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Prepublished online as a Blood First Edition Paper on June 7, 2002; DOI 10.1182/blood-2002-01-0031.
NEOPLASIA
From the Stem Cell Biology Program at the James Graham
Brown Cancer Center, University of Louisville, KY; Department of
Pathology and Laboratory Medicine, University of Pennsylvania,
Philadelphia; Children's Hospital Laboratory of Cell and Tissue
Engineering, Jagiellonian University, Cracow, Poland; and Department of
Medicine, University of Alberta and Canadian Blood Services, Edmonton,
Canada.
We hypothesized that the CXC chemokine receptor-4
(CXCR4)-stromal-derived factor-1 (SDF-1) axis may be involved in
metastasis of CXCR4+ tumor cells into the bone marrow and
lymph nodes, which secrete the The Tumor cells expressing functional CXCR4 could use similar mechanisms
for "homing" to the bone marrow to those used by CXCR4+
hematopoietic cells. Involvement of the CXCR4-SDF-1 axis in the metastasis of breast cancer to the SDF-1-rich environment of the bone
marrow and lymph nodes has been reported.10 To assess
whether the CXCR4-SDF-1 axis plays a role in the metastasis of other
tumor cells into bone marrow and lymph nodes, we phenotyped a number of
human tumor cell lines for expression of CXCR4, using immunostaining and fluorescence-activated cell sorter (FACS) analysis. We
found that it was not detectable in most cell lines derived from common human solid tumors (melanoma, breast and lung cancer), but was highly
expressed on the surface of rhabdomyosarcoma (RMS) cells.
There are 2 major histologic subtypes of RMS, alveolar (ARMS) and
embryonal (ERMS). Clinical evidence indicates that ARMS is more
aggressive and associated with a significantly worse outcome than
ERMS.11-20 Genetic characterization of RMS has identified markers that show excellent correlation with histologic subtype. Specifically, ARMS is characterized by the
t(2;13)(q35;q14) translocation in 70% of
cases14 or the variant t(1;13)(p36;q14) translocation in a
smaller percentage of cases.17 These translocations
disrupt the PAX3 or PAX7 genes on chromosome 2 or
1, respectively, and the FKHR gene on chromosome 13, and
generate PAX3-FKHR or PAX7-FKHR fusion genes.
These fusion genes encode fusion proteins, PAX3-FKHR and PAX7-FKHR,
which function as novel transcription factors. The PAX3-FKHR and
PAX7-FKHR fusion proteins demonstrate enhanced transcriptional activity
compared with wild-type PAX3 and PAX7 and are postulated to play an
important role both in survival and cell-cycle dysregulation of ARMS cells.
It is well known that RMS cells, particularly ARMS, may infiltrate the
marrow and, because they can resemble hematologic blasts, may sometimes
be misdiagnosed as acute leukemia cells.11-13 The "contamination" of bone marrow by these cells may compromise its use for autologous transplantation.21 In this study our
hypothesis was that the metastasis of RMS cells into bone marrow is
mediated by the CXCR4-SDF-1 axis.
We focused on the biologic responses of CXCR4+ ARMS and
ERMS cell lines to stimulation by exogenous SDF-1, such as
phosphorylation of signaling proteins, proliferation, survival,
adhesion, expression of MMPs and their tissue inhibitors (TIMPs),
chemotaxis, and chemoinvasion. Our findings implicate the CXCR4-SDF-1
axis in the metastatic behavior of RMS cells.
Cell lines
We also investigated 7 RMS cell lines, including 5 ARMS lines (RH5,
RH28, RH30, RHRKMP-4, and CW9019), and 2 ERMS lines (RD and SMS-CTR).
RMS cells used for experiments were cultured in RPMI-1640 medium
(Sigma, St Louis, MO), supplemented with 100 IU/mL penicillin,
10 µg/mL streptomycin, and 50 µg/mL neomycin (Gibco, Grand Island,
NY) in the presence of 10% heat-inactivated fetal calf serum (Gibco).
The ERMS cell line, RD, transfected with the PAX3-FKHR gene,
was cultured in the presence of the selective agent geneticin (G-418)
as described.14,16,19 The cells were plated at an initial
cell density of 2.5 × 104 cells per Corning flask
(Costar-Corning, Cambridge, MA). In cell cultures the medium
was changed every 48 hours, and cells were cultured in a humidified
atmosphere with 5% CO2 at 37°C.
FACS analysis
Evaluation of adhesion molecules The expression of adhesion molecules on RMS cells was evaluated by FACS. Cells were stained with specific anti-PECAM-1, ICAM-1, VCAM-1, E-selectin, VLA-5, and VLA-4 antibodies detected with PE-conjugated secondary PE-goat antimouse MoAbs as described previously.23 The following antibodies were used for this study: 4G6 (immunoglobulin G2b [IgG2b], mouse antihuman PECAM-1) generously provided by Dr S. Albelda24; R6.5 (BIRR-1), a murine IgG2a MoAb directed against extracellular domain 2 of the ICAM-1 molecule, from Boehringer Ingelheim Pharmaceuticals (Ridgefield, CT25); 4B9, an IgG1 MoAb directed against human VCAM-1, from Dr R. Lobb, Biogen, Cambridge, MA26; and ES2 (IgG1-k mouse antihuman-E-selectin MoAb), provided by Dr R. McEver, University of Oklahoma.27 Antibodies against 6 1 integrin were purchased from Pharmingen (San Diego, CA).
Phosphorylation of intracellular pathway proteins Western blots were done on extracts prepared from RMS cell lines (1 × 107 cells) that were kept in RPMI medium containing low levels of bovine serum albumin (BSA; 0.5%) to render the cells quiescent. The cells were divided and stimulated with optimal doses of SDF-1 or SDF-1 (500 ng/mL) for 1 minute to 2 hours at 37°C and
then lysed (for 10 minutes) on ice in M-Per lysing buffer (Pierce, Rockford, IL) containing protease and phosphatase inhibitors (Sigma). Subsequently, the extracted proteins were separated on either a 12% or
15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and the fractionated proteins were transferred to a
nitrocellulose membrane (Schleicher & Schuell, Keene, NH) as previously
described.23 Phosphorylation of the intracellular kinases,
44/42 mitogen-activated protein kinase (MAPK) (Thr 202/Tyr 204) and
AKT, and STAT-1, -3, -5, and -6 proteins was detected by using
commercial mouse phosphospecific MoAb (p44/42) or rabbit phosphospecific polyclonal antibodies for each of the remainder (all
from New England Biolabs, Beverly, MA) with horseradish peroxidase (HRP)-conjugated goat antimouse IgG or goat antirabbit IgG as a
secondary antibody (Santa Cruz Biotech, Santa Cruz, CA) as
described.7,23 Equal loading in the lanes was evaluated by
stripping the blots and reprobing with appropriate MoAbs: p42/44
anti-MAPK antibody clone no. 9102, anti-AKT antibody clone no. 9272, anti-STAT-3 no. 9132 (New England Biolabs), anti-STAT-1 no. sc-464
and anti-STAT-6 no. sc-1689 (Santa Cruz Biotech), and anti-STAT-5 no.
89 (Transduction Laboratories, Lexington, KY). The membranes were
developed with an enhanced chemiluminescence (ECL) reagent
(Amersham Life Sciences, Little Chalfont, United Kingdom), dried, and
subsequently exposed to film (HyperFilm; Amersham Life Sciences).
Detection of SDF-1 by Western blot analysis SDF-1 protein was detected by using specific rabbit polyclonal antibody (R&D Systems) that was detected with HRP-conjugated goat antimouse IgG or goat antirabbit IgG as a secondary antibody (Santa Cruz Biotech) as previously described.23Isolation of mRNA and RT-PCR The RMS cells were lysed in 200 µL RNAzol (Biotecx Labs, Houston, TX) plus 22 µL chloroform as described.28 Briefly, mRNA (0.5 µg) was reverse-transcribed with 500 U Moloney murine leukemia virus reverse transcriptase and 50 pmol ODN primer complementary to the 3' end of the reported chemokine sequences of SDF-1/CXCL12 (5'-CAC ATG TTG AAC CTC TTG TTT AAA AGC-3'). The resulting cDNA fragments were amplified by using 5 U Thermus aquaticus polymerase and primers specific for the 5' end of SDF-1/CXCL12 (5'-AAC GCC AAG GTC GTG GTC GTG CTG-3').-Actin mRNAs were amplified
simultaneously by using specific primers as reported
previously.28,29 The predicted size of the reverse
transcriptase-polymerase chain reaction (RT-PCR) product for
SDF-1/CXCL12 is 281 bp. Amplified products (10 µL) were
electrophoresed on a 2% agarose gel and transferred to a nylon filter.
The specificity of the amplified products was further confirmed by
Southern blotting (data not shown).
RNase protection assay For analysis of CXCR4 mRNA levels, total mRNA was isolated from cells as described previously.30 An aliquot of total cellular mRNA (10 µg) was hybridized with both [32P]UTP-labeled CXCR4 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) control antisense probes and digested with ribonucleases (RPAII Kit; Ambion Incorporated, Austin, TX). Following denaturing polyacrylamide gel electrophoresis, protected bands were quantified by phosphorimaging (Molecular Dynamics). After normalizing for the uridine content of each riboprobe, the ratio of CXCR4 mRNA to GAPDH mRNA was calculated for each sample as described.30Cell proliferation and apoptosis Cells were plated in culture flasks at an initial density of 104 cells/cm2 in the presence or absence of SDF-1 (300 ng/mL). The cell number was calculated at 12, 24, 36, 48, 60, and 72 hours after culture initiation. In some experiments the
cultures were prolonged up to 7 days. At the indicated time points
cells were harvested from the culture flasks by trypsinization, and the
number of cells was determined with the use of a Bürker
hemocytometer (Buffalo, NY). Apoptosis was evaluated by the
Annexin-V binding assay and intracellular staining for activated
caspase-3, as described.28
Calcium flux studies Briefly, cells were incubated for 30 minutes at 30°C with 1 to 2 µM Fura-2/AM (Molecular Probes, Eugene, OR). After incubation, the cells were washed once, resuspended in loading buffer without fetal bovine serum (FBS), stimulated with SDF-1 (500 ng/mL), and analyzed within 1 hour as described.28
Fluorescent staining of the actin cytoskeleton For the visualization of the actin, cytoskeleton cells were cultured for 12 hours on glass coverslips in RPMI-1640 medium supplemented with 10% FBS in the presence (300 ng/mL SDF-1) or absence of SDF-1. Subsequently, the cells were fixed in 3.7% paraformaldehyde/Ca- and Mg-free PBS for 15 minutes, permeabilized by
0.1% Triton X-100 in PBS for 1 minute at RT, and stained with TRITC-phalloidin at a concentration of 500 ng/mL for 1 hour. The stained cells were examined by using confocal laser scanning
microscopy. All measurements were performed with BioRad MRC 1024 (Hemel
Hempstead, United Kingdom) based on an inverted Nikon Diaphot 300 microscope (Nikon, Canagawa, Japan). The confocal system was
equipped with a 60 × PlanApo 1.4 NA oil immersion objective lens, an
air-cooled 25 mW output Argon-Krypton laser, 3 fluorescence detection
channels, and a 3-color transmitted nonconfocal light
detector. Laser light (568 nm) attenuated to 1% of the
maximum intensity was introduced into the sample. TRITC fluorescence
was collected by using 568 nm excitation. Fluorescence light
passed through a 488/568/645 dichromic filter (T1 BioRad filter block),
a 560 dichromic long pass filter (T2A BioRad filter block), and a 580LP
filter (Croma, VT) and was detected by a photomultiplier (Biorad PMT2).
For each image 3 scans in slow acquisition mode or photon-counting mode were collected with the use of Kalman filtering.
Time-lapse monitoring of the locomotion of individual cells The images of human RMS cells migrating on plastic at 37°C were evaluated with an inverted Hund Wetzlar microscope by using phase-contrast optics (Wetzlar, Germany). Analysis of cell migration was begun 2 hours after seeding, when the cells were already dispersed over the plastic. The locomotion images were recorded with a Hitachi CCD camera (sensitivity, 10 3 1 ×;
Tokyo, Japan) as described.31-34 CW9019, RH30, SMS-CTR, or A204 cells were plated to Corning flasks at a density of 2 to 2.5 × 104 cells/cm2 and were mock-treated or
prestimulated by SDF-1 (300 ng/mL) 30 minutes before recording. The
cell trajectories were constructed from 40 subsequent cell centroid
positions recorded for 200 minutes at 5-minute time intervals. The cell
trajectories were presented in circular diagrams, and the length of the
cell tracks was calculated in addition to the final displacement. Cell
tracks (100) were recorded under the conditions described earlier for
each cell line.
The parameters of cell movement were calculated for each cell by using procedures written in the Mathematica language as described.31-34 The following parameters were determined: (1) the total length of the cell trajectory (in micrometers); the trajectory was a sequence of n straight-line segments, each corresponding to cell centroid translocation within one time interval between 2 successive images; (2) the total length of cell displacement from the starting point to the final cell position (in micrometers); (3) the ratio of cell displacement length to cell trajectory length, here called the coefficient of movement efficiency (CME); (4) the average speed of cell movement defined as total length of cell trajectory/time of recording; and (5) the average velocity of cell displacement calculated from the final cell displacement in a given time.31-34 Transmembrane chemotaxis Cells were seeded into 6-well plates in RPMI medium containing 10% FBS. After adhesion to the dish bottom, the medium was changed and cells were made quiescent for 48 hours with 0.5% BSA in RPMI. The directional movement of cells toward the gradient of SDF concentration across an 8-µm pore polycarbonate membrane was evaluated. The membrane was covered with 50 µL fibronectin (50 µg/mL) and laminin (20 µg/mL) for 2 hours at 37°C and overnight at +4°C. The solution was discarded before assay. Cells were detached with 0.5 mM EDTA (ethylenediaminetetraacetic acid), washed in RPMI medium, resuspended in RPMI medium with 0.5% BSA, and seeded into the upper chamber of a Transwell insert (Costar Transwell; Costar-Corning) at a density of 105 in 200 µL. The lower chamber was filled with SDF at 300 ng/mL concentration, and the 0.5% BSA RPMI medium was used as a negative control. To determine whether the migration was stimulated by the gradient of the chemoattractant, in some samples SDF was also added into the upper chamber to equalize the difference in concentration between chambers. After 48 hours, the insert was removed from the transwell, cells remaining in the upper chamber were scraped off with cotton wool, and the cells that had transmigrated were counted either on the lower side of the membrane or on the bottom of the transwell.Adhesion of RMS cells to fibronectin and laminin Cells were made quiescent for 48 hours with 0.5% BSA in RPMI before incubation with SDF-1 for 24 hours (300 ng/mL). The cells were metabolically labeled overnight with 35S-methionine;
detached with 0.5 mM EDTA; washed twice and suspended in 50 mM Tris
(tris(hydroxymethyl)aminomethane)-Cl buffer, pH 7.4, containing 150 mM
NaCl, 0.5 mM CaCl2, 0.1% glucose, and 1% BSA; and added
directly onto the protein-coated wells (5 × 104/well)
for 30 minutes. The wells were coated with BSA (4 µg/mL), fibronectin
(10 µg/mL), and laminin (20 µg/mL) for 2 hours at 37°C and
overnight at +4°C and blocked with BSA for 2 hours before the
experiment. Following incubation at 37°C, the plates were vigorously
washed 3 times, and adherent cells were dissolved by using 2% SDS. The
SDS solutions were then counted for 35S in a liquid
scintillation counter. The results were normalized against 10% input
for each cell line.
Adhesion to HUVECs RMS cells were labeled before assay with the fluorescent dye calcein-AM and were subsequently added (for 5 minutes) to the 96-well plates covered by human umbilical vein endothelial cells (HUVECs) that had been pretreated with SDF-1 (5 µg/mL).
After the nonadherent cells had been discarded, the cells that adhered to the HUVECs were lysed, and fluorescence was measured by using a
spectrofluorometer as described.35
MMP/TIMP expression To evaluate MMP and TIMP activities, RMS cells were incubated for 24 hours in serum-free media in the absence (control) or presence of SDF-1 (300 ng/mL), and zymography and reverse zymography were
carried out as previously described by us.36 To evaluate gene expression for MMPs and TIMPs in these cells, total RNA was extracted (as described previously).36,37 The conversion
of mRNA to cDNA was carried out using avian myeloblastosis virus reverse transcriptase (AMVRT) (Seigaku America, Ijamsville,
MD), and PCRs were carried out following the "primer dropping"
method. Sequences for human MMP-2, MMP-9, TIMP-1, and TIMP-2 were
obtained from Genbank (Los Alamos, NM) and were used to design primer
pairs, as described by us previously.37
Chemoinvasion assay The ability of various malignant cells to invade the reconstituted basement membrane Matrigel is regarded as an important measure of their metastatic potential. Two ARMS cell lines, RH30 and RH28, were evaluated in a chemoinvasion assay as originally described38 and modified by us.39 Briefly, cells that were preincubated either with the MMP inhibitor o-phenanthroline (0.5 mM, 30 minutes) or in control conditions (media) were loaded onto the upper compartments of Boyden chambers (105 cells/chamber; BD Biosciences, Bedford, MA) and incubated for 3 hours. Cells that invaded the Matrigel barrier toward media alone or toward an SDF-1 gradient (300 ng/mL)
were counted on the undersides of filters after fixation and staining
with crystal violet. A chemoinvasion index was calculated as the ratio
of the number of cells invading the Matrigel toward an SDF-1 gradient to the number of cells invading toward media alone.
Blockade of CXCR4-SDF-1 axis using T140 Some of the adhesion and directional migration experiments were performed on cells preincubated for 30 minutes at 37°C in the presence of 1 µM T140-truncated polyphemusin analog (a gift from Dr N. Fuji, Kyoto University, Japan) or preincubated with anti-CXCR4 (10 mg/mL). In the chemotaxis experiments, cultured bone marrow stroma cells were pretreated with anti-SDF-1 (100 µg/mL; R&D Systems) as described.28Statistical analysis All results are presented as mean ± SEM. Statistical analysis of the data was performed using the nonparametric Mann-Whitney test, with P < .05 considered significant.
CXCR4 is highly expressed in ARMS but not ERMS cell lines First, we phenotyped several human breast and lung cancer as well as melanoma cell lines for expression of CXCR4 and found it to be expressed at a low level on only 1 (HTB 22) of 6 breast cancer cell lines (Table 1). In this cell line less than 15% of cells stained positive for CXCR4. All 5 lung cancer cell lines investigated in this study as well as 8 melanoma cell lines were negative for CXCR4 expression as determined by FACS. In contrast, CXCR4 was expressed on 7 of 7 human RMS cell lines tested (Table 1; Figure 1).
Because the RMS cell lines we investigated included both ARMS and ERMS cell lines, we attempted to correlate the expression of CXCR4 on these cell lines with their histologic phenotype (ARMS versus ERMS). We observed that all 5 ARMS (RH5, RH28, RH30, RHRKMP-4, and CW9019) cell lines stained highly positive for CXCR4 (> 90% of cells). CXCR4 was expressed at lower levels in about 20% of SMS-CTR and RD ERMS cells, and 2 other non-RMS sarcoma lines, A673 and A204, were negative for its expression as determined by FACS (Figure 1). CXCR4 expression increases after transfection of the ERMS cell line with PAX3-FKHR All ARMS cell lines used in our studies displayed translocations typical of ARMS: t(2;13) (RH5, RH28, RH30, and RHRKMP-4) and t(1;13) (CW9019), known to be associated with the fusion protein products PAX3-FKHR and PAX7-FKHR, respectively.11-20Thus, having found a strong correlation between the ARMS phenotype and
the expression of CXCR4, we sought to determine whether the
PAX3-FKHR fusion gene regulates the expression of CXCR4. To address this issue, the ERMS cell line RD, which constitutively expresses a low level of CXCR4, was stably transfected with an expression vector containing the PAX3-FKHR fusion gene cDNA.
We found that RD cells transfected with this fusion gene (Figure 2) expressed substantially more CXCR4, as
determined by FACS, with expression increasing from 20% to 95%, and
as determined by RNase protection, with CXCR4 mRNA expression
increasing by 3 orders of magnitude. In addition, in agreement
with the studies shown below for ARMS and ERMS cell lines,
RD2/PAX3-FKHR cells demonstrated increased response to SDF-1 by (1)
MAPK p42/44 phosphorylation, (2) calcium flux, and (3) directional
chemotaxis (Figure 2).
SDF-1 induces phosphorylation of MAPK p42/44 Next, we turned our attention to the putative role of the CXCR4-SDF-1 axis in regulating the biology of RMS cells. We determined whether stimulation of CXCR4 by SDF-1 induces phosphorylation of MAPK p42/44 and serine-threonine kinase AKT, which had been reported by others and by us to be involved in signaling from activated CXCR4.7,23,40,41 We found that 4 ARMS cell lines (RH28, RH30, CW9019, RH5) and one ERMS cell line (SMS-CTR) responded to SDF-1 by phosphorylation of MAPK p42/44, and an example of this is shown in Figure 3. In contrast, SDF-1 did not stimulate phosphorylation of either serine threonine kinase AKT or STAT-1 to -6 proteins (data not shown).
SDF-1 does not influence proliferation of RMS cell lines Further, we selected 2 ARMS cell lines (RH30, CW9019) and one ERMS cell line (SMS-CTR) that responded to SDF-1 stimulation by phosphorylation of MAPK p42/44 to determine whether their proliferation is affected by SDF-1. We stimulated them with this chemokine or not (control) under serum-free conditions or in media supplemented with 2% BCS. We found that during 72 hours the RMS cell lines proliferated intensively in both types of media (Figure 4). The kinetics of their proliferation were similar and were not affected by the presence of SDF-1 in the culture, even if the cells were cultured up to 7 days (data not shown).
Because the biology of various tumors may be regulated by
autocrine/paracrine axes, we asked whether RMS cells express the CXCR4
ligand, the SDF-1 induces locomotion of individual RMS cells Next, we studied whether SDF-1 influences the locomotion on plastic dishes of 2 selected human ARMS cell lines (CW9019 and RH30), one ERMS (SMS-CTR), and, as a control, a non-RMS sarcoma cell line A204 (not expressing CXCR4), using time-lapse monitoring of movement of individual cells. Figure 5 shows the trajectories of RMS cell migration in the absence (left panel) or presence (right panel) of SDF-1 in the culture medium. Analysis of
these trajectories and mean values and standard errors for the
parameters of cell locomotion are summarized for CW9019, RH30, SMS-CTR,
and A204 cells in Table 2.
Analysis of the time-lapse recording showed that the RMS
cells migrated extensively in the presence of SDF-1, and SDF-1
increased the final cell displacement and the average velocity of cell
displacement (Figure 5). The trajectories of CW9019 cells were
significantly changed in medium containing SDF-1 (Figure 5B), and the
distance of the cell displacement increased almost 1.5 times (Table 2). The trajectories of RH30 cells migrating in the control medium and in
the presence of SDF-1 SDF-1 alters the actin cytoskeleton Examination of the actin cytoskeleton organization by confocal microscopy revealed striking differences between the chemokine-treated and the untreated cells. RMS cells grown in control medium displayed well-developed bundles of F-actin arranged in parallel to the long axis of the cell (Figure 6A,E,I). Incubation of all RMS cells in the SDF-1-containing medium for 12 hours induced a change in the organization of actin filaments and significantly increased both the number and thickness of F-actin bundles (Figure 6C,G,K).
SDF-1 increases migration through fibronectin- or laminin-covered transwell membranes Next, we investigated directed migration of RMS cells through transmembranes covered with fibronectin or laminin (Figure 7). We selected for this study all the cell lines that had responded to SDF-1 stimulation by phosphorylation of MAPK p42/44 (Figure 3). The CXCR4-null A204 cell line was used as a negative control.
We observed that SDF-1 statistically increased the chemotactic activity of the ARMS cell lines RH30, RH28, and CW9019. Interestingly, RH30, which showed the highest spontaneous locomotion on plastic dishes in medium without SDF-1 (Figure 7) and Table 2), owed the strongest chemotaxis to SDF-1, especially through fibronectin-covered membranes. In contrast, the ERMS cell line SMS-CTR, in which locomotion activity on plastic dishes was strongly induced after the addition of SDF-1, showed relatively weak chemotaxis to SDF-1 across transwell membranes covered by fibronectin or laminin. In addition, the RD (ERMS) cell line did not show chemotaxis to SDF-1 (Figure 7E). SDF-1 increases adhesion to fibronectin and laminin We subsequently decided to investigate whether SDF-1 regulates expression/activation of integrins on human RMS. By using FACS analysis, we did not find any change in the level of expression of VLA-4, VLA-5, PECAM-1, or ICAM-1 on RMS cells after incubation with SDF-1 for 24 hours (data not shown). However, we observed that SDF-1 affected the adhesion of RH30, RH28, and CW9019 to fibronectin and laminin (Figure 8A) and that this adhesion correlated with their migration through fibronectin- or laminin-covered transwell membranes (Figure 7).
SDF-1 stimulates MMP-2 secretion in selected ARMS cell lines Extensive experimental data show that MMPs actively contribute to cancer progression and metastasis and that relatively benign cells acquire malignant properties when MMP activity is increased or TIMP activity is diminished.42 Also, it was previously demonstrated by us that SDF-1 stimulates MMP-9 and MMP-2 in normal hematopoietic CD34+ cells, and we postulated that, through this action, SDF-1 modulates homing of hematopoietic progenitors.6-9 In this study we evaluated whether RMS cells express MMPs and whether SDF-1 stimulates their secretion and/or has any effect on TIMP production. We observed MMP-2 as well as TIMP-1 and TIMP-2 transcripts in all the RMS cell lines tested (Table 3). After SDF-1 stimulation pro-MMP-2 activity (as measured by zymography) increased in RH5 and RH28 but not in the other cell lines tested, and pro-MMP-9 activity was not affected (Table 3). However, by using reverse zymography we found that SDF-1 stimulation diminished TIMP-1 and TIMP-2 protein secretion in all RMS lines, with the exception of SMS-CTR (Table 3).
MMP inhibitor diminishes SDF-1-directed chemoinvasion Because tumor cell invasion is one of the characteristics of highly metastatic cells, we evaluated this feature by using a chemoinvasion Matrigel assay. We found that the invasive capability of RH30 and RH28 cell lines increases 2- to 3.5-fold in the presence of an SDF-1 gradient (Figure 8B). Furthermore, we present evidence that a synthetic inhibitor of MMPs, o-phenanthroline, inhibits invasion of these cell lines by approximately 50%, further suggesting that MMPs play a role in the aggressive behavior of ARMS cells (Figure 8B).T140 inhibits adhesion of RMS cells to SDF-1-pretreated HUVECs and directional chemotaxis to bone marrow stroma Finally, we used T140, which is a recently described specific blocking agent for CXCR4,43-46 in an experiment to see whether this molecule affects the metastatic behavior of RMS cells. In one set of experiments we found that T140 inhibited SDF-1-directed adhesion of RH30 and CW 9019 cells to HUVECs (Figure 9A) and chemotaxis of RH30 and RH28 cells toward established cultures of bone marrow stroma fibroblasts (Figure 9B). Similarly, the chemotaxis of RMS cells toward bone marrow stromal cultures was inhibited significantly by preincubating RMS cells with anti-CXCR4 or bone marrow stroma cultures with anti-SDF-1 (data not shown), suggesting that SDF-1 is a major stromal-derived factor that attracts RMS cells.
Discussion RMS is the most common soft-tissue sarcoma of adolescence and childhood and accounts for 5% of all malignant tumors in patients younger than 15 years.11-20 Most of these tumors originate in the head and neck region, the urogenital tract, and the extremities. The 2 major subtypes of RMS (ARMS and ERMS) share a common myogenic differentiation but are not histogenetically related.12,15 Accumulated clinical evidence suggests that ARMS is more aggressive than ERMS. Almost all the reported cases associated with marrow involvement, including those presenting as possible acute leukemia, have been of the ARMS type.13Recently, the involvement of the CXCR4-SDF-1 axis in metastatic cancer has been postulated.47 In this study we did not, however, observe the CXCR4 protein in most lung and breast cancer and melanoma cell lines, but we found it to be highly expressed on the surface of 7 of 7 human RMS cell lines. Hence, we hypothesized that the CXCR4-SDF-1 axis could potentially have an important effect on the metastasis of RMS cells into bone marrow. Bone marrow involvement at diagnosis of RMS in children and adolescents is a poor prognostic sign and represents a continuing challenge to current treatment modalities.21 Moreover, patients with RMS frequently undergo treatment with high-dose chemotherapy combined with autologous bone marrow/peripheral blood stem cell transplantation, and contamination of the transplanted grafts with tumor cells can affect patients' survival rates.21 In this study we found that CXCR4 was expressed by both ARMS and ERMS cells, but its expression was much higher (> 90%) on the former. Thus, CXCR4 expression correlated with the more aggressive ARMS subtype that is associated with poor clinical outcome and a propensity for bone marrow metastasis. Moreover, after stable transfection of the ERMS cell line RD with a PAX3-FKHR gene expression construct, these modified RD cells expressed significantly more CXCR4 mRNA and protein than control transfectants. Examination of the CXCR4 promoter sequence reveals several potential PAX3 binding sites; thus, we hypothesize that the CXCR4 gene may be a direct transcriptional target of wild-type PAX3 (which is expressed by embryonal RMS cells) and the alveolar RMS-specific PAX3-FKHR fusion protein. Furthermore, PAX3-FKHR+ RD cells responded to SDF-1 by phosphorylation of MAPK p42/44, calcium flux, and directional chemotaxis. The regulation of CXCR4 expression in RMS cells may be even more complex. For example, one report indicates that CXCR4 expression in T lymphocytes is regulated by the protooncogene c-myc.48 Because RMS cells often display aberrant expression of N-myc and c-myc, and as N-myc and c-myc expression has been reported to correlate with the metastatic potential of ARMS cells,49 the role of myc protooncogenes in coregulating, together with PAX3 and PAX7, the expression of CXCR4 in human RMS cells needs further investigation. In our studies we focused on those RMS cell lines that responded to stimulation by SDF-1 by phosphorylation of MAPK p42/44. However, despite the fact that MAPK p42/44 has been shown to be involved in regulating cell proliferation and survival,50 we did not find any effect of SDF-1 on proliferation of RMS cell lines, although SDF-1 stimulated phosphorylation of MAPK p42/44 in these cells. Although we did not observe any effect of SDF-1 on proliferation or survival of RMS cells, we found that SDF-1 stimulated processes related to cell metastatic/invasive behavior, which is an integrated sequence of events, including induction of cell polarity, migration, matrix degradation, and adhesion. Accordingly, we demonstrated that SDF-1 may induce motility of RMS cells, their polarity (appearance of the leading edge), and cytoskeletal rearrangements (formation of stress fibers) as well as enhance production of certain metalloproteinases. We believe that together all these processes may contribute to the egress of RMS cells from the tumor. In further support of this idea, we present evidence that all RMS cell lines tested express MMP-2 and that SDF-1 stimulates its secretion in some of them. We also observed in most of the RMS cell lines that SDF-1 down-regulated the secretion of the natural MMP tissue inhibitors TIMP-1 and TIMP-2. Thus, simultaneous up-regulation of MMP-2 and/or down-regulation of TIMP-1 and TIMP-2 further support the role of the CXCR4-SDF-1 axis in the metastatic behavior of RMS. This concept is further supported by our findings, which show that the synthetic MMP inhibitor o-phenanthroline significantly reduced the chemoinvasion of these cells in Matrigel invasion assays. The ability of various malignant cells to penetrate Matrigel (which consists of collagen type IV, laminin, and heparin sulfate proteoglycans in a structure representative of most basement membranes, including those of blood and lymphatic vessels) is an indicator of their metastatic potential in vivo.37,38,51 Further, this assay allows discrimination between invasive and noninvasive cell populations.51 Moreover, we also demonstrated that SDF-1 increases adhesion of RMS cells to endothelium and that they follow a directional chemotaxis toward SDF-1 secreted by bone marrow stroma. Thus, all these SDF-1-induced processes such as adhesion to endothelium, migration though basement membrane, and directional chemotaxis toward bone marrow stroma may influence the invasiveness of circulating RMS cells and their "homing" from the peripheral blood into the SDF-1-rich environment of the bone marrow or lymph nodes. Moreover, we found that the responsiveness of RMS cells to SDF-1 correlated with the level of CXCR4 surface expression, the presence of the PAX3-FKHR fusion gene, and the ARMS phenotype. Generally, the biologic effects of SDF-1 on RMS cells observed here are consistent with those reported by us and others for human hematopoietic cells6-8,22,23 and involve primarily cell interactions with the microenvironment and their migration rather than cell proliferation and/or survival.6-9,22,23 However, the possibility that SDF-1 influences the metastatic potential of RMS in synergy/cooperation with other factors cannot be excluded. A potential candidate is, for example, the scatter factor. Both the scatter factor/hepatocyte growth factor and SDF-1 are highly expressed by bone marrow and lymph node stromal cells, and their respective receptors c-met52 and CXCR4, as shown here, are highly expressed by RMS cells. Furthermore, c-met has also been shown to be a direct transcriptional target of wild-type PAX3 and PAX3-FKHR fusion protein.52 Hence, we postulate that, although SDF-1 primarily directs/attracts RMS cells to bone marrow and lymph nodes, the scatter factor, alone or in combination with other factors, stimulates proliferation and survival of metastasizing cells. We also demonstrated that blocking of the CXCR4 receptor in RMS cells by the specific small-molecule inhibitor T140 perturbed adhesion of these cells to the HUVECs and inhibited directional chemotaxis toward bone marrow fibroblast cultures. These findings indicate the CXCR4-SDF-1 axis plays a significant role in the metastasis of the cells to the bone marrow. Because T140 is well tolerated in vivo,53 we suggest that it could be used as an inhibitor of metastasis of CXCR4+ cells. We are currently testing this hypothesis in an in vivo murine model. On the basis of our observations we conclude that the CXCR4-SDF-1 axis very likely plays an important role in tumor dissemination and metastasis to bone marrow, particularly of RMS. Hence, molecular strategies aimed at inhibiting this axis, eg, using small-molecule inhibitors,54-56 may lead to therapies that complement conventional radiotherapy or chemotherapy in preventing dissemination of RMS cells into hematopoietic organs.
We thank Lisa Ross, Ryan Reca, and Jacek Kijowski for their technical assistance, and Patsy Cotterill for editorial assistance. J.L. was on leave of absence from SMM, School of Molecular Medicine, Warsaw, Poland.
Submitted January 4, 2002; accepted May 21, 2002.
Prepublished online as Blood First Edition Paper, June 7, 2002; DOI 10.1182/blood-2002-01-0031.
Supported by grant R01 HL61796-01 and KBN 3P05E10122 (M.Z.R.) and R01 CA64202 (F.G.B.) from the National Institutes of Health and by Canadian Blood Services (CBS) R&D grant (A.J.-W.).
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.
Presented at the Annual Meeting of the American Society of Hematology, Orlando, FL, December 7-11, 2001. Reprints: Mariusz Z. Ratajczak, Stem Cell Biology Program at James Graham Brown Cancer Center, University of Louisville, 529 S Jackson St, Louisville, KY 40202; e-mail: mzrata01{at}louisville.edu.
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C. Laverdiere, B. H. Hoang, R. Yang, R. Sowers, J. Qin, P. A. Meyers, A. G. Huvos, J. H. Healey, and R. Gorlick Messenger RNA Expression Levels of CXCR4 Correlate with Metastatic Behavior and Outcome in Patients with Osteosarcoma Clin. Cancer Res., April 1, 2005; 11(7): 2561 - 2567. [Abstract] [Full Text] [PDF]
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A. C.W. Zannettino, A. N. Farrugia, A. Kortesidis, J. Manavis, L. B. To, S. K. Martin, P. Diamond, H. Tamamura, T. Lapidot, N. Fujii, et al. Elevated Serum Levels of Stromal-Derived Factor-1{alpha} Are Associated with Increased Osteoclast Activity and Osteolytic Bone Disease in Multiple Myeloma Patients Cancer Res., March 1, 2005; 65(5): 1700 - 1709. [Abstract] [Full Text] [PDF]
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S. Scala, A. Ottaiano, P. A. Ascierto, M. Cavalli, E. Simeone, P. Giuliano, M. Napolitano, R. Franco, G. Botti, and G. Castello Expression of CXCR4 Predicts Poor Prognosis in Patients with Malignant Melanoma Clin. Cancer Res., March 1, 2005; 11(5): 1835 - 1841. [Abstract] [Full Text] [PDF]
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E. Perissinotto, G. Cavalloni, F. Leone, V. Fonsato, S. Mitola, G. Grignani, N. Surrenti, D. Sangiolo, F. Bussolino, W. Piacibello, et al. Involvement of Chemokine Receptor 4/Stromal Cell-Derived Factor 1 System during Osteosarcoma Tumor Progression Clin. Cancer Res., January 15, 2005; 11(2): 490 - 497. [Abstract] [Full Text] [PDF]
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M. Wysoczynski, R. Reca, J. Ratajczak, M. Kucia, N. Shirvaikar, M. Honczarenko, M. Mills, J. Wanzeck, A. Janowska-Wieczorek, and M. Z. Ratajczak Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient Blood, January 1, 2005; 105(1): 40 - 48. [Abstract] [Full Text] [PDF]
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E. De Falco, D. Porcelli, A. R. Torella, S. Straino, M. G. Iachininoto, A. Orlandi, S. Truffa, P. Biglioli, M. Napolitano, M. C. Capogrossi, et al. SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells Blood, December 1, 2004; 104(12): 3472 - 3482. [Abstract] [Full Text] [PDF]
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M. Wysoczynski, K. Jankowski, K. Miekus, M. Kucia, A. Janowska-Wieczorek, J. Ratajczak, and M. Z. Ratajczak Leukemia Inhibitory Factor: A Newly Identified Chemoattractant and Regulator of Metastasis of Rhabdomyosarcomas and Neuroblastomas to Bone Marrow. Blood (ASH Annual Meeting Abstracts), November 16, 2004; 104(11): 1278 - 1278. [Abstract]
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R. Reca, K. Jankowski, G. Przybylski, A. Janowska-Wieczorek, and M. Z. Ratajczak CXCR4 Is a PAX Family Transcription Factor Regulated Gene. Blood (ASH Annual Meeting Abstracts), November 16, 2004; 104(11): 4205 - 4205. [Abstract]
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 J.-P. Spano, F. Andre, L. Morat, L. Sabatier, B. Besse, C. Combadiere, P. Deterre, A. Martin, J. Azorin, D. Valeyre, et al. Chemokine receptor CXCR4 and early-stage non-small cell lung cancer: pattern of expression and correlation with outcome Ann. Onc., April 1, 2004; 15(4): 613 - 617. [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. R. Cardones, T. Murakami, and S. T. Hwang CXCR4 Enhances Adhesion of B16 Tumor Cells to Endothelial Cells in Vitro and in Vivo via {beta}1 Integrin Cancer Res., October 15, 2003; 63(20): 6751 - 6757. [Abstract] [Full Text] [PDF]
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R. Reca, D. Mastellos, M. Majka, L. Marquez, J. Ratajczak, S. Franchini, A. Glodek, M. Honczarenko, L. A. Spruce, A. Janowska-Wieczorek, et al. Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1 Blood, May 15, 2003; 101(10): 3784 - 3793. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
) and in the presence of SDF-1
) and lower (
