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[Cancer Research 62, 7357-7363, December 15, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

SPARC Is a Key Schwannian-derived Inhibitor Controlling Neuroblastoma Tumor Angiogenesis1

Alexandre Chlenski, Shuqing Liu, Susan E. Crawford, Olga V. Volpert, George H. DeVries, Amy Evangelista, Qiwei Yang, Helen R. Salwen, Robert Farrer, James Bray and Susan L. Cohn2

Departments of Pediatrics [S. L. C.] and Pathology [S. E. C.] and The Robert H. Lurie Comprehensive Cancer Center [A. C., S. L., O. V. V., A. E., Q. Y., H. R. S., J. B.], Northwestern University, Feinberg School of Medicine, Chicago, Illinois 60611, and the Department of Cell Biology, Neurobiology, and Anatomy, Loyola University of Chicago Stritch School of Medicine, Chicago, Illinois 60153 [G. H. D., R. F.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuroblastoma (NB), a common pediatric neoplasm, consists of two main cell populations: neuroblastic/ganglionic cells and Schwann cells. NB tumors with abundant Schwannian stroma display a more benign clinical behavior than stroma-poor tumors. Recent studies suggest that Schwann cells influence NB tumor growth via secreted factors that induce differentiation, suppress proliferation, and inhibit angiogenesis. Two angiogenesis inhibitors, pigment epithelium-derived factor and tissue inhibitor of metalloproteinase-2, have been detected in Schwann cell secretions. Here, we isolated another Schwann cell-derived secreted inhibitor of angiogenesis, a 43-kDa protein identified as SPARC (secreted protein acidic and rich in cysteine), an extracellular matrix protein. We found SPARC to be critical for the antiangiogenic phenotype of cultured Schwann cells. We also show that purified SPARC potently inhibits angiogenesis and significantly impairs NB tumor growth in vivo. SPARC may be an effective candidate for the treatment of children with clinically aggressive, Schwannian stroma-poor NB tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NB,3 a common pediatric neoplasm that arises from neural crest tissue, has a broad spectrum of clinical behavior (1, 2, 3, 4) . Although numerous factors including stage (5) , patient age (6) , tumor histology (7) , molecular markers (8 , 9) , and genetic abnormalities (10, 11, 12, 13) have been shown to be predictive of outcome in children with NB, the mechanisms responsible for the highly variable clinical behavior of NB remain largely unknown. Several recent studies implicate angiogenesis in the regulation of NB growth. In primary NB tumors, high vascular index correlates with MYCN amplification, metastases, and poor outcome, whereas low tumor vascularity is associated with a better prognosis, localized stage, and favorable histology (14) . Advanced-stage NB is associated with high levels of angiogenic stimuli and {alpha}vß3 and {alpha}vß5 integrins, both markers of active angiogenesis (15 , 16) . Overexpression of exogenous MYCN results in enhanced malignant growth of NB cells and reduced levels of activin A, an inhibitor of angiogenesis (17) . Expression of the neurotrophin receptor TrkA also causes down-regulation of angiogenesis stimulators and impaired tumorigenicity in a mouse xenograft model (18) . Furthermore, preclinical studies have shown effective reduction of NB tumor growth in vivo by a variety of antiangiogenesis agents (16 , 19, 20, 21, 22, 23) .

NB tumors consist of two main cell populations, neuroblastic/ganglionic cells and Schwann cells (24) . The ratio of these cell types varies according to tumor maturation. Immature NB tumors are composed of undifferentiated neuronal cells and a paucity of Schwannian stroma, whereas larger, ganglion-like cells and abundant Schwannian stroma are seen in maturing NB tumors. The Schwann cells are thought to be normal cells that infiltrate the NB tumor (25) , although a recent report suggests that the Schwann cells may be malignant (26) . The favorable prognostic impact of the presence of Schwannian stroma has been emphasized in the pathological classification system of Shimada et al. (24) and the International NB Pathology Classification System (7) . It has been speculated that Schwann cells influence NB tumor growth by secreting molecules that serve as antiproliferative and differentiating factors for neuronal cells (25 , 27 , 28) . Schwann cells also produce a spectrum of angiogenesis inhibitors (29 , 30) , suggesting that Schwann cells may also influence NB growth by restricting angiogenesis.

The angiogenesis inhibitors PEDF and TIMP-2 have been previously found in media conditioned by Schwann cells, and both factors appear to contribute to the antiangiogenic activity of the SCM (29 , 30) . In this study, we isolated an additional angiogenic inhibitor in SCM, identified as SPARC. SPARC, also known as osteonectin, BM-40, and 43K protein, is a highly conserved calcium-binding matricellular glycoprotein (31, 32, 33) . SPARC is spatially and temporally regulated during development, and it is transiently expressed in derivatives of the three primitive germ layers in mouse embryos (34) . This glycoprotein is highly expressed in bone and in basement membranes as well as in a variety of cell types associated with remodeling tissues and high cellular turnover (35) . Although its precise function is unclear, SPARC plays a modulatory role in cell-matrix interactions (36) . SPARC induces cell rounding, blocks cell spreading and adhesion, and inhibits endothelial cell migration (37, 38, 39) . SPARC also appears to contribute to vascular morphogenesis and cellular differentiation (36) . However, the contradictory reports regarding the role of SPARC in cell growth and tumor formation (40, 41, 42) suggest that its effects are cell type specific and may be dependent on concentration, extracellular matrix components, and the ability of the cell to proteolyze SPARC (42, 43, 44) .

We report that SPARC expression is inversely correlated with the degree of malignant progression in NB tumors. We also demonstrate that SPARC is one of the key contributors to the antiangiogenesis activity of the SCM. Purified SPARC blocks angiogenesis in vitro and in vivo and significantly impairs NB tumor growth in vivo. Our observations stress the cumulative nature of the angiogenic equilibrium in NB tumors and suggest that a shift in the angiogenic balance may be prompted by even slight disturbances in the complex array of inhibitors and stimuli in the endothelial cell microenvironment.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and CM Collection.
Primary human Schwann cells were purified from adult nerves or from Schwannian stroma-dominant NB tumors and expanded as described previously (29 , 30 , 45) . Tumor-derived Schwann cells were maintained at 5% CO2 in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum (US Biotechnologies, Inc., Parkerford, PA), 50 ng/ml recombinant human heregulin ß1 (R&D Systems, Minneapolis, MN), 1% penicillin/streptomycin, 2.5 µg/ml amphotericin, 0.5 µM isobutylmethylxanthine (Sigma, St. Louis, MO), and 0.5 µM forskolin (Sigma). NB cell lines used in this study have been described previously (46, 47, 48, 49) , with the exception of NBL-L and NBL-R, MYCN-amplified lines established in our laboratory from clinically aggressive NB tumors. NB cells were grown at 5% CO2 in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and antibiotics. For some experiments, 10 µM all-trans-RA (Sigma) or 6 µM BrdUrd (Sigma) was added where indicated, and the cells were harvested at the indicated time intervals. CM from NB cell lines and SCM were collected as described previously (29 , 30) .

Isolation of SPARC from SCM.
Concentrated SCM were dialyzed against PBS and fractionated on a HiTrap Q-Sepharose column (Amersham, Piscataway, NJ) with a 0.1–1.0 M NaCl gradient in 20 mM Tris-HCl (pH 8.0). Fractions that blocked endothelial cell chemotaxis or induced endothelial cell apoptosis were dialyzed against loading buffer, fractionated using a HiTrap heparin-Sepharose column (Amersham; 0.1–1.0 M NaCl gradient), and subjected to identical functional assays. Fractions were analyzed by SDS-PAGE followed by silver staining. A common 43-kDa band in the inhibitory flow-through fractions was cut from a Coomassie Blue-stained gel and submitted for sequence analysis at the Harvard University Microchemistry Facility.

SPARC Expression Analysis.
To examine SPARC mRNA expression levels, total RNA was isolated from cultured cells using Trizol reagent (Life Technologies, Inc.) according to the manufacturer’s instructions, and 1 µg of total RNA was reversed transcribed using Superscript II (Life Technologies, Inc.). To detect the SPARC transcripts, semiquantitative RT-PCR was performed using template diluted 1:100 and the sense primer 5'-CTGCCTGCCACTGAGGGTTCC-3' and antisense primer 5'-TCCAGGCAGAACAACAAACCATCC-3'. ß-Actin was used as a loading control with template diluted 1:1000 and sense primer 5'-TGTTGGCGTACAGGTCTTTGC-3' and antisense primer 5'-GCTACGAGCTGCCTGACGG-3'. All targets were amplified for 30 cycles at an annealing temperature of 60°C. SPARC mRNA levels were also analyzed using real-time RT-PCR as described previously (50) . For the quantitative real-time RT-PCR experiments, the primer pair 5'-TCTTCCCTGTACACTGGCAGTTC-3' (sense) and 5'-AGCTCGGTGTGGGAGAGGTA-3' (antisense) was used with the probe Fam-CAGCTGGACCAGCACCCCATTGA-QSY7. SPARC protein levels in CM were examined by Western blots. Briefly, CM were concentrated 50-fold using 5K cutoff centrifugal filter devices (Millipore, Bedford, MA). Total protein (10 µg) was electrophoresed in a 4–20% SDS-PAGE gradient gel and transferred to a nitrocellulose membrane (Bio-Rad, Richmond, VA) using standard techniques (51) . After transfer, the blots were stained with Ponceau S (Sigma) to confirm equal loading. Membranes were blocked with 5% nonfat dry milk for 1 h and then incubated for 2 h with anti-osteonectin antibody (referred to hereafter as anti-SPARC antibody; Zymed, San Francisco, CA) at a 1:2000 dilution. The membranes were washed three times with PBT (PBS with 0.1% Tween 20) and then incubated for 2 h with a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody (Kirkegaard and Perry Laboratories, Gaithersburg, MD). The bound antibody complexes were detected using the LumiGLO chemiluminescence reagent (KPL).

Immunohistochemistry Studies.
Histological sections of human NB and ganglioneuromas (mature Schwannian stroma-rich NB) were immunostained using a mouse anti-SPARC monoclonal antibody (Zymed). Briefly, paraffin-embedded NB tumor tissue fixed in 10% buffered formalin was sliced into 4-µm-thick sections, rehydrated in graded alcohols, and rinsed in PBS. Antigen retrieval was performed with 0.01 M citrate buffer (pH 6.0) in a boiling steamer (20 min). Sections were incubated overnight with primary antibody (1:1600 dilution) at 4°C and developed with peroxidase labeled-dextran polymer followed by diaminobenzidine (Envision Plus System; DAKO Corp., Carpinteria, CA). Sections were counterstained with Gill’s hematoxylin. A human Schwannoma sample was included in each assay as a positive control, and staining without primary antibody was used as a negative control. Cytoplasmic patches of brown color were scored as SPARC positive. Rat antimouse monoclonal CD31 (PECAM-1) antibody (1:100 dilution; Research Diagnostics Inc., Flanders, NJ) was used to highlight endothelial cells on frozen tumor sections. MVD was quantified by counting 10 consecutive fields at x200 magnification, and the average MVD counted in the 10 fields was reported as MVD/mm2.

In Vitro Angiogenic Assay.
Migration assays were performed with human umbilical vein endothelial cells [National Cancer Institute Preclinical Repository (Bethesda, MD) and VEC Technologies (Rensselaer, NY)] as described previously (29) . Test substances were assayed in Opti-MEM media (Life Technologies, Inc.) with or without 3 ng/ml bFGF (National Cancer Institute Preclinical Repository). Purified human platelet osteonectin (referred to hereafter as SPARC) was obtained from Calbiochem (San Diego, CA). To generate dose-response curves, the data were normalized as percentage of maximum migration using the difference between bFGF/Opti-MEM-induced migration and background migration in Opti-MEM alone as 100% control. For some assays, neutralizing anti-SPARC antibody (Zymed) or isotype-matched control antibody was added to the media at 10 µg/ml after dialysis against PBS. Control mouse IgG was obtained from Lab Vision (Fremont, CA).

Endothelial Cell Apoptosis Assay.
Bovine adrenal capillary endothelial cells were treated overnight with SCM or control Opti-MEM. Dialyzed anti-SPARC antibody (Zymed) and control antibody were added at 10 µg/ml in some assays. Apoptotic cells were visualized using the ApopTag in situ Apoptosis Detection kit (InterGen, Gaithersburg, MD). Each assay was performed in triplicate, and the percentage of apoptotic cells was calculated as the number of green terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells with DNA fragmentation divided by the total number of Hoechst-counterstained nuclei.

In Vivo Angiogenesis Assay.
Female Fischer 344 rats (Harlan, Madison, WI) were used to perform rat corneal assays using previously described methods (29 , 52) . Briefly, 5 µl Hydron pellets (IFN Sciences, New Brunswick, NJ) prepared with 25 µg/ml SPARC (Calbiochem) with or without 50 ng/ml bFGF were implanted into the corneas of anesthetized rats. Control studies were performed with pellets containing PBS with or without bFGF. Additional experiments were performed with pellets also containing 50 µg/ml anti-SPARC antibody (Zymed). After 7 days, the animals were sacrificed and perfused with waterproof drawing ink (Sanford, Bellwood, IL) by intracardiac injection. The eyes were fixed in 10% neutralized buffered formalin overnight. The corneas were examined, and dense capillaries reaching the pellet were scored as positives. Animals were treated according to NIH guidelines for animal care and use, and protocols were approved by the Animal Care and Use Committee at Northwestern University.

In Vivo Inhibition of NB Growth.
NB xenografts were grown in female 4–6-week-old homozygous athymic nude mice (Harlan) after s.c. inoculation of 5 x 106 SMS-KCNR NB cells into the right flank. Once tumors were palpable, animals were anesthetized, and ALZET osmotic pumps (Durect, Cupertino, CA) containing purified SPARC (Calbiochem; n = 3) or PBS (n = 3) were implanted s.c. SPARC was released s.c. by the pump at a rate of 62.5 ng/h. Tumor volume was measured weekly using the formula: tumor volume = (length x width2)/2 (53) . Mice were sacrificed after 3 weeks of treatment, and tumors were resected for histological analysis. Tumor volume was analyzed using Student’s t test to compare control and treatment groups.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Schwann Cells Secrete Antiangiogenic SPARC.
We have previously reported antiangiogenic activity in SCM collected from normal and NB tumor-derived Schwann cells (29) and demonstrated the presence of several angiogenic inhibitors including PEDF and TIMP-2 (29 , 30) . Seeking additional angiogenesis inhibitors in SCM, we subjected it to multiple-step chromatography and tested fractions for the ability to block bFGF-induced endothelial cell migration and cause endothelial cell apoptosis. Fractions capable of both activities contained a 43-kDa protein (Fig. 1)Citation that was identified as SPARC using sequence analysis of the tryptic peptide fragments. No contaminating sequence was detected in the SPARC band.



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  		Fig. 1. Silver-stained gel of SCM fractions collected after heparin-Sepharose chromatography. The 43-kDa protein detected in Lane 3 was identified as SPARC. Fraction 3 potently inhibited bFGF-induced endothelial cell migration as indicated at the bottom of the gel.

 
SPARC Was Expressed by Schwann Cells and Differentiated NB Cells in Vitro and in Vivo.
SPARC expression was evaluated by semiquantitative RT-PCR in Schwann cells, a panel of NB cell lines, and in phenotypically distinct subclones of NB cell lines [neuroblastic (N-type) and substrate adherent (S-type)] that exhibit different malignant potentials (Refs. 54 and 55 ; Fig. 2ACitation ). Although SPARC mRNA was detected in all NB cell lines with the exception of NBL-W-N, mRNA levels were significantly higher in the Schwann cells and nontumorigenic S-type subclones than in tumorigenic N-type subclones and NB cell lines. SPARC protein levels in CM collected from the cells paralleled the mRNA levels (Fig. 2B)Citation . NB cells can be induced to differentiate in vitro with a number of agents including all-trans-RA or BrdUrd (56, 57, 58, 59) , and real-time quantitative RT-PCR demonstrated up to a 10-fold increase in SPARC mRNA in differentiated NB cells (Fig. 2C)Citation .



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  		Fig. 2. SPARC expression in NB and Schwann cells. A, SPARC mRNA was measured in NB cell lines and Schwann cells by semiquantitative RT-PCR. Higher levels of expression were observed in nontumorigenic S-type NB subclones and Schwann cells than in tumorigenic NB cell lines and N-type subclones. B, SPARC protein was measured in media conditioned by NB cell lines, normal Schwann cells (N), and tumor-derived Schwann cells (T) by Western blot analysis. Higher levels of secreted protein were detected in the nontumorigenic S-type subclones and Schwann cells compared with tumorigenic NB cell lines. C, SPARC expression, measured by real-time quantitative RT-PCR, was up-regulated in SMS-KCNR NB cells induced to differentiate with RA or BrdUrd. D, SPARC is not detected by immunoperoxidase staining in an undifferentiated, Schwannian stroma-poor NB (magnification, x400; inset, x600). Immunoperoxidase staining of a ganglioneuroma (Schwannian stroma-dominant tumor; E) and differentiating NB (F) demonstrates SPARC expression in Schwann cells (arrows) and differentiating neuroblasts/ganglion cells [arrowheads; (magnification, x200; insets, x600)].

 
To investigate whether SPARC was expressed within NB tumors, histological sections from NB tumors displaying varying degrees of differentiation and abundance of Schwannian stroma and from ganglioneuromas were stained with antibody against human SPARC. Schwannian stroma-poor tumors were composed predominantly of neuroblasts and showed minimal or no staining for SPARC (Fig. 2D)Citation . Conversely, in maturing and mature tumors, SPARC could be detected not only in Schwann cells (Fig. 2E)Citation but also in differentiating neuroblasts/ganglion cells (Fig. 2F)Citation .

SPARC in SCM Inhibited Migration and Induced Apoptosis of Endothelial Cells.
The previously reported inhibition of bFGF-induced endothelial cell migration by SCM (29) was SPARC dependent because migration was largely restored in the presence of neutralizing antibody against SPARC (Fig. 3A)Citation . We also showed that SPARC-dependent induction of endothelial cell apoptosis by SCM was effectively neutralized by the same antibody (Fig. 3B)Citation . Consistent with earlier studies (38) , purified SPARC blocked bFGF-induced endothelial cell migration in a dose-dependent manner at concentrations ranging from 0.05 to 5 µg/ml (Fig. 3C)Citation . However, endothelial cell migration inhibition was not observed at higher concentrations of SPARC. Biphasic responses have similarly been reported with the angiogenesis inhibitor thrombospondin-1 (60 , 61) . SPARC also triggered endothelial cell apoptosis, with maximal induction at 20 µg/ml (Fig. 3D)Citation .



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  		Fig. 3. SPARC inhibits angiogenesis in vitro. A, neutralizing anti-SPARC antibody reverses SCM inhibition of bFGF-induced endothelial cell migration. No effect is seen with control antibody. bFGF-induced endothelial cell migration is observed in the Opti-MEM control. B, terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells (top panels) with Hoechst-counterstained nuclei (bottom panels). Protection of endothelial cells from SCM-induced apoptosis was observed in the presence of anti-SPARC neutralizing antibody but not control antibody. The percentage of endothelial cell apoptosis for each experiment is shown in the bar graph. C, purified SPARC inhibits bFGF-induced endothelial cell migration with an ED50 of ~2 nM. D, purified SPARC triggers endothelial cell apoptosis in a dose-dependent manner with maximal response at 20 µg/ml.

 
SPARC Inhibited Angiogenesis and Impaired Tumor Growth in Vivo.
Purified SPARC blocked bFGF-induced angiogenesis in vivo in the rat corneal neovascularization assay (Fig. 4Citation ; Table 1Citation ). Furthermore, the addition of the anti-SPARC antibody fully restored angiogenesis by bFGF, indicating that this inhibitory effect was indeed due to SPARC. Angiogenesis was not observed when SPARC was tested alone (Fig. 4)Citation . To our knowledge, the ability of SPARC to inhibit angiogenesis has not previously been tested in a rat corneal model.



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  		Fig. 4. SPARC inhibits angiogenesis in vivo. Top panels, SPARC inhibits bFGF-induced angiogenesis in the rat corneal assay. Addition of anti-SPARC antibody restored angiogenesis by bFGF. Bottom panels, in the absence of bFGF, control media, purified SPARC, or purified SPARC plus anti-SPARC neutralizing antibody did not induce angiogenesis in rat corneal assays.

 

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  		Table 1 In vivo antiangiogenic activity of SPARC

 
The effect of SPARC on NB growth in vivo was tested in a mouse xenograft model where SPARC was delivered continuously for 3 weeks using osmotic pumps. During the first 2 weeks, tumor growth was completely arrested in the SPARC-treated group, whereas in the control animals carrying PBS-charged pumps, the volume of the tumors doubled every 5–6 days (Fig. 5, A and B)Citation . During the third week of treatment, a slight increase in tumor size was observed in the treatment group. After 3 weeks of treatment with SPARC, the average tumor volume was significantly smaller than that observed in control animals (152 ± 44 mm3 versus 919 ± 317 mm3; P = 0.03). Histological comparison revealed decreased vascularity in SPARC-treated tumors (MVD = 23/mm2) compared with control tumors (MVD = 47/mm2) as assessed by the number of structures that stained positively with an anti-CD31 antibody (Fig. 5, C and D)Citation .



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  		Fig. 5. SPARC inhibits NB tumor growth in vivo. A and B, short-term delivery of SPARC by osmotic pumps impairs the rate of NB xenograft tumor growth compared with control animals. C and D, immunohistochemistry staining of the SMS-KCNR NB xenograft tumors with anti-CD31 antibody demonstrates decreased numbers of blood vessels in NB xenografts from the SPARC-treated mice compared with control-treated animals (magnification, x200).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Schwann cells secrete substances that promote NB cell survival and differentiation and inhibit angiogenesis (25 , 27 , 29 , 30) . It is thought that cross-talk between neuroblasts and Schwann cells is responsible for the more benign nature of Schwannian stroma-rich/stroma-dominant NB tumors (25 , 29) . Seeking factors produced by Schwann cells that could contribute to this clinically less aggressive tumor phenotype, we identified a factor present in SCM that was capable of inhibiting angiogenesis. SPARC, a known member of the matricellular protein family, appears to be a key contributor to the cumulative antiangiogenic activity produced by Schwann cells. We confirmed a known ability of SPARC to block chemotaxis of endothelial cells induced by angiogenic stimuli (38) and report its previously unknown function to induce endothelial cell apoptosis.

Previous studies have indicated that SPARC contributes to the regulation of tumor formation, although its role appears to be cell type specific. SPARC expression is down-regulated in rat and chick embryo fibroblasts transformed with c-Jun and v-Src (40 , 62) , and its reintroduction counteracts tumorigenesis by these cells (41) . In addition, SPARC slows in vitro growth, induces apoptosis, and reduces tumorigenicity of ovarian cancer cells (44) . In contrast, suppression of SPARC expression abrogates the tumorigenicity of melanoma cells (42) .

SPARC is also involved in angiogenesis. SPARC blocks the G1 to S transition of endothelial cells manifested by decreased proliferation rates (63 , 64) and antagonizes the activity of the potent angiogenic activators bFGF and VEGF (38 , 39) . SPARC-null mice display substantially higher tumor invasion and angiogenesis compared with their wild-type littermates (65) , indicating that SPARC may interfere with tumor angiogenesis. However, other studies point to proangiogenic activity of SPARC (66) : it was found at high levels in breast cancer and colon cancer (67 , 68) , in metastatic melanoma (69) , and in invasive meningiomas (70) . Our studies suggest that disparate data regarding the role of SPARC in tumor growth and angiogenesis may be explained by its biphasic effect on the endothelial cells, where higher concentrations appear inactive or even stimulatory. Such biphasic effects are not uncommon for antiangiogenic factors and are shared by at least two more inhibitors, thrombospondin-1 (60 , 61) and PEDF.4 , 5

Our study revealed an inverse correlation between SPARC expression levels and the degree of malignant progression in NB tumors. Whereas tumorigenic cell lines and N-type subclones showed low or no detectable levels of SPARC mRNA and secreted protein, nontumorigenic S-type NB subclones and NB cells induced to differentiate in vitro expressed SPARC at high levels. In maturing and mature NB tumors rich in Schwannian stroma, Schwann cells and differentiated neuroblasts/ganglion cells showed strong positive staining for SPARC, whereas little to no staining was detected in undifferentiated, Schwannian stroma-poor tumors. Furthermore, we demonstrated antiangiogenic activity of SPARC in a mouse NB xenograft model.

The mechanisms underlying these highly variable cell type-specific activities of SPARC remain unknown. However, similar to thrombospondin-1 (60 , 61) , concentration-dependent activity of SPARC may be explained if two distinct receptors are present on vascular endothelium, where a low-affinity receptor activated by higher ligand concentrations conveys a proangiogenic function of SPARC, and a high-affinity receptor is antiangiogenic. SPARC function may also be altered via posttranslational modifications. SPARC subspecies secreted by normal fibroblasts and by melanoma tumors differ in size and glycosylation pattern (69) . It is tempting to speculate that turnover rates and receptor affinity of SPARC produced by malignant melanoma cells may be disparate from those of SPARC from normal stroma. Specific cleavage of SPARC by tumor cells may be glycosylation dependent and lead to an altered function. This hypothesis appears more feasible because the peptides from distinct structural domains of SPARC affect diverse cell phenotypes including growth rate, cell shape, matrix attachment, and angiogenic potential (66 , 69 , 71) . Changes in any of these functions alone may lead to decreased angiogenesis and slow down tumor progression. However, it is likely that it is the ability of SPARC, shared with the majority of the angiogenesis inhibitors (reviewed in Refs. 72 and 73 ), to induce apoptosis in the endothelial cells of remodeling vasculature that allows it to interrupt angiogenesis and to delay tumor progression.

Our data point to SPARC as a key contributor to the antiangiogenic activity of factors secreted by the Schwann cells. However, other inhibitors of angiogenesis including PEDF and TIMP-2 also appear to be involved in the cross-talk between Schwann cells and the neuronal component of NB tumors (29 , 30 , 74) . Our data clearly demonstrate that the angiogenic balance is complex: it is not a result of a single inducer-inhibitor combination but a composite value determined by all of the inducers and inhibitors present (reviewed in Ref. 75 ). Changes in angiogenic phenotype may occur gradually (76) or in a single step (77 , 78) . In Schwannian stroma-rich/stroma-dominant NB tumors, a spectrum of angiogenic inhibitors appears to be important for maintaining the net inhibitory phenotype, possibly because the nature of the angiogenic balance in NB tumors is quite complex. In addition to its effect on angiogenesis, PEDF also promotes survival and differentiation of the neuronal component of the tumors and thus creates a positive feedback loop (30) , whereas TIMP-2 suppresses tumor cell invasion (79) . A better understanding of the complex regulation of NB angiogenesis will hopefully lead to the development of new therapies in which inhibitors of angiogenesis may be used in concert to improve the outcome of children with clinically aggressive, Schwannian stroma-poor NB.


   FOOTNOTES
 
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.

1 Supported in part by the Neuroblastoma Children’s Cancer Society, Friends for Steven Pediatric Cancer Research Fund, the Elise Anderson Neuroblastoma Research Fund, the North Suburban Medical Research Junior Board, and the Robert H. Lurie Comprehensive Cancer Center NIH National Cancer Institute Core Grant 5P30CA60553. Back

2 To whom requests for reprints should be addressed, at Children’s Memorial Hospital, Division of Hematology/Oncology, Box 30, 2300 Children’s Plaza, Chicago, IL 60614. Phone: (773) 880-4562; Fax: (773) 880-3053; E-mail: scohn{at}northwestern.edu Back

3 The abbreviations used are: NB, neuroblastoma; PEDF, pigment epithelium-derived factor; TIMP-2, tissue inhibitor of metalloproteinase-2; SCM, Schwann cell-conditioned media; RA, retinoic acid; BrdUrd, 5-bromo-2'-deoxyuridine; CM, conditioned media; MVD, microvascular density; bFGF, basic fibroblast growth factor; RT-PCR, reverse transcription-PCR. Back

4 D. W. Dawson, unpublished data. Back

5 R. Schodlu et al., Augmentation of choroidal neovascularization in a laser induced mouse model by PEDF, in preparation. Back

Received 7/12/02. Accepted 10/10/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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