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Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences and The Ela Kodesz Institute for Research on Cancer Development and Prevention, Tel Aviv University, Tel Aviv, Israel;
Pediatric Hemato-Oncology Department, Chaim Sheba Medical Center, Sackler School of Medicine, Tel Aviv University, Tel-Hashomer, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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to up-regulate CXCR4 expression on the tumor cells. The
reduction in CXCR4 expression following short term exposure to
recombinant human SDF-1 can be recovered as a result of de novo
receptor synthesis. Recombinant human SDF-1 induces the migration of
CXCR4-expressing SH-SY5Y neuroblastoma cells in CXCR4- and
heterotrimeric G protein-dependent manners. Furthermore, SH-SY5Y cells
interact at multiple levels with bone marrow components, as evidenced
by the fact that bone marrow-derived constituents promote SH-SY5Y cell
migration, adhesion to bone marrow stromal cells, and proliferation.
These results suggest that SH-SY5Y neuroblastoma cells are equipped
with adequate machinery to support their homing to the bone marrow.
Therefore, the ability of neuroblastoma tumors to preferentially form
metastases in the bone marrow may be influenced by a set of complex
CXCR4-SDF-1 interactions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanisms responsible for selecting the bone marrow as a preferential metastatic site by different malignancies are not well understood. In our efforts to better understand the mechanisms that mediate the homing of tumor cells to the bone marrow, we focused on neuroblastoma tumors. Neuroblastoma is an embryonal malignant tumor of neural crest origin that most commonly arises in the adrenal gland. It is a common solid tumor in children, the median age of diagnosis being 2 yr. In children diagnosed with localized disease or before 1 yr of age, the disease is often curable. In contrast, older children with disseminated disease (International Neuroblastoma Staging System stage IV) have a poor outcome (4, 5, 6, 7, 8, 9).
Metastatic dissemination of neuroblastoma occurs by hemogenous and lymphatic pathways. The hemogenous spread is most frequently noted in bone marrow, bone, liver, and skin (4, 5, 6, 7, 8, 9). Bone marrow involvement by neuroblastoma cells can be detected by a standard morphologic examination of aspirates and biopsies in approximately 65% of children diagnosed with stage IV disease. Recent findings demonstrated that quantification of neuroblastoma cells in bone marrow during induction chemotherapy provides prognostic information that can identify patients with a very high risk disease (6, 7). Moreover, a sequential molecular detection of minimal residual disease in bone marrow of neuroblastoma patients indicated that persistence of minimal residual disease in the bone marrow might predict poor prognosis in advanced neuroblastoma (8).
Bone marrow metastasis, as a key prognostic factor for neuroblastoma, illustrates the need for better understanding the mechanisms involved in bone marrow metastasis formation by neuroblastoma cells. It is, therefore, extremely important to study the chain of events leading to the homing of cancer cells to the bone marrow as well as the interactions between bone marrow-derived microenvironmental factors and tumor cells. A comprehensive understanding of these relationships may lead to the development of new therapeutic modalities with the capacity to block the metastatic cascade.
The bone marrow-seeking nature of neuroblastoma cells suggests that specific stimuli support the homing of the tumor cells to the bone marrow and their ability to establish metastases at that site. The working hypothesis of our study was that neuroblastoma cells may use a mechanism similar to that used by hemopoietic stem cells (HSC)3 to home to the bone marrow. The homing of HSC to the bone marrow is mediated via an interaction between the CXCR4 receptor expressed by the HSC and the corresponding ligand, stromal cell-derived factor-1 (SDF-1), a CXC chemokine secreted by bone marrow stromal cells (10, 11, 12, 13, 14, 15). In similarity to HSC, it is possible that neuroblastoma cells use a CXCR4-SDF-1-based interaction to preferentially home to the bone marrow.
Bone marrow metastasis formation requires not only the homing of the tumor cells to the bone marrow, but also successful establishment of micrometastases at this site. Key events in this process may be increased adhesion to bone marrow cells, followed by proliferation of the tumor cells. HSC engraftment and homing require adhesion-mediated arrest of the cells on the bone marrow microvasculature. The integrins LFA-1, very late Ag 4 (VLA-4), and VLA-5 were implicated in this step (16, 17, 18, 19, 20). Most importantly, recent findings showed that SDF-1 activates these integrins, thus facilitating HSC transendothelial/stromal migration and engraftment of NOD/SCID mice (15). Bone marrow metastasis formation by neuroblastoma cells may be mediated by similar processes.
Previous studies demonstrated the expression of CXCR4 on a few neuroblastoma cell lines (21, 22, 23). In our study we identified CXCR4 expression as a possible general characteristic of neuroblastoma cells. We have further analyzed the regulation of neuroblastoma-expressed CXCR4 by SDF-1 and characterized the ability of SH-SY5Y neuroblastoma cells to interact at multiple levels, including that of migration, with bone marrow constituents.
Our results suggest that neuroblastoma cells are equipped with a bone marrow homing system similar to that of HSC, and that SDF-1 may mediate the establishment of bone marrow metastasis by neuroblastoma cells. Moreover, a SDF-1-induced down-regulation of CXCR4 suggests that following the homing of the tumor cells through a SDF-1-based gradient to the bone marrow, the expression of CXCR4 is down-regulated by SDF-1 at the metastatic site. The combined effects of SDF-1 on adhesion and reduced CXCR4 expression may facilitate bone marrow metastases formation and prevent the exit of the tumor cells to other SDF-1-expressing sites. Together, these results suggest that the ability of neuroblastoma cells to home to the bone marrow may be influenced by an equilibrium that exists between a set of different CXCR4-SDF-1 interactions that occur at the site of primary tumor and at the bone marrow.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human neuroblastoma cell lines (SH-SY5Y, CHP126, NHB, LAI55N, KELLY, SK-NMC, NBL-WN, and SK-NSH) were maintained in RPMI 1640 supplemented with 20% FCS, 100 U/ml streptomycin, 12.5 U/ml nystatin, 100 U/ml penicillin, 2 mM L-glutamine, 1 mM sodium pyruvate, a 1/100 dilution of nonessential amino acids (all purchased from Biological Industries, Beit Ha’emek, Israel), and 5 x 10-5 M 2-ME (Sigma, St. Louis, MO). Jurkat T lymphocytes from acute T cell leukemia were maintained under similar conditions. The MBA 2.1 mouse bone marrow stromal cell line was a gift from Prof. D. Zipori (Department of Immunology, Weizmann Institute of Science, Rehovot, Israel), and was maintained in DMEM supplemented with 10% FCS, 100 U/ml streptomycin, 12.5 U/ml nystatin, 100 U/ml penicillin, and 2 mM L-glutamine.
Flow cytometric analysis
Cells (5 x 105) were incubated for 45 min at 4°C with a primary Ab (10 µg/ml mouse anti-human CXCR4 mAb 12G5 (R&D Systems, Minneapolis, MN), 40 µg/ml mouse anti-VLA-4 mAb (Serotec, Oxford, U.K.), or 20 µg/ml mouse anti-VLA-5 mAb (Serotec)). Following a wash with cell sorter medium (CSM: RPMI 1640 supplemented with 5% FCS and 0.01% sodium azide), the cells were incubated for 45 min at 4°C with FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Following an additional wash, Ag expression on 5000 live cells was determined using a Becton Dickinson FACSort (Mountain View, CA) and CellQuest software. Baseline staining was obtained by adding CSM to the cells instead of primary Ab.
Determination of SDF-1 production by RT-PCR
Total RNA was isolated from cells using the EZ RNA kit (Biological Industries). mRNAs were reverse transcribed with Expand Reverse Transcriptase (Roche, Basel, Switzerland) for 45 min at 42°C, and the resulting cDNA was subjected to PCR with a thermal cycler (the human SDF-1 reaction: 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min for two cycles, followed by 41 cycles of 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min; the mouse SDF-1 reaction: 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min for 43 cycles). For human SDF-1 the forward primer (5'-GGGGGAATTCCATGAACGCCAAGGTCGTGGTC-3') annealed to nt 78–99 of the mRNA, and the reverse primer (5'-GGGGTCTAGAGGGCATGGATGAATATAAGCTGC-3') annealed to nt 551–573 of the mRNA. For mouse SDF-1 the forward primer (5'-CACTTTCACTCTCGGTCCAC-3') annealed to nt 1–20 of the mRNA, and the reverse primer (5'-GCTCCTCCTGTAAGTTCCTC-3') annealed to nt 381–400 of the mRNA.
Immunoslot analysis of SDF-1 production
SH-SY5Y cells were plated in a six-well plate for 48 h in growth medium. MBA 2.1 cells were plated in 25-cm2 tissue culture flasks for 72 h in growth medium. The supernatants were collected, and different volumes were slot blotted on nitrocellulose (Schleicher & Schuell, Dassel, Germany) and subjected to overnight incubation with 6 µg/ml goat anti-human SDF-1 (R&D Systems) that recognizes both human and murine SDF-1. Following washings, the nitrocellulose was incubated with 0.22 µg/ml biotin-conjugated donkey Abs to goat IgG, then with 1/10,000 dilution of streptavidin-HRP (Jackson ImmunoResearch Laboratories). Chemiluminescence signal was detected using an ECL detection system (Amersham, Little Chalfont, U.K.).
SDF-1 neutralization
SH-SY5Y cells were plated in a 24-well plate (3 x
105 cells/well) for 8–12 h in growth medium
supplemented with 15% FCS. To neutralize SDF-1 activity, 20 µg/well
goat IgG against human SDF-1 (R&D Systems) was added to the well.
The procedure was repeated for a total of five times at 8-h intervals.
The controls included PBS in a similar volume to that of the Abs and 20
µg/well (in a similar volume) of normal goat IgG (R&D Systems). Four
hours after the last addition, the expression of CXCR4 was determined
by flow cytometric analysis as described above. Statistical analysis
was performed using Student’s t test.
Induction of reduction in CXCR4 expression by short term exposure
to recombinant human SDF-1 (rhSDF-1)
All the experimental steps were performed while the cells were
in suspension. Cells (5 x 105) were
incubated with different concentrations of rhSDF-1 (R&D Systems)
diluted in BSA medium (RPMI 1640 containing 5% BSA and 25 mM HEPES),
while no rhSDF-1 was added to control tubes. The cells were
incubated at 37°C for 1 h, washed in CSM containing 0.02%
sodium azide, and stained as described above. Baseline staining was
obtained by adding CSM to the cells instead of anti-CXCR4 Ab.
Analysis performed at 4°C indicated that the preincubation of the
cells with SDF-1 did not prevent the Abs from binding to CXCR4 on the
cell surface of the cells. Statistical analysis was performed using
Student’s t test.
Induction of CXCR4 re-expression on the plasma membrane
The procedure used to determine receptor re-expression was
similar to that used to induce reduction in CXCR4 expression, except
cells were allowed to recover following rhSDF-1 (100 ng/ml)-induced
reduction in receptor expression. Receptor recovery was performed by
washing the cells and incubating them in chemokine-free medium for
2 h at 37°C. To determine whether receptor re-expression was the
result of receptor de novo synthesis, receptor recovery was performed
in the presence of 10 µg/ml cycloheximide (Sigma). Following the
incubation, the cells were washed and labeled as described above.
Statistical analysis was performed using Student’s t
test.
CXCR4 cloning and transfection
CXCR4 was cloned from SH-SY5Y cells using the RT-PCR method as described above. The cDNA was subjected to 32 cycles of PCR with a thermal cycler (94°C for 1 min, 54°C for 1 min, and 72°C for 1 min for two cycles, followed by 30 cycles of 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min). The forward primer (5'-GGAAGCTTCATGGAGGGGATCAGTATATAC-3') annealed to nt 87–109 of the mRNA, and the reverse primer (5'-GGTCTAGATTAGCTGGAGTGAAAACTTG-3') annealed to nt 1127–1147 of the mRNA. The primers were designed to include restriction sites at their ends (HindIII and XbaI for the forward and reverse primers, respectively) to enable cloning to pcDNA3. The CXCR4-expressing vector and the control vector were transfected by electroporation with Electro cell manipulator 830 (BTX-Genetronics, San Diego, CA) into SH-SY5Y cells that lost expression of the endogenic receptor.
Migration assays
The migration of SH-SY5Y cells was assessed by a 48-well
microchemotaxis chamber technique as previously described
(24). Briefly, the lower compartment of the chamber was
loaded with aliquots of BSA medium or with different concentrations of
rhSDF-1 and MBA 2.1-derived conditioned media diluted in BSA medium.
The MBA 2.1-derived conditioned media were produced by plating the
cells to 
80% confluence, followed by two washings in serum-free
DMEM and the addition of serum-free DMEM containing 0.1% BSA to the
cells for 24–48 h. The conditioned media used in the migration assays
were x10 concentrated by Vivaspin 4 (cutoff 5 kDa; Vivascience,
Binbrook, U.K.), followed by 1/1 to 1/5 dilution in BSA medium. The
upper compartment of the chamber was loaded with cells (resuspended in
BSA medium). To determine whether SH-SY5Y migration was heterotrimeric
G protein-mediated, the cells were preincubated with 100 ng/ml
pertussis toxin (PTx; Sigma) for 2 h at 37°C, washed, and loaded
in the chemotaxis chamber. The two compartments were separated by a
5-µm pore size polycarbonate filter (Osmonics, Livermore, CA) coated
with 50 µg/ml rat collagen type I (Collaborative Biomedical Products,
Bedford, MA). Following 6-h incubation at 37°C the filter was
removed, fixed, and stained with a Diff-Quik kit (Dade Behring,
Düdingen, Switzerland). Cells migrating through to the
underside of the filter were counted in five fields (x160–x400) by
light microscopy in triplicate. Statistical analysis was performed
using Student’s t test.
Adhesion assays
MBA 2.1 mouse bone marrow cells (2 x
104/well) and HUVEC (3 x
104/well) were plated for 24 h in a 96-well
plate at a density compatible with monolayer confluence. Activated
HUVEC were established by preincubation with 100 U/ml TNF- and 100
U/ml IFN-
for 4 h at 37°C (25, 26). To allow
rhSDF-1 binding to glycosaminoglycans, cells (in HUVEC, following
activation) were preincubated with 1 ng/ml rhSDF-1 for 1 h,
followed by washing (27, 28). To determine SH-SY5Y
neuroblastoma cell adhesion to MBA 2.1 cells or HUVEC, SH-SY5Y cells
were washed in serum-free medium, incubated in 2 mg/ml HRP solution
(Sigma) for 15 min at 37°C, washed thoroughly, resuspended in
serum-free RPMI, and added to the 96-well plate (1 x
104/well). Following 1-h incubation at 37°C,
unattached cells were removed, and remaining cells were washed three
times with prewarmed PBS. An HRP substrate buffer (containing 0.5 mg/ml
o-phenylenediamine dihydrochloride, 100 mM sodium citrate,
0.5% Triton X-100, and 0.0075%
H2O2) was added for 15 min
in darkness. A 1.25 M H2SO4
solution was added to arrest the colorimetric reaction, and OD was
measured at 490 nm. Statistical analysis was performed using Student’s
t test.
Proliferation assays
MBA 2.1 bone marrow stromal cells were plated in
25-cm2 flasks for 72 h in 5 ml growth
medium. The MBA 2.1-derived conditioned medium was centrifuged and
diluted in fresh medium, as indicated in Fig. 10
. To determine the
ability of MBA 2.1 cell-derived supernatants to affect SH-SY5Y
neuroblastoma cell proliferation, SH-SY5Y cells were plated for 24
h in a 96-well plate in growth medium (104
cells/well). Following a wash of SH-SY5Y cells in serum-free medium,
dilutions of MBA 2.1-derived conditioned medium were added to the
plated SH-SY5Y cells for 48 h at 37°C. The control SH-SY5Y cells
that were not exposed to MBA 2.1-derived conditioned medium were
treated with the same fresh growth medium that was used to dilute the
MBA 2.1-derived conditioned medium. After several washes with PBS, an
alkaline phosphatase substrate buffer (containing 3 mg/ml
p-nitrophenyl phosphate disodium, 50 mM sodium acetate, and
0.4% Triton X-100) was added for 1 h. A 1-M NaOH solution was
added to arrest the reaction and to promote color development. OD was
measured at 405 nm. Statistical analysis was performed using Student’s
t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When metastatic neuroblastoma is diagnosed, bone marrow
micrometastases are usually present (4, 5, 6, 7, 8). The basic
similarity between the pathological process of bone marrow metastasis
formation and the normal process of hemopoietic homing to the bone
marrow led to the specific examination of different components
necessary for this process. The receptor CXCR4 plays a pivotal role in
bone marrow homing of hemopoietic cells (10, 11, 12, 13, 14, 15). A
prerequisite for the usage of a similar mechanism by neuroblastoma
cells is the expression of CXCR4. Previous studies demonstrated the
expression of CXCR4 on a few neuroblastoma cell lines: on SK-NSH and
its noradrenergic subclone SH-SY5Y and on CHP100 (21, 22, 23).
To test the generalized nature of this phenomenon, we determined the
expression of CXCR4 in eight different neuroblastoma cell lines,
including SK-NSH and SH-SY5Y. Flow cytometric analysis indicated that
CXCR4 was expressed by all cell lines (Fig. 1). A variability in receptor expression
was observed in different independent experiments performed on most
cell lines. This phenomenon was characteristic not only for the
percentage of cells expressing CXCR4, but also for the mean
fluorescence values (ranging from 10–50 arbitrary units for different
experiments in which >10% of the cells expressed CXCR4). This
provided evidence for the instability of receptor expression by these
cells. The highest levels of CXCR4 expression, in terms of percentage
of positive cells and mean fluorescence values (ranging between 26–40
arbitrary mean fluorescence values), were observed in the SH-SY5Y
neuroblastoma cell line.
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CXCR4 expression by neuroblastoma cells is down-regulated by neuroblastoma-derived SDF-1
The observed instability of CXCR4 expression in neuroblastoma cell
lines raised the possibility of a regulatory mechanism, in which
neuroblastoma cell-derived SDF-1 down-regulates the expression of CXCR4
on the cells. Similar findings were observed for expression of the
chemokine receptor CCR2 on monocytes and its ligand, monocyte
chemoattractant protein-1 (29). In light of such a
possibility, the expression of SDF-1 by SH-SY5Y cells was determined.
To this end, RT-PCR analysis, using RNA extracted from SH-SY5Y
neuroblastoma cells, was performed. RNA from MBA 2.1 murine bone marrow
stromal cells served as a positive control. Primers for the PCR
reaction were chosen from the edges of the coding regions or from the
5'- or 3'-untranslated region in close proximity to the coding region.
The sizes of the products derived from the RT-PCR reaction performed on
these cells were as expected: 512 and 400 bp for the human SDF-1 (in
SH-SY5Y cells) and murine SDF-1 (in MBA 2.1 cells), respectively (Fig. 2A). These results demonstrate
the presence of SDF-1 mRNA transcripts in SH-SY5Y cells (and in the
control MBA 2.1 cells). SDF-1 expression was also determined at the
protein level by immunoslot analysis of supernatants derived
from SH-SY5Y cells (Fig. 2B). Similarly to control MBA 2.1
cells, SDF-1 was secreted by SH-SY5Y neuroblastoma cells. The protein
secreted by SH-SY5Y cells was further confirmed as SDF-1 by Western
blot analysis (data not shown).
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to increase CXCR4 expression by
blocking the activity of SH-SY5Y-derived SDF-1. To this end, SH-SY5Y
cells were grown for 36 h in the presence of neutralizing Abs
against human SDF-1, followed by determination of CXCR4 expression
by flow cytometry. The cells used for this analysis were SH-SY5Y cells
that expressed minimal levels of the receptors due to a prolonged
growth in culture. The expression level of CXCR4 on these cells
following growth in the presence of goat IgG neutralizing Abs against
human SDF-1 was compared with that of untreated cells and cells
grown in the presence of similar concentrations of normal goat IgG. As
shown in Fig. 3 the neutralization of
SH-SY5Y-derived SDF-1 resulted in a highly significant increase in the
percentage of cells expressing CXCR4, from 18 ± 2% positive
cells in untreated cells to 47 ± 1.8% positive cells in SH-SY5Y
cells treated by neutralizing Abs against SDF-1
(p < 0.001). In contrast, treatment of SH-SH5Y
cells by control nonrelevant goat Abs did not induce the expression of
CXCR4 (17.7 ± 5.2% positive cells for CXCR4 expression).
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CXCR4 is re-expressed on SH-SY5Y neuroblastoma cells following SDF-1 removal
The results presented in Fig. 3
demonstrate that the continuous
growth of SH-SY5Y cells in the presence of tumor cell-derived SDF-1
results in down-regulation of CXCR4 expression by the tumor cells. To
better understand the regulation of CXCR4 by SDF-1, we characterized
the ability of CXCR4 to be re-expressed following SDF-1-induced
down-regulation.
To this end, SH-SY5Y neuroblastoma cells were exposed to different
concentrations of rhSDF-1 for 1 h at 37°C, followed by
determination of receptor expression by flow cytometry. The Jurkat T
lymphocyte line, derived from acute T cell leukemia and expressing
innate CXCR4 (30, 31), was used as a positive control. As
shown in Fig. 4, a 1-h short-term
exposure of the cells to rhSDF-1 induced a potent reduction of CXCR4
expression on both cell lines. The percentage of CXCR4-expressing
SH-SY5Y cells, not exposed to rhSDF-1 ranged in different
experiments from 52–72%. Following exposure to 10 ng/ml rhSDF-1,
the percentage of CXCR4-expressing cells dropped to 34–64%, while
exposure to 500 ng/ml rhSDF-1 resulted in 16–36% CXCR4-expressing
cells. The wide dose range of rhSDF-1 (10, 50, 100, and 500 ng/ml)
used in these experiments demonstrated a dose-dependent reduction of
CXCR4 expression, similar to the dose-dependent response observed in
Jurkat T cells (Fig. 4 and data not shown). The SDF-1-induced
reduction in CXCR4 expression on SH-SY5Y cells was significant at
rhSDF-1 concentrations of 50 ng/ml and above (upon normalization of
values, p < 0.05). Analyses performed at 4°C
indicated that the binding of SDF-1 to CXCR4 did not interfere with
CXCR4 recognition by the Abs to CXCR4 (data not shown), thus precluding
the possibility that the SDF-1-induced reduction in CXCR4 expression
resulted from the fact that the SDF-1 present in the system inhibits
binding of the Abs to CXCR4.
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-induced reduction in CXCR4 expression was followed by
ligand removal and recovery of the cells at 37°C for 2 h in a
chemokine-free medium. As shown in Fig. 5, following this procedure, CXCR4
partial re-expression on SH-SY5Y cells was observed. The percentage of
CXCR4-expressing SH-SY5Y not exposed to rhSDF-1 ranged in different
experiments from 20–63%. Exposure of the cells to 100 ng/ml
rhSDF-1 resulted in decrease in the percentage of CXCR4-expressing
cells to 8–17% (upon normalization of values, p <
0.05). However, following the recovery process the percentage of such
cells was elevated and ranged from 16–36% (p
< 0.05). The partial receptor re-expression was noted in all
experiments performed.
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). Treatment of SH-SY5Y cells with
cycloheximide during the recovery process resulted in inhibition of
receptor recovery (the number of CXCR4-positive cells remained low,
7–18%; p < 0.05), indicating that CXCR4
re-expression on SH-SY5Y neuroblastoma cells following SDF-1-induced
reduction in CXCR4 expression was the result of de novo receptor
synthesis. These results therefore indicate that CXCR4 on neuroblastoma cells may be re-expressed following SDF-1-induced down-regulation. CXCR4 re-expression required ligand removal and resulted from de novo receptor synthesis. This observation suggests that CXCR4 may be regulated in vivo by alternate processes of SDF-1-induced down-regulation or re-expression following SDF-1 removal.
CXCR4-expressing SH-SY5Y neuroblastoma cells migrate to SDF-1 in CXCR4- and heterotrimeric G protein-dependent manners
A prerequisite for a CXCR4-SDF-1-mediated homing of neuroblastoma cells to the bone marrow is the migration of neuroblastoma cells in response to SDF-1. To evaluate the SDF-1-induced migration of CXCR4-expressing SH-SY5Y cells, modified Boyden chamber analysis was established. In preliminary analyses a weak and unstable migration of SH-SY5Y cells to SDF-1 was observed. The instability of the response may very well be due to CXCR4 down-regulation in the course of the migration assay, induced by SDF-1 that is secreted by the tumor cells. Nevertheless, to allow for determination of the ability of SH-SY5Y-expressed CXCR4 to mediate migratory responses we established a SH-SY5Y-based cell system in which receptor transcription was driven by a CMV promoter. In the event that SDF-1-induced down-regulation of CXCR4 occurs at the transcriptional level, it is possible that the expression of transfected CXCR4 may not be down-regulated by SDF-1. To this end, we selected for SH-SY5Y cells that lost the expression of endogenous CXCR4 due to extended growth in culture and transfected them with either a CXCR4-expressing vector or a sham control vector. The resulting cells therefore included cells that expressed stable levels of CXCR4 (termed SH-SY5YHigh CXCR4 cells), and cells that did not express CXCR4 (termed SH-SY5YLow CXCR4 cells). These two cell lines may also allow us to directly elucidate the specific role of CXCR4 in the migration of neuroblastoma cells to SDF-1.
First, to determine whether the transfected CXCR4 expressed by
SH-SY5YHigh CXCR4 cells was biologically active
in a manner similar to that of the endogenous receptor, a
rhSDF-1-mediated down-regulation of CXCR4 on the CXCR4-transfected
cells (SH-SY5YHigh CXCR4 cells) was performed in
conditions similar to those used previously (on parental SH-SY5Y
cells). The results shown in Fig. 6
indicate that the transfected CXCR4 expressed on
SH-SY5YHigh CXCR4 cells is regulated in a
similar manner to that of the endogenous receptor (Fig. 6 vs Fig. 4).
Recombinant human SDF-1 induced a dose-dependent reduction in the
expression of the transfected CXCR4 (Fig. 6; upon normalization of
values, p < 0.05 for down-regulation induced by 10
ng/ml and p < 0.01 for reduction induced by 500 ng/ml
SDF-1). These results indicate that the transfected CXCR4 is
biologically active in a similar manner as endogenous CXCR4, thus
enabling the use of SH-SY5YHigh CXCR4 cells for
further analyses.
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in a dose-dependent manner
(p < 0.001 for migration of the cells to all
rhSDF-1 concentrations vs migration to control medium; Fig. 7A). Optimal migration of
SH-SY5YHigh CXCR4 cells was induced by 50–400
ng/ml rhSDF-1. In contrast, SH-SY5YLow CXCR4
cells did not migrate toward 100 or 200 ng/ml rhSDF-1 (Fig. 7A). These results indicate that the migration of SH SY5Y
neuroblastoma cells to SDF-1 was CXCR4 dependent.
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subclass of G proteins, but also by other members of the
G class (30, 38, 39, 40, 41, 42, 43, 44, 45). To further
characterize the SDF-1-induced migration of SH-SY5YHigh
CXCR4 cells, migration to rhSDF-1 was performed in the
presence of PTx, a specific inhibitor of heterotrimeric G protein
coupling to G protein-coupled receptors, potently inhibiting signaling
through Gi proteins (46). Upon
pre-exposure of SH-SY5YHigh CXCR4 cells to PTx, a
complete inhibition of rhSDF-1-induced migratory response was
observed (Fig. 7B). These results indicate that the
migratory response induced by rhSDF-1 on SH-SY5YHigh
CXCR4 cells is mediated by heterotrimeric G proteins,
possibly Gi, suggesting that the signaling
events are transmitted through the CXCR4 receptor, which is a G
protein-coupled receptor. This receptor was previously shown to
activate cellular responses by inducing the coupling of
Gi proteins to the receptor
(42, 43, 44, 45).
Therefore, our results provide evidence for a CXCR4-SDF-1-driven
migration of neuroblastoma cells that is dependent on heterotrimeric G
protein coupling and is possibly mediated by
Gi proteins. These observations suggest that
neuroblastoma cells are equipped with the required machinery that may
enable their preferential migration to the bone marrow, possibly
leading eventually to bone marrow metastasis formation by these tumor
cells.
Neuroblastoma cells interact at multiple levels with bone marrow-derived constituents
The potential contribution of the CXCR4-SDF-1 axis to bone marrow metastasis formation by neuroblastoma cells suggests that neuroblastoma cells have acquired the ability to respond to bone marrow stromal cell-derived factors and possibly to interact with the bone marrow stromal cells directly. To address such a possibility, we have determined the ability of bone marrow cell-derived constituents to induce in neuroblastoma cells properties that are required for productive establishment of bone marrow metastasis, namely migration, adhesion, and proliferation.
The SDF-1-induced migration of SH-SY5Y cells suggests that bone
marrow-derived factors are key inducers of neuroblastoma cell
migration. Support for such a possibility was derived by migration
assays in which conditioned medium derived from MBA 2.1 bone marrow
stromal cells induced the migration of parental (nontransfected)
SH-SY5Y cells that express endogenous CXCR4 (Fig. 8A). However, the migration of
parental SH-SY5Y cells to bone marrow stromal cell-derived conditioned
medium was not uniformly stable and was observed in four of six
experiments performed. Therefore, to further elucidate this issue, we
performed a similar analysis with SH-SY5YHigh
CXCR4 cells that were used in the studies described in
Figs. 6 and 7. These cells have lost the expression of the endogenous
CXCR4 and were transfected by CXCR4 cDNA to highly and stably express
this receptor. As described above, the transfected CXCR4 may be under a
different transcriptional regulation than the endogenous receptor,
possibly making it less susceptible to SDF-1-induced regulation. In
these experiments the migration of SY5YHigh CXCR4
cells in response to bone marrow stromal cell-derived conditioned
medium was determined. These experiments (Fig. 8B) indicate
that the migration of SH-SY5Y to factors that are secreted by bone
marrow stromal cells was highly potent and stable. These results
demonstrate that under conditions of stable expression and complete
functionality of CXCR4, neuroblastoma cells potently migrate to bone
marrow-derived constituents, suggesting a partial role for CXCR4 in
this process.
|
Similarly to studies of neuroblastoma cells (47, 48, 49), it
was shown in our experiments that SH-SY5Y express the VLA-4 and VLA-5
integrins (data not shown). As a key event in the formation of bone
marrow metastases, the adhesion of SH-SY5Y to bone marrow stromal or
endothelial cells was evaluated. To determine the contribution of SDF-1
to this process, adhesion assays were performed on stromal and
endothelial cells that were either uncoated or coated, by rhSDF-1.
To this end, MBA 2.1 stromal cells and HUVEC were plated for 24 h,
washed, and incubated for 1 h with 1 ng/ml rhSDF-1. It has been
shown that rhSDF-1 binds to glycosaminoglycans on cell membranes and
in extracellular matrix under these conditions (27, 28).
Parental (nontransfected) SH-SY5Y cells were then added to the cells
and allowed to adhere for 1 h (see Materials and
Methods). The results of these experiments (Fig. 9) indicate that rhSDF-1 significantly
augmented SH-SY5Y cell adhesion to bone marrow stromal cells (upon
normalization of values, p < 0.05), while the adhesion
of neuroblastoma cells to vascular endothelium (either not activated or
activated by TNF- and IFN-) remained unaffected by SDF-1.
|
The final step necessary for neuroblastoma metastasis formation at the
bone marrow is the ability of neuroblastoma cells to use growth factors
that are available at the metastatic site and thrive under the existing
conditions, leading to elevated cell proliferation (50).
Previous studies indicated that conditioned medium from bone marrow
cells supports the proliferation of SK-NSH neuroblastoma cells. The
proliferative effect was primarily induced by conditioned medium
derived from nonadherent bone marrow cells (hemopoietic cells) and only
minimally by conditioned medium derived from adherent bone marrow cells
(stromal cells) (51, 52). To further evaluate the validity
of these observations to our cell system, we analyzed the ability of
MBA 2.1 bone marrow stromal cell-derived conditioned medium to induce
SH-SY5Y cell proliferation. The results, demonstrated in Fig. 10, indicate that bone marrow stromal
cell-derived conditioned medium enhanced the proliferation of parental
(nontransfected) SH-SY5Y neuroblastoma cells (upon normalization of
values, p < 0.05). This finding provides further
evidence for the significant role of the bone marrow microenvironment
in supporting the final step of the sequential and complex process of
bone marrow metastasis formation by neuroblastoma cells.
|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we addressed the possible roles of the above three mechanisms in the preferential formation of bone marrow metastases by neuroblastoma cells. Our research was based on the hypothesis that neuroblastoma cells use HSC-like properties to home to the bone marrow, to adhere to specific cells at this site, and to proliferate at this metastatic site. Recent studies indicate that HSC engraftment and repopulation in the bone marrow are dependent on a SDF-1-mediated attraction of CXCR4-expressing CD34+ stem cells to the bone marrow. Furthermore, SDF-1 was demonstrated to induce firm adhesion and transendothelial migration, which were dependent on the LFA-1 and VLA-4 integrins (10, 11, 12, 13, 14, 15).
In view of these findings we focused in our study on the possibility that the homing of neuroblastoma cells to the bone marrow and the ability of the tumor cells to establish bone marrow metastases are CXCR4-SDF-1-dependent processes. The possible role of CXCR4-SDF-1 interactions in bone marrow metastasis formation by neuroblastoma cells was supported by previous findings on the expression of CXCR4 by the neuroblastoma cell lines SK-NSH, its noradrenergic subclone SH-SY5Y, and CHP100 (21, 22, 23). Our study supports the likelihood of a CXCR4-SDF-1-dependent preferential establishment of bone marrow metastases by neuroblastoma cells. This is indicated by the following novel findings:
1) The expression of CXCR4 is a possible general characteristic of neuroblastoma cells. This observation suggests that the expression of CXCR4 is of key importance for the pathogenesis of this disease. However, on most neuroblastoma cell lines instability of CXCR4 expression was noted, indicating that the expression of this receptor is under tight regulation.
2) SH-SY5Y neuroblastoma cells express not only CXCR4, but also its ligand, SDF-1, suggesting that neuroblastoma-derived SDF-1 may regulate the expression of CXCR4 by neuroblastoma cells. A recent publication by Bajetto et al. (53) demonstrated that both CXCR4 and SDF-1 are expressed by cortical type I astrocytes, cortical neurons, and cerebellar granule cells. Since neuroblastoma cells are of neural crest origin, these observations raise the possibility that the expression of both CXCR4 and its ligand by neuroblastoma cells may be a general characteristic of neuroblastoma cells.
3) CXCR4 expression on SH-SY5Y neuroblastoma cells is tightly regulated by neuroblastoma-derived SDF-1. The exposure of a continuous culture of SH-SY5Y cells to neutralizing Abs to SDF-1 resulted in up-regulation of CXCR4 expression on these cells. These results indicate that tumor cell-derived SDF-1 negatively regulates, possibly through autocrine circuits, the expression of CXCR4 on neuroblastoma cells, suggesting that a similar scenario takes place under in vivo conditions.
4) CXCR4 is re-expressed on SH-SY5Y neuroblastoma cells following SDF-1
removal. A prominent and dose-dependent reduction in CXCR4 expression
was noted following short term exposure of SH-SY5Y cells to rhSDF-1.
In similarity to SDF-1-induced CXCR4 down-regulation in hemopoietic
cells (as confirmed by confocal analysis (54, 55)), this
SDF-1-induced reduction of CXCR4 expression by SH-SY5Y cells may be the
result of CXCR4 internalization.
Of major importance is the fact that following the short term
rhSDF-1-induced reduction in CXCR4 expression, re-expression of the
receptors on SH-SY5Y neuroblastoma cells was noted after removal of the
ligand and recovery of the cells at 37°C. The recovery of CXCR4 was
partial and resulted from de novo synthesis of the receptor. In
contrast to other chemokine receptors, little evidence is available to
support high level of CXCR4 re-expression on hemopoietic cells
following ligand-induced internalization/down-regulation (32, 33, 34, 54, 55). CXCR4 re-expression on hemopoietic cells following
SDF-1-induced down-regulation was the result of receptor recycling and
was partial. The quick re-expression of CXCR4 on SH-SY5Y neuroblastoma
cells that results from de novo receptor synthesis (following ligand
removal) suggests that the receptor is undergoing a very rapid turnover
once the concentration of ligand at the tumor cell proximity is
reduced. Our findings therefore indicate that CXCR4 on neuroblastoma
cells undergoes a unique and a restricted process of regulation.
5) SH-SY5Y neuroblastoma cells migrate in response to SDF-1 in CXCR4-
and heterotrimeric G protein-dependent manners. To date, the
functionality of neuroblastoma-expressed CXCR4 was demonstrated in a
study describing a SDF-1-induced rapid and transient calcium flux in
SK-NSH neuroblastoma cells (21). Our study is the first to
show that in neuroblastoma cells SDF-1 transmits signals that give rise
to the actual and final readout of chemokine-induced activities, namely
migration. This activity was manifested only by SH-SY5Y cells that were
transfected to express CXCR4 and not by cells transfected by the sham
vector, indicating that the ability of neuroblastoma cells to migrate
to SDF-1 is CXCR4 dependent. In similarity to HSC, T cells, B cells,
neurons, and astrocytes, SDF-1-induced signaling was sensitive to PTx,
indicating that the process is mediated by heterotrimeric G proteins,
possibly by Gi proteins (12, 30, 42, 43, 44, 45, 46). These results suggest that the signaling events are
transmitted through the CXCR4 receptor, a G protein-coupled receptor.
Neuroblastoma cells may therefore use the CXCR4-SDF-1-based machinery
to facilitate their homing to the SDF-1-expressing bone marrow.
6) Neuroblastoma cells interact at multiple levels with bone marrow-derived constituents. Our analysis demonstrates that neuroblastoma cells undergo a complex set of interactions with bone marrow constituents. These interactions occur at multiple levels, including migration in response to bone marrow-derived factors (possibly dependent in part on CXCR4), SDF-1-mediated adhesion to bone marrow cells, and promoted proliferation induced by bone marrow stromal cell-derived components. These interactions provide neuroblastoma cells with the adequate set of requirements that may eventually allow them to form metastases in the bone marrow. These results further indicate that neuroblastoma cells indeed use mechanisms similar to those of HSC that serve to promote their adhesion and suggest that the CXCR4-SDF-1 interaction may support in a preferential manner the establishment of bone marrow metastasis formation by neuroblastoma cells.
The observations made in our study support the idea that, similar to
HSC, the interactions between neuroblastoma-expressed CXCR4 and bone
marrow-derived SDF-1 may be major events in directing neuroblastoma
cells to the bone marrow, supporting their ability to establish
micrometastases at this site. SDF-1, as a major constituent of the bone
marrow may be a key player in such a process not only by mediating the
homing of the tumor cells to the bone marrow, but also by promoting
their adhesion to bone marrow stroma. Furthermore, bone marrow stromal
cell-derived factors were shown to induce neuroblastoma migration in a
manner possibly dependent on CXCR4. In view of these results, it is
important to note that CXCR4/SDF-1 interactions were suggested recently
to account for metastasis formation in other tumors. A study by Muller
et al. (56) demonstrated the involvement of CXCR4 in
breast cancer metastasis formation at sites that highly express
SDF-1. Moreover, mechanisms mediated by CXCR4/SDF-1 interactions
were suggested to take part in trafficking and localization of multiple
myeloma cells in the bone marrow (57).
The SDF-1-induced down-regulation of CXCR4 expression has two important
implications. First, since CXCR4 down-regulation was shown to be
induced by neuroblastoma-derived SDF-1 (Fig. 3), it is possible that in
vivo the expression of the receptor is reduced on tumor cells that
constitute the primary tumor and express SDF-1. Second, the ability of
SDF-1 to induce down-regulation of CXCR4 expression on neuroblastoma
cells (as also manifested by short term exposure to rhSDF-1) implies
that specific regulation of CXCR4 may occur at the bone marrow. The
bone marrow is a key SDF-1-producing site. Therefore, the possibility
exists that the tumor cells home to the bone marrow in a manner that is
dependent on a gradient based on SDF-1 as well as on additional
chemotactic factors. Once at the bone marrow, the high concentrations
of SDF-1 may induce CXCR4 down-regulation, thereby preventing migration
of the tumor cells to any other SDF-1-expressing sites. Combined with
the SDF-1-induced adhesion of neuroblastoma cells to bone marrow
stromal cells and with the promotion of neuroblastoma cell
proliferation that was mediated by bone marrow-derived factors, the
process may result in a preferential establishment of bone marrow
metastases.
In the present report we have provided insight into the regulation of CXCR4 by SDF-1, its re-expression following SDF-1 removal, the CXCR4-mediated migration of SH-SH5Y neuroblastoma cells to SDF-1, and the ability of bone marrow-derived constituents to support neuroblastoma cell migration (possibly through CXCR4), adhesion (through SDF-1), and proliferation. These observations suggest that the ability of neuroblastoma cells to form bone marrow metastasis is regulated by a complex interplay between CXCR4 and SDF-1. A model depicting the mechanisms involved in neuroblastoma bone marrow metastasis formation should take into account the multifaceted set of interactions that exist between CXCR4 and SDF-1. According to such a model, the expression of CXCR4 in the primary tumor is down-regulated by tumor cell-derived SDF-1. However, upon detachment of tumor cells from the primary tumor, the tumor cells may circulate away from the primary tumor in a microenvironment that is essentially depleted of SDF-1. Under these circumstances, CXCR4 may be rapidly re-expressed on the cell membrane as a result of de novo synthesis. As the process proceeds, the tumor cells may encounter a bone marrow-derived SDF-1 chemotactic gradient and therefore may be attracted to the bone marrow. At the bone marrow microenvironment, the tumor cells are exposed again to high SDF-1 concentrations, which induce CXCR4 down-regulation. The reduction in CXCR4 expression may prevent the exit of the tumor cells to other SDF-1-producing metastatic sites. Combined with the activity of bone marrow-derived components that promote migration, adhesion, and proliferation of the neuroblastoma cells, the process eventually results in the efficient establishment of bone marrow metastases.
On the whole, our results suggest that neuroblastoma cells may use mechanisms similar to those of HSC to home to the bone marrow and preferentially establish metastases at this site. CXCR4-SDF-1 interactions may be key determinants of this process, supporting many of the sequential events that are involved in bone marrow metastasis formation. Our results illustrate the possible complexity of the neuroblastoma metastatic process and suggest that further investigations should be performed to characterize the roles of CXCR4 and SDF-1 in this process. Better understanding of the role of CXCR4-SDF-1 interactions in the preferential establishment of bone marrow metastases by neuroblastoma cells may lead the way to the design of novel therapeutic tools for the treatment of neuroblastoma patients.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Adit Ben-Baruch, Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel-Aviv 69978, Israel. E-mail: aabb{at}post.tau.ac.il
3 Abbreviations used in this paper: HSC, hemopoietic stem cells; CSM, cell sorter medium; PTx, pertussis toxin; SDF-1, stromal cell-derived factor-1; rhSDF-1, recombinant human SDF-1; VLA, very late Ag.
Received for publication April 18, 2001. Accepted for publication August 6, 2001.
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G. G. Vaday, S.-B. Hua, D. M. Peehl, M. H. Pauling, Y.-H. Lin, L. Zhu, D. M. Lawrence, H. D. Foda, and S. Zucker CXCR4 and CXCL12 (SDF-1) in Prostate Cancer: Inhibitory Effects of Human Single Chain Fv Antibodies Clin. Cancer Res., August 15, 2004; 10(16): 5630 - 5639. [Abstract] [Full Text] [PDF]
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B.-C. Lee, T.-H. Lee, S. Avraham, and H. K. Avraham Involvement of the Chemokine Receptor CXCR4 and Its Ligand Stromal Cell-Derived Factor 1{alpha} in Breast Cancer Cell Migration Through Human Brain Microvascular Endothelial Cells Mol. Cancer Res., June 1, 2004; 2(6): 327 - 338. [Abstract] [Full Text] [PDF]
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 Y. S. Haviv, W. J. van Houdt, B. Lu, D. T. Curiel, and Z. B. Zhu Transcriptional targeting in renal cancer cell lines via the human CXCR4 promoter Mol. Cancer Ther., June 1, 2004; 3(6): 687 - 691. [Abstract] [Full Text] [PDF]
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J. F. Ji, B. P. He, S. T. Dheen, and S. S. W. Tay Interactions of Chemokines and Chemokine Receptors Mediate the Migration of Mesenchymal Stem Cells to the Impaired Site in the Brain After Hypoglossal Nerve Injury Stem Cells, May 1, 2004; 22(3): 415 - 427. [Abstract] [Full Text] [PDF]
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S. Tavor, I. Petit, S. Porozov, A. Avigdor, A. Dar, L. Leider-Trejo, N. Shemtov, V. Deutsch, E. Naparstek, A. Nagler, et al. CXCR4 Regulates Migration and Development of Human Acute Myelogenous Leukemia Stem Cells in Transplanted NOD/SCID Mice Cancer Res., April 15, 2004; 64(8): 2817 - 2824. [Abstract] [Full Text] [PDF]
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R. A. Bartolome, B. G. Galvez, N. Longo, F. Baleux, G. N. P. van Muijen, P. Sanchez-Mateos, A. G. Arroyo, and J. Teixido Stromal Cell-Derived Factor-1{alpha} Promotes Melanoma Cell Invasion across Basement Membranes Involving Stimulation of Membrane-Type 1 Matrix Metalloproteinase and Rho GTPase Activities Cancer Res., April 1, 2004; 64(7): 2534 - 2543. [Abstract] [Full Text] [PDF]
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A. Corcione, N. Arduino, E. Ferretti, L. Raffaghello, S. Roncella, D. Rossi, F. Fedeli, L. Ottonello, L. Trentin, F. Dallegri, et al. CCL19 and CXCL12 Trigger in Vitro Chemotaxis of Human Mantle Cell Lymphoma B Cells Clin. Cancer Res., February 1, 2004; 10(3): 964 - 971. [Abstract] [Full Text] [PDF]
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K. Jankowski, M. Kucia, M. Wysoczynski, R. Reca, D. Zhao, E. Trzyna, J. Trent, S. Peiper, M. Zembala, J. Ratajczak, et al. Both Hepatocyte Growth Factor (HGF) and Stromal-Derived Factor-1 Regulate the Metastatic Behavior of Human Rhabdomyosarcoma Cells, But Only HGF Enhances Their Resistance to Radiochemotherapy Cancer Res., November 15, 2003; 63(22): 7926 - 7935. [Abstract] [Full Text] [PDF]
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 J. B. Rubin, A. L. Kung, R. S. Klein, J. A. Chan, Y. Sun, K. Schmidt, M. W. Kieran, A. D. Luster, and R. A. Segal A small-molecule antagonist of CXCR4 inhibits intracranial growth of primary brain tumors PNAS, November 11, 2003; 100(23): 13513 - 13518. [Abstract] [Full Text] [PDF]
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A. Letsch, U. Keilholz, G. Assfalg, V. Mailander, E. Thiel, and C. Scheibenbogen Bone Marrow Contains Melanoma-reactive CD8+ Effector T Cells and, Compared with Peripheral Blood, Enriched Numbers of Melanoma-reactive CD8+ Memory T Cells Cancer Res., September 1, 2003; 63(17): 5582 - 5586. [Abstract] [Full Text] [PDF]
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 R. J. Phillips, M. D. Burdick, M. Lutz, J. A. Belperio, M. P. Keane, and R. M. Strieter The Stromal Derived Factor-1/CXCL12-CXC Chemokine Receptor 4 Biological Axis in Non-Small Cell Lung Cancer Metastases Am. J. Respir. Crit. Care Med., June 15, 2003; 167(12): 1676 - 1686. [Abstract] [Full Text] [PDF]
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T. Papayannopoulou, G. V. Priestley, H. Bonig, and B. Nakamoto The role of G-protein signaling in hematopoietic stem/progenitor cell mobilization Blood, June 15, 2003; 101(12): 4739 - 4747. [Abstract] [Full Text] [PDF]
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G. Dontu, W. M. Abdallah, J. M. Foley, K. W. Jackson, M. F. Clarke, M. J. Kawamura, and M. S. Wicha In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells Genes & Dev., May 15, 2003; 17(10): 1253 - 1270. [Abstract] [Full Text] [PDF]
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S. Barbero, R. Bonavia, A. Bajetto, C. Porcile, P. Pirani, J. L. Ravetti, G. L. Zona, R. Spaziante, T. Florio, and G. Schettini Stromal Cell-derived Factor 1{alpha} Stimulates Human Glioblastoma Cell Growth through the Activation of Both Extracellular Signal-regulated Kinases 1/2 and Akt Cancer Res., April 15, 2003; 63(8): 1969 - 1974. [Abstract] [Full Text] [PDF]
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 J. H. Hwang, J. H. Hwang, H. K. Chung, D. W. Kim, E. S. Hwang, J. M. Suh, H. Kim, K.-H. You, O-Y. Kwon, H. K. Ro, et al. CXC Chemokine Receptor 4 Expression and Function in Human Anaplastic Thyroid Cancer Cells J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 408 - 416. [Abstract] [Full Text] [PDF]
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T. Murakami, W. Maki, A. R. Cardones, H. Fang, A. Tun Kyi, F. O. Nestle, and S. T. Hwang Expression of CXC Chemokine Receptor-4 Enhances the Pulmonary Metastatic Potential of Murine B16 Melanoma Cells Cancer Res., December 1, 2002; 62(24): 7328 - 7334. [Abstract] [Full Text] [PDF]
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C. J. Scotton, J. L. Wilson, K. Scott, G. Stamp, G. D. Wilbanks, S. Fricker, G. Bridger, and F. R. Balkwill Multiple Actions of the Chemokine CXCL12 on Epithelial Tumor Cells in Human Ovarian Cancer Cancer Res., October 15, 2002; 62(20): 5930 - 5938. [Abstract] [Full Text] [PDF]
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T. Hideshima, D. Chauhan, T. Hayashi, K. Podar, M. Akiyama, D. Gupta, P. Richardson, N. Munshi, and K. C. Anderson The Biological Sequelae of Stromal Cell-derived Factor-1{alpha} in Multiple Myeloma Mol. Cancer Ther., May 1, 2002; 1(7): 539 - 544. [Abstract] [Full Text] [PDF]
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R. S. Taichman, C. Cooper, E. T. Keller, K. J. Pienta, N. S. Taichman, and L. K. McCauley Use of the Stromal Cell-derived Factor-1/CXCR4 Pathway in Prostate Cancer Metastasis to Bone Cancer Res., March 1, 2002; 62(6): 1832 - 1837. [Abstract] [Full Text] [PDF]
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, Cells preincubated without
rhSDF-1
, cells preincubated with rhSDF-1