Abstract
Dermatofibrosarcoma protuberans (DFSP) and giant cell fibroblastoma (GCF) are recurrent, infiltrative skin tumors that presently are treated with surgery. DFSP and GCF tumors are genetically characterized by chromosomal rearrangements fusing the collagen type Iα1 (COLIA1) gene to the platelet-derived growth factor B-chain (PDGFB) gene. It has been shown that the resulting COL1A1/PDGF-B fusion protein is processed to mature PDGF-BB. Autocrine PDGF receptor stimulation has therefore been predicted to contribute to DFSP and GCF tumor development and growth. Here we demonstrate presence of activated PDGF receptors in primary cultures derived from six different DFSP and GCF tumors. Three of the primary cultures were further characterized; their in vitro growth displayed an increased sensitivity to treatment with the PDGF receptor tyrosine kinase inhibitor STI571, as compared with normal fibroblasts. Transplantable tumors, displaying a DFSP-like histology, were established from one of the DFSP primary cultures. Treatment of tumor-bearing severe combined immunodeficient mice with STI571 reduced tumor growth. The growth-inhibitory effects in vitro and in vivo occurred predominantly through induction of tumor cell apoptosis. Our study demonstrates growth-inhibitory effects of PDGF receptor antagonists on human DFSP- and GCF-derived tumor cells and demonstrates that autocrine PDGF receptor stimulation provides antiapoptotic signals contributing to the growth of these cells. These findings suggest targeting of PDGF receptors as a novel treatment strategy for DFSP and GCF.
INTRODUCTION
DFSP 7 and GCF are skin tumors of intermediate malignancy, of which surgical removal is presently the sole effective treatment (1) . The high recurrence rate often necessitates multiple disfiguring excisions, and therefore, the development of additional pharmacological treatment procedures is warranted. The large majority, if not all, of DFSP and GCF tumors are characterized by chromosomal rearrangements, fusing the COLIA1 gene to the PDGFB gene (2 , 3) . It has therefore been proposed that autocrine PDGF receptor stimulation contributes to the development and growth of DFSP and GCF. Recent studies using cells transfected with genomic DNA encompassing DFSP- and GCF-derived fusion genes have confirmed that the fusion genes encode proteins that are processed to mature PDGF-BB and thereby lead to autocrine PDGF receptor stimulation (4, 5, 6) .
Various types of PDGF and PDGF receptor antagonists have been developed over recent years, and these candidate drugs have allowed investigations of the role of PDGF receptor activation in various pathological situations (7) . Encouraging results have been observed in animal models of restenosis, glomerulonephritis, and lung fibrosis. PDGF antagonists have also been used in cellular and animal models of malignancies associated with autocrine PDGF receptor activation. PDGF receptor-dependent growth has been demonstrated in s.c. and intracranially grown glioblastomas (8 , 9) . In mouse models of chronic myelomonocytic leukemia as well, PDGF antagonists have been used to establish a causal link between activated PDGF receptors and disease development (10) .
STI571 is a selective low-molecular-weight inhibitor of the PDGF receptor tyrosine kinase that displays in vivo activity after oral administration (11) . At the concentrations required for inhibition of the PDGF receptor, STI571 also blocks the cytosolic c-Abl and v-Abl tyrosine kinases, as well as the leukemia-associated Tel-Abl, Tel-PDGFβR, and Bcr-Abl tyrosine kinases (12 , 13) . The efficient blocking of Bcr-Abl by STI571 has led to clinical trials using this compound for treatment of chronic myeloid leukemia (14 , 15) .
A dependency on PDGF receptor signaling in fibroblasts transformed by transfection of DFSP-derived fusion genes has been demonstrated previously (4, 5, 6) . To what extent this requirement for PDGF receptor signaling also occurs in the case of tumor-derived DFSP or GCF cells has not been investigated. The purpose of this study was to investigate whether autocrine PDGF receptor activation contributes to DFSP and GCF growth by assessing the STI571 sensitivity of primary tumor cells derived from DFSP and GCF tumors.
MATERIALS AND METHODS
Cell Culture.
Surgical specimens from histopathologically diagnosed DFSP (four cases) and GCF (two cases) were obtained from the Centre of Human Genetics, Leuven University. Normal fibroblasts from human skin and primary cultures of DFSP and GCF cells were obtained by standard enzymatic disaggregation of tissue (16) . The karyotype was determined by standard procedures. RT-PCR detection and characterization of COLIA1/PDGFB fusion genes was performed as described previously (17) . Cells were kept in 5% CO2 humid atmosphere at 37°C and cultured in DMEM supplemented with 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin. All in vitro experiments were performed on cells that had undergone <10 population doublings in vitro. Cell cultures from s.c. grown 149333 tumors were obtained after 17 serial in vivo passages by homogenizing tumors through a metal grid and subsequent culturing in DMEM supplemented with 10% FCS and antibiotics.
Immunoprecipitation and Immunoblotting.
The rabbit antiserum PDGFR-3, recognizing the PDGF β-receptor, has been described previously (18) . The goat PDGF β-receptor antiserum 958 (sc-432) and the monoclonal anti-phosphotyrosine antibody PY99 (sc-7020) were acquired from Santa Cruz Biotechnology, Santa Cruz, CA, and used as recommended by the supplier. Prior to harvest, cells were incubated overnight in the presence or absence of 1 μm STI571 in serum-free DMEM containing 1 mg/ml BSA. Immunoprecipitations of the PDGF β-receptor and phosphotyrosine and PDGF β-receptor immunoblotting were performed as described (4) .
In Vitro Growth Characterization.
To study the in vitro growth rate of the three low-passage DFSP cultures, cells were plated in six-well plates (5 × 104 cells/well) in DMEM supplemented with 10 or 1% FCS in the presence or absence of 1 μm STI571. Medium was changed three times/week. After trypsinization, cells were counted using a Coulter particle counter.
TUNEL staining was performed on 105 149333 cells derived from transplanted 149333 tumors and NF control cells, respectively, grown for 48 h on glass coverslips in DMEM with 1 or 10% FCS in the absence or presence of 1 μm STI571. After fixation in 2% buffered PFA, apoptotic cells were labeled with FITC using TUNEL Label (Roche) according to the protocol of the manufacturer. Omission of TdT enzyme served as negative control. After mounting in Fluoromount-G, images were captured at 200-fold magnification in a fluorescence microscope (Zeiss Axioplan 2).
Establishment of Tumor Xenografts from 149333 Cells and Treatment Studies with STI571.
The animal work in this study was approved by the local board of animal experimentation and carried out in accordance with the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines (19) . All manipulations were performed in isoflurane gas anesthesia (Forene; Abbott). Upon arrival from the supplier (M&B, Ry, Denmark), the animals were acclimated, caged in groups of five or fewer, and had their backs shaved. To establish tumors from primary DFSP, 3 × 106 low-passage 149333 cells in 50 μl of DMEM were injected s. c. in four 6–8-week-old, athymic BALB/c mice. One of the established tumors was kept as a serial transplant in Fox-Chase SCID mice. The expression of the COL1A1/PDGFB fusion gene was verified by RT-PCR at passages 4, 9, and 17.
STI571 treatment studies were performed in 6–8-week-old, female Fox-Chase SCID mice inoculated s. c. with 5 × 105 149333 cells in 50 μl of DMEM. When tumors reached a volume of 100 mm3, the animals were randomly assigned to receive 200 mg/kg per day STI571 in PBS (n = 7) or vehicle alone (n = 7). STI571 was administered by gavage in doses of 100 mg/kg at 12-h intervals in 200 μl of PBS. Tumor dimensions were measured with calipers, and tumor volume was calculated according to the formula
, where a represents the shorter and b the longer dimensions of the tumor, respectively. Three h before sacrifice, 100 mg/kg BrdUrd in 0.9% NaCl was administered by i.p. injection. Animals were sacrificed by means of a lethal dose of pentobarbitone and perfused through the left cardiac ventricle with PBS, followed by 4% PFA in PBS (pH 7.4). After excision, tumors were weighed, fixed overnight in 4% PFA, and embedded in paraffin. Sections were cut at 4 μm on Superfrost Plus slides (Histolab, Göteburg, Sweden).
Effects of STI571 on PDGF β-receptor phosphorylation in vivo were studied in SCID mice bearing s.c. 149333 tumors of 1.5 cm3. Four h before sacrifice, two animals received 100 mg/kg STI571 in PBS by oral gavage, and two animals received PBS alone. Treated and control tumors were excised, weighed, and homogenized in RIPA lysis buffer containing 100 mm Tris (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, Complete protease inhibitor mixture (Roche), and 100 μm orthovanadate. Lysates were clarified by centrifugation, and an equivalent of 100 mg of tumor tissue was subjected to immunoprecipitation with preimmune serum, followed by PDGF β-receptor immunoprecipitation and phosphotyrosine and PDGF β-receptor immunoblotting (4) .
Immunohistochemistry.
For the detection of capillary blood vessels, sections were deparaffinized and pretreated by boiling in 10 mm citrate buffer (pH 6.0) for 2 × 7 min at 750 W in a microwave oven. Tissue peroxidase activity was quenched by incubation in 3% H2O2 in PBS for 10 min, followed by blocking in 1% BSA. Immunohistochemistry was performed with a goat antimouse CD31/PECAM-1 antibody (sc-1506, 1:100; Santa Cruz Biotechnology). Positive reactions were developed using DAB (Vector) as a peroxidase substrate. Immunohistochemical detection of BrdUrd was performed on deparaffinized, citrate-pretreated sections preincubated in 55% formamide/SSC/0.1% Tween 20 for 30 min at 72°C. Staining with a monoclonal mouse anti-BrdUrd antibody (1:50; Becton Dickinson) was done on a NexES immunostainer equipped with DAB substrate kit (Ventana Medical Systems, Tucson, AZ). Omission of primary antibody, in the case of CD31/PECAM-1 staining, or replacement of the primary antibody with an irrelevant mouse IgG, in the case of BrdUrd staining, was used as a negative control. TUNEL staining for detection of apoptotic cells in tumor sections was performed as described (20) with omission of Proteinase K digestion. Nick-end labeling was carried out using TdT enzyme and digoxigenin-11-dUTP (Roche) as recommended by the manufacturer. Positive nuclei were stained with peroxidase-coupled F(ab) fragments raised against dUTP-digoxigenin (1:500; Roche) and DAB peroxidase substrate.
Sections were counterstained in Mayer’s hematoxylin, dehydrated, and coverslipped in Mountex resin (Histolab). Nuclear stainings (BrdUrd; TUNEL) were quantitated and presented as the percentage of positive nuclei of 2000 counted at ×400 magnification under a microscope (VANOX-T; Olympus). Tumor angiogenesis was assessed by stereological quantification (21 , 22) of CD31+ vascular structures counted at ×400 with an eyepiece grid of 10 × 10 squares (0.30 × 0.30 mm). The grid was placed at random in the upper left-hand corner of the section and systematically advanced in steps of 2 mm in both directions. Morphological parameters of 25–50 vision fields were quantified from each tumor. The presence of viable tissue in the uppermost square to the far right of the grid (nvc, number of grids with viable corner) was noted and used as an estimator of the fraction of viable tumor tissue and in the calculation of vascular parameters (defined in Table 2 ⇓ ).
Statistical Analysis.
Statistical analysis was performed using two-tailed Student’s t test. P < 0.05 was considered as statistically significant.
RESULTS
DFSP and GCF Cells Express Activated PDGF β-Receptor.
A set of primary cultures of DFSP and GCF cells were established from surgical specimens from six different patients. Analysis by RT-PCR revealed expression of transcripts encoding different COLIA1/PDGF-B fusion proteins in all six cultures (Table 1) ⇓ . To determine whether expression of COLIA1/PDGF-B fusion transcripts was accompanied by the presence of activated PDGF β-receptor, we investigated the steady-state level of PDGF β-receptor tyrosine phosphorylation in the six primary cultures. DFSP, GCF, and normal human fibroblast cells were seeded at equal density; after overnight serum starvation, 3 × 106 cells were lysed, and PDGF β-receptors were isolated by immunoprecipitation and subjected to phosphotyrosine immunoblotting. All DFSP and GCF cultures displayed tyrosine phosphorylated receptors in contrast to normal human fibroblast cells (Fig. 1A) ⇓ . We thus conclude that autocrine PDGF β-receptor stimulation occurs in all six analyzed primary cultures derived from DFSP and GCF.
Activation of PDGF β-receptor in primary DFSP/GCF cells. Subconfluent cultures of normal human fibroblast (NF) and primary DFSP/GCF cells were lysed, and lysates were subjected to PDGF β-receptor immunoprecipitation and SDS-PAGE. After transfer to membranes, immunoblotting with phosphotyrosine antibodies (top) and PDGF β-receptor antiserum (bottom) was performed.
Clinical characteristics and COL1A1/PDGFB fusion gene expression in DFSP/GCF-derived cell lines
Inhibition of PDGF β-Receptor Activation Causes Inhibition of in Vitro Growth of DFSP and GCF Cells.
To investigate whether autocrine PDGF β-receptor stimulation contributes to the growth of DFSP cells, the effects on in vitro cell growth of the PDGF receptor tyrosine kinase inhibitor STI571 were investigated. Analysis of PDGF receptor tyrosine phosphorylation in DFSP cells cultured in the presence of STI571 confirmed that this treatment reduced PDGF β-receptor tyrosine phosphorylation to background levels (Fig. 2A) ⇓ . Three of the DFSP primary cultures and normal human fibroblast cells were, therefore, grown in the absence or presence of 1 μm STI571 in DMEM containing 10% FCS. STI571 reduced the growth rate of DFSP cells and led to a 60–75% reduction in cell number after 20 days of treatment, as compared with untreated cells (Fig. 2B) ⇓ . Similar results were also obtained when STI571 was used at a concentration of 0.5 μm (data not shown). STI571 had a smaller effect on the growth of normal fibroblasts, leading to <20% reduction in cell number (Fig. 2B) ⇓ . We next investigated the effects of STI571 on the growth of DFSP cells and normal human fibroblast cells in DMEM containing 1% FCS. Treatment under these conditions with 1 μm (Fig. 2C) ⇓ or 0.3 μm (data not shown) of STI571 reduced the growth of DFSP cells; in contrast, the growth of normal human fibroblast cells was not affected by STI571. These results indicate that autocrine PDGF β-receptor stimulation contributes to the in vitro growth of DFSP and GCF cells.
STI571 blocks PDGF β-receptor tyrosine phosphorylation and inhibits the in vitro growth of primary DFSP/GCF cells. A, subconfluent cultures of normal human fibroblasts (NF) and primary DFSP/GCF cells, cultured in DMEM with 10% FCS in the absence or presence of 1 μm STI571, were lysed, and lysates were subjected to PDGF β-receptor immunoprecipitation and SDS-PAGE. After transfer to membranes, immunoblotting with phosphotyrosine antibodies (top) and PDGF β-receptor antiserum (bottom) was performed. IB, immunoblot; P-Tyr, phosphotyrosine; IP, immunoprecipitation. B, normal fibroblasts (NF) and cells from three of the DFSP/GCF primary cultures were plated in six-well plates at a concentration of 5 × 104 cells/well in DMEM containing 10% FCS and grown in the absence (○) or presence (▪) of 1 μm STI571. At the indicated times, cells were trypsinized, and cell numbers were determined with a Coulter counter. C, normal fibroblasts (NF) and DFSP cells were plated in six-well plates at a concentration of 5 × 104 cells/well in DMEM containing 1% FCS and grown in the absence or presence of 1 μm STI571 for 12 days. Cells were trypsinized, and cell numbers were determined with a Coulter counter. Each experiment was performed four times with similar results.
Cells from DFSP Primary Cultures Form Transplantable s.c. Tumors That Are Sensitive to STI571 Treatment.
After s.c. implantation of low-passage 149333 cells, three of four mice displayed tumor take. One tumor from first-generation animals was propagated as a serial transplant in immunodeficient mice and has, at the time of writing, been xenografted for >20 passages. Several attempts at establishing the other above-mentioned GCF/DFSP cell lines as xenografts in immunodeficient mice were unsuccessful. This might be because of the intermediate degree of malignancy ascribed to GCF/DFSP. RT-PCR analysis using primers surrounding the breakpoint confirmed persistent expression of the COLIA1/PDGFB fusion gene at passages 4, 9, and 17 (data not shown).
To investigate the dependence of autocrine PDGF receptor stimulation for growth of the 149333 tumors, a treatment study on serially transplanted 149333 tumors using the PDGF receptor kinase inhibitor STI571 was performed. Peroral treatment with 200 mg/kg per day STI571 reduced 149333 tumor volume to one-third the size of control tumors (Fig. 3A) ⇓ . This difference was also reflected in postmortem tumor weight (0.7 ± 0.1 g versus 2.0 ± 0.3 g, mean and SD; P < 0.001). In a similar experiment, peroral administration of 100 mg/kg per day STI571 once daily resulted in a 2-fold reduction of tumor volume at the end of treatment (data not shown). To confirm that STI571 treatment indeed reduced PDGF receptor activation, the tyrosine phosphorylation status of tumor-derived PDGF receptors was examined. PDGF β-receptors were extracted from tumors 4 h after administration of 100 mg/kg STI571. As shown in Fig. 3B ⇓ , treatment with STI571 decreased tyrosine phosphorylation of PDGF β-receptors in 149333 tumors, as compared with PBS controls, without affecting expression levels or the ratio of mature and precursor form of the PDGF β-receptor.
Treatment with STI571 inhibits 149333 tumor growth and PDGF receptor phosphorylation in vivo. A, Fox-Chase SCID mice were inoculated s.c. with 5 × 105 149333 cells in DMEM. Peroral treatment with 200 mg/kg per day STI571 (□; n = 7) or PBS (♦; n = 7) was started when tumors reached a volume of 100 mm3. After 9 days of treatment, tumors of STI571-treated animals were only one-third the size of control tumors. Data are means; bars, SD. ∗∗∗, P < 0.001 (Student’s t test). B, lysates from two control (PBS) and two STI571-treated tumors were subjected to consecutive immunoprecipitations (IP) with preimmune serum, followed by PDGF β-receptor antiserum. After separation in 7% SDS-PAGE and transfer to blotting membranes, the PDGF β-receptor immunoprecipitations were analyzed by immunoblotting using antibodies against phosphotyrosine (upper panel) and antisera against the PDGF β-receptor (lower panel). The two bands seen in the lower panel represent the Mr 180,000 mature receptor and Mr 160,000 precursor, respectively. IB, immunoblot; P-Tyr, phosphotyrosine.
STI571 Reduces in Vivo and in Vitro Growth of DFSP Cells by Induction of Apoptosis.
To identify the mechanism behind STI571-induced inhibition of 149333 tumor growth, sections from STI571 and control treated tumors were immunostained to assess proliferation (BrdUrd), capillary blood vessel density (CD31), and apoptosis (TUNEL; Fig. 4A ⇓ ). Tumor cells in S-phase of the cell cycle were labeled by i.p. injection of BrdUrd and visualized by immunostaining with anti-BrdUrd antibodies. Microscopic quantification showed similar levels of tumor cell proliferation in STI571-treated tumors as compared with vehicle control (Fig. 4B) ⇓ . Control and STI571-treated tumors were also subjected to TUNEL labeling for detection of tumor cell apoptosis. As shown in Fig. 4B ⇓ , treatment with STI571 resulted in a 3-fold increase in tumor cell apoptosis as compared with vehicle control.
STI571 induces apoptosis in 149333 tumors in vivo. STI571-treated (n = 7) and control (n = 7) tumors were sectioned and immunostained to assess proliferation, capillary blood vessel density, and apoptosis. A, representative vision fields from STI571-treated and control tumors stained for proliferation (BrdU; upper panels), apoptosis (TUNEL; middle panels), and capillary endothelial cells (CD31; lower panels). Arrowheads, TUNEL-positive tumor cells. Bar, 20 μm. B, proliferating and apoptotic cells were scored under a microscope. Indices are expressed as percentage positive nuclei of 2000 counted cells. Data are means; bars, SD. ∗∗∗, P < 0.001 (Student’s t test).
The effect of STI571 treatment on apoptosis in vitro was examined by TUNEL staining of 149333 DFSP cells, derived after 17 in vivo passages in SCID mice, and normal human fibroblasts subjected to STI571 treatment in high (10%) or low (1%) serum concentration. As shown in Fig. 5 ⇓ , addition of STI571 to 149333 cells in low serum concentration induced apoptosis. In contrast, apoptosis was not induced in normal fibroblasts by STI571.
Treatment with STI571 induces apoptosis of 149333 cells in vitro. 149333 DFSP and normal fibroblast (NF) cells were grown for 48 h in 10% FCS or 1% FCS in the absence or presence of 1 μm STI571. Apoptotic cells were visualized using TUNEL-FITC.
Together, these results indicate that the major mechanism whereby STI571 affects the growth of 149333 tumors in vitro and in vivo is through induction of tumor cell apoptosis, rather than by reducing tumor cell proliferation.
STI571 Treatment Alters the Vascular Morphology of 149333 Tumors and Decreases Tissue Necrosis.
Potential antiangiogenic effects of STI571 were assessed by staining of capillary blood vessels in control and treated 149333 tumors by CD31/PECAM-1 immunohistochemistry and subsequent stereological quantification. The length density, volumetric density, and surface density of blood vessels were not significantly reduced in tumors treated with STI571, as compared with control-treated tumors (Table 2) ⇓ . On the contrary, mean vessel area, boundary length, and diameter were decreased by administration of STI571. This decrease is attributable to the relative absence in STI571-treated tumors of dilated, presumably venous, vascular structures present in vehicle-treated tumors (Fig. 4A) ⇓ . Notably, necrosis was virtually absent in the STI571-treated tumors. This is reflected by an increase in the fraction of viable tumor tissue by 42% as compared with PBS control. From the characterization of the vascular morphology of treated and control tumors, we conclude that antiangiogenic effects do not contribute to the antitumor effect of STI571 on 149333 tumors.
Quantification of angiogenesis and tissue viability in control and STI571-treated tumors
DISCUSSION
In this study, we have demonstrated that blockage of autocrine PDGF β-receptor stimulation in tumors derived from primary DFSP cells leads to reduced growth in vivo and in vitro. Our findings support the notion that targeting of PDGF receptors might provide a pharmacological complement to present modalities used for treatment of DFSP. Our study also presents an orthotopic model of DFSP, derived from primary DFSP cells, which should be useful for additional studies on novel DFSP therapies.
DFSP is not the only type of soft tissue sarcoma where autocrine PDGF receptor stimulation has been suggested to contribute to disease development. Coexpression of PDGF ligand and receptor has been documented in clinical samples of a variety of fibroblast-derived tumors (23 , 24) . The effects of PDGF antagonists on cell lines derived from these types of tumors have not been characterized.
Our characterization of the effects of STI571 on proliferation and apoptosis in vivo and in vitro indicated that the growth-inhibitory effects in vivo are predominately achieved through induction of apoptosis. Although PDGF stimulation of normal cells is traditionally considered to induce a proliferative response, antiapoptotic effects by PDGF stimulation have also been demonstrated in normal cells (25) . In this context, it is noteworthy that the recently reported growth-inhibitory effect of STI571 on human glioblastoma cells does not appear to occur through induction of apoptosis (9) .
Our characterization of the vascular morphology failed to provide any evidence for primary antiangiogenic effects of STI571 in this tumor model. STI571 treatment of 149333 tumors decreased the mean vessel section area, boundary length, and diameter while not significantly affecting blood vessel density. This difference reflects the relative absence in STI571-treated tumors of large-diameter vessels found in the untreated group. This vascular phenotype is clearly different from what has been observed using angiogenesis inhibitors, where the length density, volume density, and surface density of vessels are decreased (20) . Relieved stasis of tumor blood vessels because of a high rate of tumor cell apoptosis and decreased cell density might explain the effect of STI571. We conclude that the altered vascular morphology of 149333 tumors upon STI571 treatment is attributable to effects on the tumor cells rather than being direct effects on the endothelial compartment itself. Furthermore, the overall tissue viability is increased upon STI571 treatment. To what extent this change in tumor microenvironment by STI571 treatment will lead to a sensitization to the therapeutic effect of chemotherapy administered at the same time constitutes a valid purpose for future studies.
The PDGF receptor antagonist used in this study, STI571, belongs to a growing class of low-molecular-weight compounds that block PDGF receptor signaling by interfering with ATP binding to the receptor tyrosine kinase (7) . Similar to other tyrosine kinase inhibitors, STI571 does not display absolute selectivity for the PDGF receptors. A recent study characterized in some detail the activity of STI571 against PDGF receptor-related tyrosine kinases (11) . STI571 was found to block PDGF α- and β-receptor and the structurally related stem cell factor receptor, c-Kit, with similar efficiency. However, the closely related receptor for CSF-1, c-Fms, or the VEGF receptors VEGF-R1 and VEGF-R2 showed much lower sensitivity to STI571. The fact that no major side effects have been observed in the early clinical trials using STI571 in treatment of chronic myeloid leukemia makes STI571 well suited for treatment of DFSP (15) .
In conclusion, our study has provided experimental support for the notion that PDGF receptors are potential drug targets for treatment of DFSP. Obvious ways to build on these observations include investigations of the effects of STI571 on other fibroblast-derived tumors, as well as to investigate whether STI571 will show synergistic effects when coadministered with conventional chemotherapeutic drugs.
Acknowledgments
We thank Helena Hermelin and Mari-Anne Carlsson for excellent technical assistance and Ingegärd Schiller for expert secretarial assistance.
Footnotes
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 These authors contributed equally to this work.
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↵2 Present address: Department of Dermatology, Yamanashi Medical University, 1110 Shimokato, Tamaho, Nakakoma, Yamanashi 409-38, Japan.
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↵3 Present address: Department of Dermatology, Research Center M3:02, Karolinska Hospital, S-171 76 Stockholm, Sweden.
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↵4 Present address: Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115.
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↵5 Present address: Department of Genetics and Pathology, Uppsala University, S-751 85 Uppsala, Sweden.
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↵6 To whom requests for reprints should be addressed, at Ludwig Institute for Cancer Research, Box 595, S-751 24 Uppsala, Sweden. Phone: 46-18-160401; Fax: 46-18-160420; E-mail: c-h.heldin{at}licr.uu.se
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↵7 The abbreviations used are: DFSP, dermatofibrosarcoma protuberans; GCF, giant cell fibroblastoma; COLIA1, collagen type Iα1; PDGFB, platelet-derived growth factor B-chain; BrdUrd, bromodeoxyuridine; DAB, 3, 3′-diaminobenzidine; PECAM, platelet/endothelial cell adhesion molecule; PFA, paraformaldehyde; RT-PCR, reverse transcription polymerase chain reaction; SCID, severe combined immunodeficient; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; VEGF, vascular endothelial growth factor.
- Received January 19, 2001.
- Accepted June 9, 2001.
- ©2001 American Association for Cancer Research.
References
Volume 61, Issue 15
