 8
CXCR4 EXPRESSION AND ACTIVATION IN PRE-B ALL CELLS
2905
Discussion
Figure 6. Signaling inhibitors affect normal precursor-B cells similar to CD34ⴙ
cells. CB MNCs were pretreated with pertussis toxin (PTX; 100 ng/mL, 2 hours,
37°C), Toxin B (ToxB; 100 ng/mL, 20 hours, 37°C), or chelerythrine chloride (CC; 10
␮M, 30-60 minutes, 37°C) prior to in vitro migration or homing into the BM of
NOD/SCID mice. Cells that migrated or homed were gated according to their
expression of CD45, and among this population the number of CD10⫹/CD19⫹
precursor-B cells was determined. Results show average (of at least 3 experiments)
percentage ⫾ SE of in vitro migration and homing of normal precursor-B CD10⫹/
CD19⫹ cells relative to control untreated cells (⫽ 100%). *P ⬍ .05.
cells. In vitro migration was inhibited by 85%, and a 90% reduction
in homing to the BM was detected (Figure 5A-B). Contrary to that,
when normal CB or MPB CD34⫹ progenitor cells were preincubated with Toxin B, only a slight decrease (15% and 25%,
respectively) in migration was observed. Similarly, homing of CB
CD34⫹ into the BM of NOD/SCID mice was reduced by only 25%,
and a 25% increase in homing of MPB CD34⫹ cells was
documented (Figure 5A-B).
Laudanna et al39 found that CC, a broad-range protein kinase C
(PKC) inhibitor, blocks both CXCR2-mediated adhesion and
chemotaxis of neutrophils while studying another CXC chemokine,
IL-8. We found that the PKC pathway is also involved in
SDF-1/CXCR4 signaling because CC significantly inhibited SDF1–induced migration of both normal CD34⫹ cells and precursor-B
ALL cells. Moreover, pretreatment of human CB CD34⫹ and
precursor-B ALL cells with CC efficiently inhibited the homing of
65% of the cells into the BM15 (Figure 5A-B). Interestingly, both
homing and migration of MPB CD34⫹ cells were totally inhibited
following pretreatment of cells with CC. Taken together these
results demonstrate that SDF-1 stimulation leads to activation of
different signaling pathways with only a partial overlap in normal
and malignant human progenitors.
Unique SDF-1–induced signaling pathways result from the
leukemic phenotype of precursor-B ALL cells
The differences between normal human CD34⫹ cells and malignant precursor-B ALL cells may result from the fact that the
precursor-B cells are more differentiated than the CD34⫹ cells.
Alternatively, the differences may be due to a leukemic “phenotype” acquired by the malignant cells. To address this issue,
SDF-1–induced migration and homing of CB MNCs following
pretreatment with signaling inhibitors was tested. Human leukocytes were gated according to their expression of CD45, and among
this population the number of CD10⫹/CD19⫹ precursor-B cells
was determined. Similar to migration of CD34⫹ cells, SDF-1–induced
migration of normal precursor-B CD10⫹/CD19⫹ population of CB
MNCs was inhibited by PTX but was only slightly reduced following
treatment with Toxin B. Unlike the leukemic cells, yet similar to normal
CD34⫹ cells, pretreatment with PTX resulted in a slight increase in
homing of normal precursor-B cells (Figure 6). Thus, normal CD10⫹/
CD19⫹ precursor-B cells share similar signaling pathways with normal
CD34⫹ cells but differ from those of the leukemic cells, strongly
suggesting that the unique response to SDF-1 results from changes
acquired by the malignant cells and not differences between primitive
precursors and B-lineage progenitors.
Precursor-B ALL is the most common childhood malignancy and
the second most common adult acute leukemia.40-42 The leukemic
cells have the ability to infiltrate the liver, spleen, lymph nodes, and
central nervous system. Investigating the mechanisms of extramedullary dissemination of the leukemic cells is extremely important,
because specific targeting of these cells may improve treatment of
patients. Immune deficient SCID and NOD/SCID mice provide a
useful preclinical model to study in vivo malignant migration and
repopulation by human precursor-B ALL progenitors, because this
functional system can predict the patient’s prognosis.3,5,6 In the
present study we demonstrate that the migration and homing
requirements of malignant precursor-B ALL cells differ from those
required for successful homing and migration of normal CD34⫹
progenitors despite the dependence of both cell populations on
SDF-1/CXCR4 interactions.
High expression of CXCR4 by the leukemic cells is strongly
predictive for extramedullary organ invasiveness, including infiltration to the central nervous system in patients with childhood
ALL.43 We show that, similar to normal CD34⫹ progenitor cells,
SDF-1/CXCR4 interactions are essential for both in vitro migration
as well as in vivo homing of malignant precursor-B ALL cells.
Pretreatment leading to interference of SDF-1/CXCR4 interactions
demonstrates a strong correlation between receptor expression and
migratory capacity both in vitro and in vivo. These results are in
accordance with a report by Shen et al24 that showed reduction in
homing and engraftment of precursor-B ALL cells following
CXCR4 desensitization by pretreatment with high concentrations
of SDF-1.
Internal reservoirs of CXCR4 are of utmost importance for cell
motility and development, and we have recently shown that rapid
and dynamic turnover between intracellular and cell surface
expression is responsible for homing and engraftment by normal
CB CD34⫹/CXCR4⫺ sorted cells.33 We reveal a major difference in
the ratio of intracellular versus cell surface CXCR4 expression
obtained by the leukemic cells and normal CD34⫹ (both CB and
MPB) cells. The latter express relatively low levels of cell surface
CXCR4 in comparison to high intracellular levels. Contrary to that,
the leukemic cells express high levels of cell surface CXCR4 but
only low levels of intracellular receptor. The enhanced expression
of cell surface CXCR4 by the leukemic cells may explain their
unique response to low levels of SDF-1 in both migration and
homing into nonirradiated recipient mice. Eventually, this may also
lead to the rapid proliferation of the malignant cells, since
preliminary results indicate that the leukemic cells exhibit increased proliferation and survival only in response to low levels of
SDF-1. This is in contrast to normal human CD34⫹ progenitor cells
whose survival has been shown to be supported only by high levels
(100 ng/mL) of SDF-1 but not by lower levels.19
Total body irradiation is widely used clinically as well as in
experimental models as a crucial conditioning procedure preceding
stem cell transplantation. Ballen et al35 have shown that following
transplantation of CB MNCs, engraftment of the CD34⫹ population is increased when recipient mice were irradiated prior to
injection. We show that precursor-B ALL cells do not require
preconditioning of the host by sublethal irradiation to successfully
home to the BM of NOD/SCID mice. Conditioning of mice with
DNA-damaging agents such as ionizing irradiation causes an
increase in SDF-1 expression and secretion by immature mesenchymal osteoblasts, adipocytes, fibroblasts, and endothelial cells
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2906
BLOOD, 15 APRIL 2004 䡠 VOLUME 103, NUMBER 8
SPIEGEL et al
within the BM.8 We suggest that, because of their increased
sensitivity to SDF-1, the leukemic cells exhibit unique homing
abilities and are able to home to the BM of nonirradiated mice, in
which SDF-1 levels are low. However, homing of the leukemic
cells is reduced when mice are irradiated and SDF-1 levels are
increased, probably because the leukemic cells have reduced
migratory capacity toward high levels of SDF-1. Normal human
CD34⫹ cells respond poorly to low concentrations of SDF-1 and,
therefore, require irradiation that will increase SDF-1 levels in the
BM of mice that received transplants.
MMP-2 and -9 secretion in response to SDF-1 has been shown
to be involved in transmatrigel migration of immature human
CD34⫹ cells as well as in stem cell mobilization and recruitment to
injured murine liver, suggesting a possible role for MMPs in
hematopoietic cell motility.34,44,45 We show that, unlike CB CD34⫹
cells, MMP secretion by the leukemic cells is high even without
stimulation with SDF-1. This finding, together with the increased
migration of the leukemic cells in response to relatively low
concentrations of SDF-1, may explain the potential of the malignant cells to home into the BM of nonirradiated mice, because their
ability to penetrate through the ECM is optimal even without
stimulation with increased SDF-1 levels (due to irradiation).
Furthermore, substrate specificity of the different enzymes may
also account for the differences in homing capacities because
MMP-2, which is secreted by the leukemic cells, has been shown to
degrade fibronectin and various types of collagen that are not
degraded by MMP-9 (secreted by the normal CD34⫹ cells).36
The major integrins VLA-4, VLA-5, and LFA-1 expressed by
CD34⫹ cells are activated by SDF-1 and are necessary for
successful engraftment of NOD/SCID mice.22 SDF-1 and CXCR4
are involved in regulation of ␤1 integrin function in precursor-B
ALL cells.24 We found that VLA-4 has a unique and crucial role in
the homing of precursor-B ALL cells to the BM; however, homing
of normal CB CD34⫹ cells is equally dependent on all 3 major
integrins.15 A distinct role for the different integrins may also
explain the need for TBI in the homing process of normal
progenitors, because it was recently shown that irradiation alters
the expression of adhesion molecules on the lumen of BM
microvessels.46
CXCR4 is a 7-transmembrane receptor coupled to a heterotrimeric GTP-binding protein. PTX is a known inhibitor of the G␣i
subunit. We have previously shown that in vivo homing of normal
CD34⫹ cells is not inhibited by PTX.15 However, we report here
that both migration and homing of precursor-B cells were inhibited
following pretreatment with PTX. Inhibition of in vitro migration
in transwells but not homing of normal CD34⫹ cells by PTX
remains an unresolved issue. One possible explanation is that
different signaling pathways are activated in the 2 processes.
Alternatively, it is also possible that homing of PTX-treated CD34⫹
cells is mediated by internal CXCR4. Indeed we have recently
shown that homing is mediated by intracellular CXCR4 that can
rapidly be expressed on the cell surface.33 Interestingly, PTX has
been shown to up-regulate CXCR4 levels and cause mobilization
of leukocytes from the BM to the peripheral blood.47 Preliminary
results indicate that washing affects CXCR4 levels in PTXpretreated CD34⫹ cells (data not shown). Therefore, it could be that
on cell injection intracellular CXCR4 molecules in PTX-pretreated
cells that are not affected by the inhibitor are relocated to the cell
surface, thus enabling successful homing. If so, the low levels of
internal reservoirs of CXCR4 might explain the inability of
precursor-B ALL cells to home to the BM following treatment
with PTX.
Rho GTPases control a wide variety of signal transduction
pathways and have a pivotal role in regulating actin cytoskeleton
rearrangement.28 We show that small GTP binding proteins from
the Rho family are involved in the migration process of precursor-B ALL cells, because Toxin-B inhibited both in vitro migration
and in vivo homing. However, migration and homing of normal
CD34⫹ cells was significantly less affected by Toxin-B, indicating
that different signaling pathways are activated in SDF-1–induced
precursor-B ALL and immature CD34⫹ cell motility. The PKC
signaling pathway was shown to be essential in the migration and
adhesion of neutrophils induced by the CXC chemokine IL-8,
because both activities were blocked in a dose-dependent manner
by CC, a broad-range PKC inhibitor.39 CC inhibited the migration
and homing of both CD34⫹ and precursor-B ALL cells in a similar
manner, demonstrating a partial overlap in CXCR4-induced signaling in both populations involving the PKC pathway.
Our results demonstrate that in normal CD10⫹/CD19⫹ precursor-B cells the signaling pathways activated following stimulation
with SDF-1 are similar to those activated in normal CD34⫹ cells
but differ from those of leukemic precursor-B ALL cells. This
finding indicates that the differences reported here are most
probably based on changes acquired by the malignant cells and not
merely differences between primitive multilineage precursors and
B-lineage progenitors. In summary, we show that the mechanisms
of migration and homing of malignant cells differ from those
required for successful homing and migration of normal CD10⫹/
CD19⫹ precursor B and progenitor CD34⫹ cells. This is reflected
by differences in the preconditioning of recipient mice, response to
various SDF-1 concentrations, cell surface, and intracellular levels
of CXCR4, involvement of adhesion molecules and signaling
pathways activated. These findings provide new insight on leukemic cell biology and may enable the development of novel
therapeutic protocols aimed at preventing cancer cell proliferation
and dissemination while only minimally affecting normal hematopoietic stem cells in patients with ALL.
Acknowledgments
We thank Prof M. Freedman (The Hospital for Sick Children,
Toronto, Canada) for providing the cell lines A1, B1, BRE, and G2,
and Prof H. Ben-Bassat and Mrs T. Shlomai (Hadassah Medical
School, Jerusalem, Israel) for providing the Nalm-6 cells. We
would also like to thank Prof R. Henschler and Dr K. Aktories for
providing the Clostridium difficile toxin B-10463 inhibitor, and
Prof N. Fujii for providing the CXCR4 antagonists T-22 and T-140.
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2004 103: 2900-2907
doi:10.1182/blood-2003-06-1891 originally published online
November 20, 2003
Unique SDF-1−induced activation of human precursor-B ALL cells as a
result of altered CXCR4 expression and signaling
Asaf Spiegel, Orit Kollet, Amnon Peled, Loya Abel, Arnon Nagler, Bella Bielorai, Gideon Rechavi,
Josef Vormoor and Tsvee Lapidot
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