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2007;5:1015-1030. Published online October 19, 2007.Mol Cancer ResNeveen Said, Matthew J. Socha, Jeffrey J. Olearczyk, et al.SPARCNormalization of the Ovarian Cancer Microenvironment by

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Normalization of the Ovarian Cancer Microenvironmentby SPARC

Neveen Said,1 Matthew J. Socha,1 Jeffrey J. Olearczyk,1 Ahmed A. Elmarakby,1

John D. Imig,1,2 and Kouros Motamed1,3

1Vascular Biology Center and Departments of 2Physiology and3Pathology,

Medical College of Georgia, Augusta, Georgia

AbstractMalignant ascites is a major source of morbidity and

mortality in ovarian cancer patients. It functions as a

permissive reactive tumor-host microenvironment and

provides sustenance for the floating tumor cells through

a plethora of survival/metastasis-associated molecules.

Using a syngeneic, immunocompetent model of

peritoneal ovarian carcinomatosis in SP/ mice, we

investigated the molecular mechanisms implicated in

the interplay between host secreted protein acidic and

rich in cysteine (SPARC) and ascitic fluid prosurvival/

prometastasis factors that result in the significantly

augmented levels of vascular endothelial growth factor

(VEGF) and matrix metalloproteinases (MMP). Ascitic

fluidenhanced ID8 invasiveness was mediated through

VEGF via a positive feedback loop with MMP-2 and

MMP-9 and through activation of Av and B1 integrins.

Host SPARC down-regulated the VEGF-MMP axis at the

transcriptional and posttranscriptional levels. In vitro,

SPARC attenuated the basal as well as VEGF-induced

integrin activation in tumor cells. SPARC inhibited theVEGF- and integrin-mediated ID8 proliferation in vitro

and significantly suppressed their tumorigenicity

in vivo. Relative to SP+/+, SP/ ascitic fluid contained

significantly higher levels of bioactive lipids and exerted

stronger chemotactic, proinvasive, and mitogenic

effects on ID8 cells in vitro. SP/ ascites also

contained high levels of interleukin-6, macrophage

chemoattractant protein-1, and 8-isoprostane

(prostaglandin F2A) that were positively correlated with

extensive infiltration of SP/ ovarian tumors and

ascites with macrophages. In summary, our findings

strongly suggest that host SPARC normalizes the

microenvironment of ovarian cancer malignant ascitesthrough down-regulation of the VEGF-integrin-MMP

axis, decreases the levels and activity of bioactive lipids,

and ameliorates downstream inflammation.

(Mol Cancer Res 2007;5(10):101530)

IntroductionOvarian cancer is the leading cause of death among

gynecologic cancers in the United States. Metastatic

dissemination to peritoneal and pleural effusions is consid-

ered the major cause of patient morbidity and mortality in

ovarian cancer. The ability of floating ovarian cancer cells

in these malignant effusions to survive, proliferate, and

disseminate in the absence of immediate proximity to a solid

scaffold and vascular structures has been attributed to the

unique, yet poorly defined, permissive microenvironment of

these dense exudative fluids (1). The high autonomous

proliferation rate of the metastatic ovarian cancer cells, their

ability to develop alternative means of survival, as well as

the contributions of inflammatory cells present in malignant

effusions are believed to be responsible for the pluoripotent

nature of these malignant effusions (2-5). Ovarian cancer

cells and peritoneal mesothelial cells constitutively produce

bioactive lipids and cytokines that further promote moto-

genesis and mitogenesis of ovarian cancer cells in both

autocrine and paracrine fashion (6-9). The autocrine secretion

of cytokines, peptide growth factors, as well as proteolyticenzymes by malignant cells not only contributes to ascites

accumulation but also enhances their survival and dissemi-

nation (1, 10-12). Malignant ascites contains inflammatory

cells such as macrophages that further contribute to the

production of bioactive phospholipids, inflammatory cyto-

kines, and peptide growth factors in the ascitic fluid (13-15).

The interplay between different components of ascitic fluid is

complex and reflects the tumor-host interactions that support

the malignant process. Identifying and dissecting the factors

involved in the crosstalk between the components of such

microenvironments will not only improve our understanding

of the disease but will ultimately enable us to provide better

patient care.

Secreted protein, acidic, and rich in cysteine (SPARC) is a

matricellular protein involved in the modulation of cell

adhesion, motility, and interactions with components of the

extracellular matrix (16, 17). Recent reports highlight the role

of this molecule as a positive or negative modulator in the

pathogenesis of different malignancies (18-24). We and others

have shown that SPARC functions as a tumor suppressor in

ovarian cancer (25-28). As a tumor suppressor, effect of

SPARC in different cancers was attributed not only to its

counteradhesive, antiproliferative functions but also to its role

in modulating angiogenesis as well as regulation of the

production, assembly, and organization of the structural

extracellular matrix proteins including type I collagen and

fibronectin (23, 25, 29-32). Interestingly, the quality and

Received 1/1/07; revised 5/28/07; accepted 6/7/07.

Grant support: NIH grants K01-CA089689 (K. Motamed) and HL59699 (J.D.Imig).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby markedadvertisement in

accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Requests for reprints: Kouros Motamed, Vascular Biology Center, CB-3306,

Medical College of Georgia, 1459 Laney Walker Boulevard, Augusta, GA 30912.Phone: 706-721-9796; Fax: 706-721-9799. E-mail: [email protected] 2007 American Association for Cancer Research.

doi:10.1158/1541-7786.MCR-07-0001

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quantity of mature collagen bundles reported in tumor stroma

were positively correlated with levels of both tumor cell

SPARC (32, 33) and host SPARC (19, 25, 34). Thus, it was

hypothesized that SPARC-induced changes in the tumor

microenvironment were responsible, at least in part, for its

anticancer effects (32).

Herein, to assess the role of host SPARC in modulating the

ovarian cancer microenvironment, specifically ascitic fluid,

we used a syngeneic model of ovarian carcinomatosis in

immunocompetent SP/ mice by peritoneal implantation of

ID8 mouse ovarian cancer cells. The results of our in vitro and

in vivo studies have identified SPARC as a novel tumor

suppressor that normalizes the ovarian cancer microenviron-

ment, in part, through significant attenuation of the vascular

endothelial growth factor (VEGF)-integrin-MMP signaling

axis as well as the inflammatory processes that result from,

augment, and/or contribute to this axis.

ResultsSPARC Inhibits VEGF-Induced Proliferation and Survival

of Ovarian Cancer Cells In vitro and In vivo

VEGF is known to exert an autocrine as well as paracrine

mitogenic effect on a variety of tumor cells including

melanoma, prostate cancer, and ovarian cancer in vivo

(22, 35-38). Our pilot studies indicated that VEGF stimulates

the proliferation of ID8 cells in a concentration-dependent

manner (data not shown). To investigate the autocrine effect

of VEGF and to mimic the human disease, where VEGF is

constitutively up-regulated in cancer cells, we used an ID8 cell

line stably expressing VEGF164 (ID8-VEGF). This cell line has

previously been shown to dramatically accelerate tumor cell

growth and survival, tumor angiogenesis, and ascites formation

in a syngeneic mouse model of ovarian carcinomatosis

(39). ID8-VEGF exhibited higher proliferation rates than ID8

cells (Fig. 1A). In accord with our earlier report (25), the

FIGURE 1. SPARC inhibits VEGF-induced survival of ovarian cancer cells in vitro and in vivo. A. Proliferation of ID8 and ID8-VEGF cells was determinedby MTS assay. ID8-VEGF cells exhibited significantly higher rates of proliferation than ID8 cells, and both were inhibited by SPARC in a concentration-dependent manner. B. Animals injected intraperitoneally with ID8-VEGF cells exhibited decreased survival rates than those injected with ID8. SP/ miceexhibited decreased survival than SP+/+ counterparts. C. SP/ mice injected with ID8-VEGF developed larger volumes of ascitic fluid as compared withSP+/+. D. SP/ mice developed significantly larger ID8-VEGF subcutaneous tumors than SP+/+ counterparts. E. The mean vascular density (MVD) in ID8and ID8-VEGF tumors in SP/ and SP+/+ mice was determined by CD31 immunostaining. Columns, number of CD31-positive vessels counted in fivemicroscopic fields of at least four different tumor samples; bars, SE. #, P < 0.05, between ID8-VEGF tumors in SP+/+ and SP/ mice. {, P < 0.05, between

ID8 and ID8-VEGF tumors in SP/ mice.

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growth in mice (43). A reciprocal stimulation of the expression

and activity of MMP-2 by VEGF has also been reported in

ovarian cancer cell lines (44). Consistent with our previous

study (25), ascitic fluid of SP/ ovarian tumorbearing mice

showed enhanced MMP-2 and MMP-9 proteolytic activity

(data not shown). Up-regulated levels of MMP-2 and MMP-9

mRNA in SP/ tumors were concomitant with the down-

regulation of tissue inhibitor of metalloproteinase (TIMP)-1 and

TIMP-2 levels. Conversely, up-regulated TIMP-1 and TIMP-2

levels in tumors of SP+/+ mice were associated with down-

regulation of MMPs (Fig. 2A). Immunostaining of tumor

sections also confirmed the increased expression of MMP-2 and

MMP-9 and the decreased expression of TIMP-1 and TIMP-2

in SP/ tumors (Fig. 2B). In vitro, treatment of ID8 cell line

with SPARC (20 Ag/mL) for 6 h resulted in down-regulation of

MMP-2 and MMP-9 mRNA expression (Fig. 2C). However,

SPARC treatment did not have a significant effect on regulation

of TIMP-1 and TIMP-2 mRNA levels (Fig. 1C). These

results indicate that the tumor suppressor effect of SPARC is

mediated through both down-regulation of the expression and

activity of MMPs and up-regulation of their tissue inhibitors

in ID8 tumor cells in vivo and in vitro. The elevated levels

and activity of MMPs in SP/ tumor-bearing mice can also be

attributed to the significantly increased tumor-associated and

FIGURE 3. Augmentation of ID8 cell adhesion and invasion by ascitic fluid is VEGF and MMP mediated. A. ID8 cells (1 106/mL) were preincubated withsFlt2-11 (50 Ag/mL), KDR2 inhibitor IV (KDR2 I; 50 Amol/L), VEGF neutralizing antibody (VEGF-Ab; 20 Ag/mL), or vehicle control (DMSO) in DMEM-0.4%bovine serum albumin for 30 min before incubation with SP/ and SP+/+ ascitic fluid (final dilution of ascitic fluid was 1:2 in DMEM). Cells were allowed toadhere to fibronectin-coated wells of 96-well plates for 4 h. Adherent cells were stained and quantified by measuring absorbance ( OD) at 590 nm. B. ID8 cellswere treated exactly the same as in (A), and in the presence of MMP-2/MMP-9 inhibitors (MMP-2/9 I; 50 Amol/L) or with the general MMP inhibitor GM 6001(50 Amol/L) for 30 min before being allowed to invade fibronectin-coated inserts for 5 h. Columns, mean of three independent experiments done in triplicates;bars, SE. *, P < 0.05, compared with vehicle control. #, P < 0.05, between SP/ and SP+/+. C. Basal adhesion of ID8 cells to fibronectin was measured afterpretreatment with SPARC (20 Ag/mL, 2 h), VEGF neutralizing antibody, Flt2-11, and KDR2 inhibitor IV as described above. D. Basal fibronectin invasion byID8 cells after pretreatment with SPARC, VEGF neutralizing antibody, Flt2-11, and KDR2 inhibitor IV was done exactly as described in ( A). ID8-VEGF celladhesion to (E) and invasion of (F) fibronectin were tested as described above. Adhesion or invasion of ID8-VEGF cells treated with vehicle alone (control)was assigned a value of 100%. Adhesion and invasion results are expressed as percent of control. Columns, mean of three independent experiments done in

quadruplicates; bars, SE. *, P < 0.05.

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tumor-infiltrating macrophages. Interestingly, SP/ tumor

tissues and ascites exhibited a significant increase in

macrophage infiltration, as indicated by F4/80 staining relative

to their SP+/+ counterparts (Fig. 2D). In accord with our

previous study (25), these results were coincident with the up-

regulation of the expression of VEGF and its receptors in

tumor tissues and VEGF levels and activity in the ascitic fluid

of SP/ mice.

Augmentation of ID8 Cell Adhesion and Invasion by

Ascitic Fluid Is VEGF and MMP Mediated

The proinvasive properties of malignant ascites have been

shown to be mediated through the interplay of VEGF and

MMPs (2, 41, 45-47). Our previous findings that ascitic fluid

from SP/ mice enhanced the invasiveness and, to a lesser

extent, the adhesion of ID8 cells (25) led us to investigate the

possible mechanisms by which SPARC modulates the interplay

of VEGF and MMPs in the milieu of the ascitic fluid.

Consistent with our earlier report, the difference between SP+/+

and SP/ ascitic fluid induced adhesion was found to be

insignificant but markedly inhibited by a VEGF neutralizing

antibody, sFlt2-11, and a VEGFR2 (KDR) tyrosine kinase

inhibitor, suggesting an autocrine effect of VEGF produced

by ID8 cells themselves (Fig. 3A). On the other hand, SP/

ascitic fluid significantly (>75%) enhanced the invasiveness

of ID8 cells compared with SP+/+ ascitic fluid (Fig. 3B). This

FIGURE 4. Modulation of integrin-mediated proliferation, adhesion, and invasion of ID8 cells by host SPARC. A. P4G11 stimulation (10 Ag/mL DMEM-2%fetal bovine serum) increased the proliferation of ID8 cells grown on uncoated (solid columns), fibronectin-coated (checkered columns), and collagenIcoated (open columns) wells by 1- to 1.2-fold over unstimulated controls. SPARC treatment significantly decreased P4G11-induced proliferation(*, P < 0.05). MnCl2 (Mn

2+) increased ID8 proliferation by 1-fold (on uncoated wells) and f2-fold when grown on fibronectin and vitronectin (striped columns).SPARC significantly inhibited MnCl2-induced proliferation (**, P < 0.01). Columns, mean fold increase in cell proliferation, as compared with matched isotypecontrol antibody treated in DMEM-2% fetal bovine serum (assigned a value of 1); bars, SE. ID8 cells were preincubated with LM609, P1F6, and P4C10(15 Ag/mL each) or RGD peptide (10 Amol/L) for 30 min before stimulation with SP+/+ or SP/ ascitic fluid (final dilution is 1:2 in DMEM-0.4% bovine serumalbumin). Cells were allowed to adhere to fibronectin-coated wells for 4 h ( B) or invade fibronectin-coated inserts for 5 h ( C). *, P < 0.05, versus vehicle- ormatched isotype-treated SP+/+ controls. **, P < 0.05, versus vehicle- or matched isotype-treated SP/ controls. D. An ex vivo cell adhesion system showingsignificantly increased ID8-GFP adhesion to SP/ peritoneal explants compared with SP+/+. E. Adhesion of ID8-GFP cells to SP+/+ and SP/ murineperitoneal mesothelial cells is integrin mediated. ID8-GFP cells were pretreated exactly as described in ( B) and then were cocultured with primary SP+/+ andSP/ murine peritoneal mesothelial cells in 24-well plates for 5 h, as described in Materials and Methods. Columns, mean fluorescent intensity ( MFI) of sixfields per experimental condition from three independent experiments done in triplicates; bars, SE. *, P < 0.05, compared with SP+/+ vehicle- or isotype-treated controls. **, P < 0.05, compared with SP/ vehicle- or isotype-treated controls. F. Reverse transcription-PCR analysis of integrin expression in ID8peritoneal tumors grown in SP+/+ and SP/ mice. Columns, mean mRNA expression levels of integrin subunits from three different tumor samples in eachgroup following normalization to levels of rpS6 used as an internal control; bars, SE. Representative of three independent experiments.

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invasion of fibronectin-coated inserts was not only inhibited

significantly by VEGF neutralizing antibody and VEGFR

inhibitors but also by MMP inhibitors. These results suggest

that the proinvasive and, to a lesser extent, the proadhesive

effects of ascitic fluid are mediated through the paracrine effects

of VEGF and MMPs. In support of these findings, basal levels

of ID8 cell adhesion to and invasion of fibronectin were shown

to be significantly attenuated (f35% and 50%, respectively) in

the presence of exogenous SPARC (Fig. 3C and D). Inhibitors

of VEGF and VEGFR2, but not VEGFR1, showed significant

suppression of ID8 cell adhesion (Fig. 3C), whereas inhibitors

of VEGF, VEGFR, and MMP all resulted in significant

diminutions in ID8 cell invasion (Fig. 3D). These findings

suggest an autocrine effect exerted by VEGF that is produced

by ID8 cells themselves.

To further investigate the autocrine effect of VEGF, using

ID8-VEGF cell line, we found that they exhibited a significant

increase in adhesion to fibronectin (f20%) as compared with

ID8 controls. This increase in adhesion was significantlyinhibited (up to 40%) by exogenous SPARC and inhibitors of

VEGFRs but not MMPs (Fig. 3E). Furthermore, ID8-VEGF

cells exhibited markedly increased (f37%) fibronectin inva-

sion, relative to ID8 controls, which was significantly (30-70%)

inhibited by exogenous SPARC as well as by inhibitors of

VEGFRs and MMPs (Fig. 3F).

Modulation of Integrin-Mediated Adhesion and Invasion

of ID8 Cells by Host SPARC

It has been shown that adhesion of ovarian cancer cells

to the peritoneal mesothelial cells is integrin mediated (48-50).

We have previously shown that SPARC significantly inhibits

human ovarian cancer cell adhesion to and invasion ofextracellular matrix proteins and human mesothelial cells

(25), and that these functions were mediated through attenua-

tions of tumor cell surface expression and/or clustering of

av and h1 integrins (28). In the latter study, we found that

SPARC inhibited integrin-mediated proliferation of human

ovarian cancer cell lines (28). Herein, we show that activation

ofav integrin with 2.5 mmol/L MnCl2 significantly stimulated

proliferation of ID8 cells plated on fibronectin- and vitronectin-

coated wells (f2.2-fold as compared with unstimulated cells,

assigned a value of 1; Fig. 4A) as compared with uncoated

wells. Exogenous SPARC (10 Ag/mL) attenuated MnCl2-

induced proliferation by >1.5-fold. On the other hand,

activation ofh1 integrin by P4G11 exerted a significant, albeit

less pronounced, effect on ID8 proliferation whether they were

seeded on uncoated, collagen Icoated, or fibronectin-coated

plates. This effect was significantly inhibited by SPARC. These

findings led us to investigate whether ascitic fluid induced ID8

cell adhesion and invasion are also integrin mediated. We found

that ascitic fluidinduced adhesion of ID8 cells to fibronectin

was significantly suppressed by inhibitors of avh3 (LM609),

avh5 (P1F6), and h1 (P4C10), as well as by RGD peptide

(Fig. 4B). However, no significant difference was detected

between the proadhesive effects of SP/ and SP

+/+ ascitic

fluids. Conversely, the proinvasive effect of SP/ ascitic fluid

was significantly (>70%) higher than that of SP+/+ controls

(Fig. 4C). This ascitic fluid induced invasion of fibronectin

was also found to be inhibited by the aforementioned integrin

inhibitors. Next, we tested whether SP/ peritoneal mesothe-

lium supports adhesion of ID8 cells better than SP+/+ controls

and whether this adhesion is integrin mediated. Using an

ex vivo organ culture system, coincubation of freshly isolated

peritoneal explants, from either diaphragmatic peritoneum or

peritoneal lining of the anterior abdominal wall of SP/

animals, resulted in a significant (f3-fold) increase in the

adhesion of ID8-GFP cells relative to their SP+/+ counterparts

(Fig. 4D). Therefore, we tested the effect of integrin activation

and inhibition on the adhesion of ID8-GFP cells to isolated

SP/ and SP+/+ primary murine peritoneal mesothelial cells

in vitro. SP/ murine peritoneal mesothelial cells consistently

showed a significantly increased (25%) basal rate of adhesion

to ID8-GFP cells compared with SP+/+ controls (Fig. 4E).

Activation of av and h1 integrins by Mn2+ and P4G11,

respectively, resulted in a significant increase in adhesion of

ID8-GFP cells over unstimulated controls and was markedly

suppressed by function-blocking antibodies against avh3 and

h1 (Fig. 4E). ID8-GFP adhesion rates to SP/ murine

peritoneal mesothelial cells were consistently (15-25%) higher

than SP+/+ control whether integrins were activated by Mn2+

and P4G11 or inhibited by function-blocking antibodies and

RGD peptide. These results indicated that although the

adhesion of ID8 cells to murine peritoneal mesothelial cells

was integrin mediated, the difference in the adhesion rates was

correlated with the basal levels, which were mainly attributed

to the absence of SPARC in SP/ murine peritoneal

mesothelial cells. At the transcription level, absence of host

SPARC had no significant effect on integrin mRNA levels

(Fig. 4F) or protein levels (data not shown) in SP/ and SP

+/+

tumors. However, we cannot rule out differences in activation

of tumor cell surface integrins due to unavailability ofantibodies against mouse-specific, activated integrin subunits.

Taken together, these results indicated that the absence of host

SPARC augments the adhesion of ID8 cells to extracellular

matrix of the peritoneal mesothelium and that the adhesion is

also integrin mediated. This integrin-mediated adhesion is

independent of mesothelial SPARC but is hypothesized to be

due to the effect of soluble SPARC itself on integrin activation

and clustering and/or its effects on the soluble factors in ascitic

fluid that activate integrins on the cell surface of ID8 cells.

Interplay between VEGF and Integrins Influences Ligand

Recognition and the Proinvasive Properties of Ovarian

Cancer Cells

VEGF has been reported to activate the key integrins in

tumor cells as well as endothelial cells, thus influencing not

only angiogenesis but also tumor growth and metastasis

(22, 35-37, 51, 52). Ovarian cancer cells synthesize high levels

of VEGF, which not only induces tumor angiogenesis but also

functions as a permeability factor implicated in the production

and maintenance of malignant ascitic fluid, which in turn

supports tumor growth and metastasis (2, 47). Having shown

that the proadhesive and proinvasive effects of SP/ and SP

+/+

ascitic fluid on ID8 cells are both VEGF and integrin mediated,

we investigated whether SPARC itself inhibits integrin

activation and/or inhibits integrin activation by VEGF in ID8

cells. We found that ID8 cells exhibited high levels of basal

adhesion to fibronectin that was significantly augmented by

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integrin activators P4G11 and Mn2+ (Fig. 5A). ID8-VEGF

exhibited significantly (f40%) higher adhesion to fibronectin

than ID8 cells, an increase that was comparable to that of

integrin activation. SPARC inhibited both VEGF-mediated and

integrin-mediated adhesion to fibronectin by 40% to 50%,

respectively (Fig. 5A). To determine the integrins activated by

VEGF, we used integrin inhibiting antibodies and RGD peptide.

We found that the adhesion of ID8-VEGF cells to fibronectin

was inhibited (up to 40%) by function-blocking antibodies to

avh3 (LM609), avh5 (P1F6), h1 (P4C10), as well as RGD

FIGURE 5. SPARC inhibits integrin-mediated and VEGF-enhanced adhesion of ID8 cells. A. ID8 cells were preincubated with SPARC (10 Ag/mL) for2 h before stimulation with P4G11 (10 Ag/mL) or MnCl2 (2.5 mmol/L) for 30 min. ID8-VEGF cells were also pretreated with SPARC as mentioned above. Cellsuspensions were allowed to bind to fibronectin-coated wells for 4 h. Adhesion of ID8 cells treated with vehicle alone was assigned a value of 100%.Adhesion is expressed as percent change from control untreated ID8 cells, which is assigned a value of 100%. *, P < 0.05, versus vehicle- or isotype-stimulated control. **, P < 0.05, versus vehicle- or isotype-stimulated ID8 or ID8-VEGF controls. B. ID8-VEGF cells were pretreated with SPARC or integrininhibitors as described earlier and were plated on fibronectin-coated wells. Adhesion is expressed as percent change from control untreated ID8-VEGF cells,which is assigned a value of 100%. *, P < 0.05, versus ID8-VEGF control (C) cells. C. ID8 cells were preincubated with SPARC as in ( A) before stimulationwith MnCl2 (2.5 mmol/L) for 30 min. ID8-VEGF cells were similarly pretreated with SPARC. Cell suspensions were allowed to bind to vitronectin-coated wellsfor 4 h. Adhesion is expressed as percent change from vehicle controltreated ID8 cells, which is assigned a value of 100%. *, P < 0.05, versus vehicle-treated ID8 control. **, P < 0.05, versus vehicle-treated ID8 or ID8-VEGF. D. ID8-VEGF cells were pretreated with SPARC or av integrin inhibitors, asdescribed earlier, and were plated on vitronectin-coated wells. Adhesion is expressed as percent change from vehicle- or isotype control treated ID8-VEGFcells, which is assigned a value of 100%. *, P < 0.05, versus ID8-VEGF cells. E. ID8 cells were preincubated with SPARC as in ( A) before stimulation withP4G11 for 30 min. ID8-VEGF cells were similarly pretreated with SPARC. Cell suspensions were allowed to bind to collagen I coated wells for 4 h. Adhesionis expressed as percent change from control untreated ID8 cells, which is assigned a value of 100%. *, P < 0.05, versus isotype control stimulated ID8control. **, P < 0.05, versus isotype-stimulated ID8 or ID8-VEGF. F. ID8-VEGF cells were pretreated with SPARC or h1 integrin inhibitor P4C10, as describedearlier, and were then added to collagen I coated wells. Adhesion is expressed as percent change from vehicle- or isotype control treated ID8-VEGF cells,

which is assigned a value of 100%. *, P < 0.05, versus ID8-VEGF cells.



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peptide (Fig. 5B). To further identify the VEGF-activated

integrins, we first used vitronectin, the prototypic ligand of

avh3, and found that ID8-VEGF exhibited a significantly

increased (f50%) adhesion to vitronectin relative to ID8-GFP

cells (Fig. 5C) and that the observed increased adhesion is

significantly inhibited by LM609 and RGD but not by P1F6

antibodies (Fig. 5D). Next, we used collagen I as a putative

ligand for h1 integrins and found that the adhesion of ID8-

VEGF was comparable to that of P4G11-activated ID8-GFP

cells (Fig. 5E) and was significantly inhibited by P4C10

(Fig. 5F). These data indicate that VEGF regulates adhesion

and recognition of extracellular matrix molecules by ovarian

cancer cells through activation ofav (avh3, avh5, and possibly

avh1) and h1 integrin subunits.

FIGURE 6. SPARC inhibits integrin-mediated and VEGF-enhanced ID8 invasion by ID8 cells. ID8 and ID8-VEGF cell lines were treated exactly asdescribed in Fig. 4. ID8 cells pretreated with SPARC or PBS, in the presence or absence of integrin activators, were added to the upper chamber offibronectin-coated inserts (A). After 5 h, fibronectin invasion was determined by counting the cells on the undersurface of the inserts. Invasion is expressed aspercent change from vehicle- or isotype control antibody treated ID8 cells, which is assigned a value of 100%. *, P < 0.05, versus ID8 controls. **, P < 0.05,versus matched stimulated ID8 or ID8-VEGF. B. ID8-VEGF cells were pretreated with SPARC, vehicle, or isotype control antibody (C), or integrin inhibitors,and allowed to invade fibronectin-coated inserts. Invasion is expressed as percent change from control ID8-VEGF cells, which is assigned a value of 100%.*, P < 0.05, versus ID8-VEGF cells. C. ID8 cells were preincubated with SPARC or PBS in the presence or absence of MnCl 2. ID8-VEGF cells werepretreated with SPARC or PBS and allowed to invade vitronectin-coated inserts. Invasion is expressed as percent change from control ID8 cells, which isassigned a value of 100. *, P < 0.05, versus untreated ID8 control. **, P < 0.05, versus matched stimulated ID8 or ID8-VEGF. D. ID8-VEGF cells werepretreated with SPARC, isotype control antibody, or av integrin inhibitors and allowed to invade vitronectin-coated inserts. Vitronectin invasion is expressedas percent change from control ID8-VEGF cells, which is assigned a value of 100%. *, P < 0.05, versus ID8-VEGF cells. E. ID8 cells were preincubated withSPARC in the presence of h1 integrin activator P4G11 or isotype control antibody. ID8-VEGF cells were pretreated with SPARC or PBS and allowed toinvade collagen I coated inserts. Collagen I invasion is expressed as percent change from control untreated ID8 cells, which is assigned a value of 100%.*, P < 0.05, versus isotype-treated ID8 control. **, P < 0.05, versus matched stimulated ID8 or ID8-VEGF. F. ID8-VEGF cells were pretreated with isotypecontrol antibody, SPARC, or h1 integrin inhibitor P4C10, as described earlier, and were added to the upper chambers of collagen I coated inserts. Invasion

is expressed as percent change from control ID8-VEGF cells, which is assigned a value of 100. *, P < 0.05, versus ID8-VEGF cells.

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Next, we tested whether VEGF influences the integrin-

mediated invasiveness of ovarian cancer cells. Accordingly, the

effect of overexpression of VEGF164 on ID8 cell invasion was

assessed. In a transwell system, ID8-VEGF exerted a profound

increase in invasion of fibronectin, vitronectin, and collagen I

matrices. As shown in Fig. 6A, fibronectin invasion by ID8-

VEGF was significantly (f60%) higher than that of ID8-GFP

and was comparable to that induced by activating h1 integrins

(using P4G11) and av integrins (using Mn2+). This invasion

was inhibited substantially (up to 40%) by LM609, RGD

peptide, P1F6, P4C10, as well as SPARC (Fig. 6B). To

delineate the identity of the activated integrins, we used

vitronectin and collagen I as substrates. We found that basal

invasion of vitronectin by ID8 cells was significantly increased

(>55%) by Mn2+ and inhibited (>60%) by SPARC (Fig. 6C).

ID8-VEGF exhibited a significant (>40%) increase in vitro-

nectin invasion as compared with ID8-GFP. Vitronectin

invasion by ID8-VEGF was significantly inhibited (up to

40%) by LM609 and RGD peptide but not by P1F6 (Fig. 6D),

suggesting that avh3 is involved in VEGF-mediated invasion

of ID8 cells. Similarly, invasion of collagen I by ID8 cells

was markedly (f40%) augmented by h1 integrin activating

antibody P4G11, comparable to that of levels found in ID8-

VEGF (Fig. 6E). Collagen I invasion by ID8-VEGF was

significantly (up to 35%) inhibited by P4C10 and SPARC

(Fig. 6F). These results suggest that the constitutive activity of

integrins on ID8 cells was induced by a VEGF-dependent

autocrine loop through activation of av and h1 integrins.

SPARC attenuated not only the constitutive activity of integrins

in tumor cell lines but also their activated levels induced by

specific integrin activators and VEGF.

Modulation of the Activity of Bioactive Lipids in the Ascitic

Fluid by Host SPARC

We and others have recently shown that biologically active

lipids, mainly lysophosphatidic acid (LPA), in the heat-

insensitive fraction of mesothelial cell conditioned medium

exert a chemotactic and proinvasive effect on human ovarian

cancer cells in vitro (9, 27). In the latter study, we also reported

that exogenous SPARC significantly inhibited LPA-mediated

invasiveness and proliferation of human ovarian cancer cells.

These findings prompted us to test whether the proinvasive and

FIGURE 7. Modulation of the activity of bioactive lipids in the ascitic fluid by host SPARC. Pooled SP+/+ and SP/ ascitic fluids with equivalent proteinconcentration were heated at 95jC for 10 min to inactivate the biologically active peptides and proteins. The effect of heated and unheated samples onfibronectin invasion (A) or migration (B) of ID8 cells was measured as described earlier. The chemotactic effect of heated and unheated SP/ and SP+/+

ascitic fluids was tested by adding 600 AL of ascitic fluid to the bottom chamber of fibronectin-coated transwell inserts. ID8 cells (1 105/100 AL of DMEM-0.4% bovine serum albumin) were added to the upper chamber. After 5 h, cells that migrated to the undersurface of the inserts were stained and counted.*, P < 0.05, compared with unheated SP+/+ ascitic fluid. **, P < 0.05, between heated and unheated ascitic fluid. The proliferative effect of SP/ and SP+/+

ascitic fluids on ID8 cells was determined by MTS assay (C). *, P < 0.05, compared with unheated SP+/+ ascitic fluid. **, P < 0.05, between heated andunheated ascitic fluid. Cell proliferation of ID8 cells in response to indicated concentrations of LPA, in the presence and absence of SPARC (10 Ag/mL), wasassessed by MTS assay (D). Columns, mean of three independent experiments done in quadruplicates; bars, SE. *, P < 0.05, compared with vehicle control.**, P < 0.05, compared with corresponding LPA stimulation.



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chemotactic effects of SP+/+ and SP

/ ascitic fluids are

mediated by bioactive lipids, especially LPA. We found that the

proinvasive activity of ascitic fluids from intraperitoneal

tumorbearing SP+/+ and SP

/ mice was significantly

decreased (f23% and 27%, respectively) after heating at

95jC for 10 min (Fig. 7A). A significant (f2-fold) difference

in the proinvasive activity was also observed between heated

and unheated SP+/+ and SP

/ ascitic fluids. Similarly, we

found that the ascitic fluid from SP/ mice exerted a

significantly more potent chemotactic activity effect on ID8

cells than that of SP+/+ mice (Fig. 7B). As anticipated, heating

significantly decreased this chemotactic effect (>33%). Fur-

thermore, the mitogenic effect of ascitic fluids on ID8 cells was

also significantly (f35%) inhibited by heating and revealed a

statistically significant difference between the proliferative

capacities of SP+/+ and SP

/ ascitic fluids (Fig. 7C). These

results indicate that bioactive lipids representf60% to 65% of

the activity of the ascitic fluid. The major identified component

of these bioactive lipids was LPA, which influences almostevery aspect of ovarian cancer progression (7, 9). In agreement

with our recent report (27), LPA was also shown to induce

proliferation of ID8 cells directly in a concentration-dependent

manner, and addition of exogenous SPARC (10 Amol/L)

significantly diminished the proliferation induced by low

concentrations (5-10 Amol/L) of LPA but had no significant

effect when higher concentrations (>20 Amol/L) of LPA were

used (Fig. 7D). These data suggest that the significantly

augmented proinvasive, chemotactic, and mitogenic effects of

SP/ ascitic fluid on ID8 cells are mediated, at least in part, by

the biologically active lipids in the ascitic fluid, of which LPA

is well recognized. Moreover, these pronounced effects

contribute to the increased tumor burden in SP/ mice, which

in turn increases LPA production not only from tumor cells but

also from mesothelial cells and other stromal components of the

ascitic fluid.

SPARC Ameliorates the Peritoneal Tumor Induced

Inflammation

Ovarian cancer cells have been reported to producecytokines that further promote tumor survival and invasiveness

FIGURE 8. SPARC ameliorates peritoneal tumor induced inflammation. Determination of the level of MCP-1 (A), 8-isoprostane (B), and IL-6 (C) inSP/ and SP+/+ ascitic fluids by ELISA. Columns, mean of eight SP/ and five SP+/+ ascitic fluid samples; bars, SE. *, P < 0.01, compared with SP+/+. D.Chemotactic effect of ascitic fluid on ID8 cells was determined as described in legend of Fig. 7B. IL-6 (50 ng/mL) was added with SP+/+ ascitic fluid and IL-6neutralizing antibody (IL-6 Ab, 50 Ag/mL) was preincubated with SP/ ascitic fluid for 30 min before being added to the bottom chambers. *, P < 0.05,compared with vehicle control treated cells. **, P < 0.01, compared with SP+/+. E. The proinvasive activity of IL-6 in the ascitic fluid was tested in afibronectin invasion assay. ID8 cells (1 105/100 AL ascitic fluid) were added to the upper chamber of fibronectin-coated inserts in the presence and absenceof IL-6 and IL-6 neutralizing antibody as described earlier. The bottom chambers contained DMEM-5% fetal bovine serum. Columns, mean of threeindependent experiments done in triplicates; bars, SE. *, P < 0.05, compared with control untreated ascitic fluid. **, P < 0.01, compared with SP+/+. F. Cellproliferation of ID8 cells in response to indicated concentrations of IL-6, in the presence and absence of SPARC (10 Ag/mL), was assessed by MTS assay.Columns, mean of three independent experiments done in quadruplicates; bars, SE. *, P < 0.05, compared with control cells that are not stimulated (NS). **,

P < 0.05, compared with the corresponding IL-6 stimulation.

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(53). High interleukin-6 (IL-6) levels in the ascitic fluid of

ovarian cancer patients were correlated with poor prognosis

(6). Other studies have highlighted the crucial role of IL-6 in

the pathogenesis of ovarian cancer (54, 55). In the present

study, we found significantly higher levels of macrophage

chemoattractant protein-1 (MCP-1; >2.5-fold) in SP/ ascitic

fluid (Fig. 8A), and these levels coincided with the extensive

macrophage infiltration in SP/ tumors and ascites shown in

Fig. 2D, highlighting a potent anti-inflammatory effect for

SPARC. We also found that the levels of arachidonic acid

inflammatory mediator, 8-isoprostane, were significantly

higher (4.3-fold) in SP/ ascitic fluid compared with SP

+/+

(Fig. 8B), indicating the hypoxic stress in the ascitic fluid

microenvironment, which correlates with LPA, tumor burden,

as well as inflammation. Consistently, ascitic fluid from SP/

mice contained significantly (f2-fold) higher levels of IL-6

as compared with SP+/+ fluid (Fig. 8C). These data suggest

that SPARC, through down-regulation of MCP-1, decreases

tumor infiltration by macrophages, and hence decreases IL-6and 8-isoprostane proinflammatory mediators in the ascitic

fluid. Furthermore, our in vitro data indicated that the

augmented chemotactic (Fig. 8D) and proinvasive (Fig. 8E)

effects of SP/ over SP+/+ ascitic fluid were, in part, due to

the high IL-6 content, as confirmed by restoration of these

activities in SP+/+

ascitic fluid by exogenous IL-6 and

neutralizing them by IL-6 neutralizing antibody in SP/

ascites. Moreover, IL-6 exerted a concentration-dependent

increase in ID8 proliferation, which was shown to be inhibited

by exogenous SPARC (Fig. 8F).

DiscussionOf the numerous growth factors involved in the patho-

genesis of ovarian cancer, the angiogenic growth factor VEGF

plays a central role in the development of exudative asci-

tic fluidthe hallmark of peritoneal ovarian carcinomatosis

(41, 56). Cross talk between VEGF and integrin cell adhesion

receptors is believed to play a critical role in modulation of

tumor growth, survival, vascularization, invasion, and metas-

tasis (51). The metastatic potential of tumor cells has been

correlated with up-regulation of VEGF and VEGF-induced

cell-surface expression and clustering of integrins including

avh3, avh5, and h1 (35-37, 51, 57). Consistent with these

reports, our findings in this study provide evidence that (a) the

proadhesive and proinvasive effects of ascitic fluid are

mediated, in part, through VEGF binding to its cognate

receptors and activation of av and h1 integrins in ovarian

cancer cells; (b) host SPARC negatively regulates MMP-2 and

MMP-9 levels and activity in peritoneal tumors and ascitic

fluid; (c) the enhanced adhesion of ovarian cancer cells to

SP/ peritoneal mesothelial cells and their augmented

invasion of fibronectin in response to SP/ ascitic fluid are

mediated, in part, through the inhibitory effect of SPARC on

integrin expression and activation of ovarian cancer cells,

either directly or by modulation of levels and/or activity of

soluble factors in the ascitic fluid; (d) SPARC inhibits VEGF-

and integrin-mediated ovarian cancer cell proliferation in vitro,

as well as VEGF-mediated mitogenic and tumorigenic effects

in vivo; and (e) host SPARC ameliorates the peritoneal

tumor induced inflammatory response that accompanies

ovarian cancer through attenuation of levels and activity of

bioactive lipids, IL-6, MCP-1, and 8-isoprostane proinflam-

matory mediators (Fig. 9).

VEGF has been shown to activate MMP-2 in ascitic fluid

during peritoneal dissemination of ovarian cancer (58).Moreover, VEGF-regulated ovarian cancer invasion and

migration was recently reported to involve expression and

activation of MMP-2, MMP-7, and MMP-9 (59). Conversely,

MMP-2 and MMP-9 have been implicated in formation of

ascites through induction of the release of VEGF by ovarian

carcinoma cells (41), suggesting the existence of a positive

autocrine feedback loop between VEGF and MMPs. A link

between integrins and the up-regulated MMPs in SP/ ascites

may be suggested based on the earlier findings that avh3integrin binds MMP-2 on the surface of melanoma and

endothelial cells and both colocalize in caveolae and promote

tumor growth and angiogenesis in vivo (60). avh3 integrin

also cooperates with MMP-9 by increasing migration of

metastatic breast cancer cells, as avh3 integrin does not adhere

to native, intact collagen but it does adhere to collagen that

has undergone proteolytic degradation due to exposure of an

RGD binding site (61). Furthermore, av and h1 integrins,

through outside in and inside out integrin signaling, have

been reported to induce the transcription of MMP genes in a

variety of cancer cell lines, including ovarian cancer (refs. 62, 63

and the references reviewed therein). This well-orchestrated

interplay of VEGF-integrin-MMP axis in ascitic fluid favors the

proinvasive and migratory phenotype of ovarian cancer cells

after temporary ligand recognition and binding. It is therefore

plausible to suggest that perturbation of this axis at the

transcriptional and posttranscriptional levels by SPARC attenu-

ates ovarian cancer cell motility, invasiveness, and metastasis.

FIGURE 9. Hypothesized roles of SPARC in modulating signalingpathways involved in the pathogenesis of ovarian cancer. The pathwaysinvolved in peritoneal ovarian carcinomatosis are complex, interrelated,often synergistic, and mostly converge on stimulation of tumor cellsurvival, proliferation, and invasion. This schema summarizes the majorsignaling pathways involved in the pathogenesis of ovarian carcinomatosisand their proposed regulation by SPARC (SP). These various roles ofSPARC were hypothesized based on our findings in the present study aswell as our published studies using human cells and mouse tumor models.PGs, prostaglandins; ROS, reactive oxygen species; FN, fibronectin; VN,vitronectin.



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LPA has been identified as an ovarian cancer promoting

factor because high levels of this bioactive lipid have been

reported in both plasma and ascites of ovarian cancer patients

with stage I disease, suggesting that LPA promotes early events

in ovarian carcinoma dissemination (64). Up-regulation of

potent prosurvival, proangiogenic, as well as proinflammatory

cytokines and chemokines has been reported downstream of

LPA activity in vivo and in vitro (6, 7, 65, 66). LPA is

constitutively produced by peritoneal mesothelial cells and has

been shown to be crucial for the chemotactic activity of shed

ovarian cancer cells (9, 27). Moreover, LPA enhances motility

and invasiveness of ovarian cancer cells, in part, through up-

regulation and activation of MMPs in ovarian cancer cells (67).

Through binding to its cognate receptor, LPA has been shown

to synergize with signals induced by h1 integrin and activation

of EGF or platelet-derived growth factor receptors to modulate

cellular migration (68). Accordingly, our results in the present

study suggest that LPA- and VEGF-induced integrin activation

may function via convergent signal transduction pathways topromote MMP expression and/or processing, resulting in

significant augmentations in the migratory and invasive

behavior of ovarian cancer cells. Moreover, the mitogenic and

prosurvival effects of LPA have been attributed to its direct

effect on cell cycle progression, up-regulation of VEGF,

transactivation of receptor tyrosine kinases (e.g., EGFR and

VEGFRs), and induction of an inflammatory response (66). Our

recent report on significant attenuation of the LPA-mediated

crosstalk between mesothelial cells and human ovarian cancer

cell lines by exogenous addition or ectopic overexpression of

SPARC (27), as well as significant diminutions in proliferation,

migration, and invasion of bioactive lipids in ascites of ovarian

tumorbearing SP+/+

mice, highlights the important role ofSPARC in suppression of LPA-mediated events in ovarian

carcinoma.

In ovarian cancer, tumor cells are not the only source of

MMPs and proinflammtory cytokines. Tumor-associated mono-

cytes/macrophages are recruited from peripheral blood mono-

cytes in response to chemokines produced by tumor cells,

mainly through the action of MCP-1 (CCL2; refs. 69, 70). As a

consequence, tumor-associated monocytes/macrophages under-

go marked phenotypic changes with activation of hypoxia-

inducible factor 1a, dramatically up-regulating the expression

of a large number of genes encoding mitogenic, proangiogenic,

and prometastatic cytokines, enzymes, and bioactive lipids

(70, 71). Interestingly, in vivo and in vitro studies have shown

that VEGF, MMPs, and IL-6 in the tumor microenvironment

inhibit the differentiation and maturation of dendritic cells and

switch their differentiation toward the macrophage lineage

(72, 73). Accordingly, the infiltration of malignant ascitic fluid

and solid peritoneal tumors with macrophages in our syngeneic

ovarian cancer model was directly correlated with the levels of

ascitic fluid VEGF, MMPs, MCP-1, and IL-6. Our results that

SP/ tumor-bearing mice had higher ascitic fluid MCP-1 and

IL-6 levels and more extensive macrophage infiltration than

SP+/+ mice further validate a negative regulatory role for host

SPARCin thedevelopmentof ovarian carcinomatosis. In support

of these findings, we have recently shown that IL-6 and MCP-1

production by human ovarian cancer cell lines is enhanced by

LPA stimulation and down-regulated by SPARC (27).

In this study, further evidence for the anti-inflammatory

role of host SPARC is provided by the dramatic increase in

8-isoprostane (prostaglandin F2a) levels in SP/ ascitic

fluid, compared with SP+/+ counterparts. Isoprostanes,

including 8-isoprostane, are produced by the action of

cyclooxygenase enzymes (Cox-1 and Cox-2) on arachidonic

acid via a free radicalcatalyzed mechanism of phospholipid

peroxidation, and have been associated with oxidative stress

and high levels of LPA (74, 75). LPA has recently been

shown to up-regulate Cox-1 and Cox-2 expression in ovarian

cancer, with subsequent up-regulation of MMPs and growth

factors, enhancing ovarian cancer cell survival and invasive-

ness (66). Furthermore, in a recent study by Rask et al. (76),

the expression of Cox-1 and Cox-2 in ovarian cancer, as well

as their downstream prostaglandin E2, was associated with

the high grade of ovarian cancer, suggesting an important

role in their malignant transformation and progression. The

observation that Cox-1 and Cox-2, as well as prostaglandin

E2, were expressed in the tumor cells as well as in stroma ledthe authors to suggest a paracrine signaling mechanism

mediated by growth factors, cytokines, and possibly prosta-

glandins due to the interactions between tumor cells and

stromal cells (fibroblasts and immune cells). Moreover,

cytosolic phospholipase A2 (cPLA2) has been implicated in

LPA production in ascitic fluid and was associated with the

invasive metastatic phenotype (77). Recently, it has been

reported that under the hypoxic conditions encountered in

ovarian cancer ascitic fluid, LPA up-regulates cytosolic

phospholipase A2 and augments the metastatic phenotype

through a hypoxia-inducible factor 1a dependent pathway

(78). Taken together, we can speculate that the up-regulation

of 8-isoprostane in SP/

ascitic fluid is attributed to theactivation and up-regulation of cLPA and the downstream Cox

enzymes by LPA in the hypoxic microenvironment created by

the rapidly proliferating tumor cells and other infiltrating

inflammatory cells. To the best of our knowledge, this is the

first report implicating the involvement of 8-isoprostane

(prostaglandin F2a) in the ascitic fluid of ovarian cancer and

its down-regulation by host SPARC. The anti-inflammatory

effects of SPARC have previously been implicated in SP/

mice as evidenced by decreased leukocyte infiltration in

bleomycin-induced peritonitis (79), attenuated 12-O-tetradeca-

noylphorbol-13-acetate induced skin inflammation (24), as

well as lung and pancreatic tumors, and have been attributed,

at least in part, to abundance of less dense, immature collagen

fibrils and a more malleable extracellular matrix (19, 31).

Lastly, SPARC has also been shown to negatively regulate the

availability and/or activity of proinflammatory/angiogenic

cytokines including platelet-derived growth factor (80),

fibroblast growth factor-2 (81), and VEGF (82). Collectively,

these data strongly suggest a negative regulatory role for host

SPARC in the inflammatory process that accompanies ovarian

cancer.

In summary, we have provided evidence that SPARC,

through interference with the VEGF-integrin-MMP axis as

well as that of the inflammatory mediators/intermediates in

the malignant ascitic fluid, plays a crucial role in the norma-

lization of the reactive microenvironment of peritoneal ovarian

carcinomatosis. These findings, in combination with the

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established antiproliferative, proapoptotic, and antimetastatic

properties of SPARC in ovarian cancer, underscore its

therapeutic potential as a promising novel inhibitor of

peritoneal ovarian carcinomatosis.

Materials and MethodsCell Culture

ID8, a cell line that was derived from spontaneous in vitro

malignant transformation of C57BL/6 mouse ovarian surface

epithelial cells (83) and was stably transfected with a retroviral

vector expressing green fluorescent protein (GFP) or VEGF164plus GFP (ID8-VEGF; ref. 39), was kindly provided by

Dr. George Coukos (Abramson Family Cancer Research

Institute, University of Pennsylvania, Philadelphia, PA). Cells

were maintained in DMEM, supplemented with 2 mmol/L

L-glutamine (Sigma), 5% fetal bovine serum (Atlanta Bio-

logicals), and 100 units/mL penicillin and 100 Ag/mL

streptomycin (Sigma).

Reagents and Antibodies

SPARC from mouse parietal yolk sac was purchased from

Sigma. Bovine osteonectin was purchased from Haematologic

Technologies. Collagen I from rat tail, human plasma fibronec-

tin, and vitronectin were from Sigma. Integrin-activating and

integrin-blocking antibodies, rabbit anti-VEGF neutralizing

antibody, rabbit anticytokeratin 5, anticytokeratin 18, and

anti-vimentin antibodies were from Chemicon. Polyclonal

goat antibodies against MMP-2, MMP-9, TIMP-1, TIMP-2,

and integrins were from Santa Cruz Biotechnology, Inc. Rat

anti-mouse macrophage marker F4/80 was from Serotec,

Inc. Rat anti-mouse CD31 was from Chemicon. MMP inhibitor

kit II and VEGF inhibitors were from Calbiochem. Lysopho-

spahtidic acid (LPA) and RGD peptides were purchased from

Sigma. Recombinant murine IL-6 and anti-mouse IL-6 anti-

bodies were from RayBiotech. All other chemicals were of

analytic grade and were purchased from Sigma and Fisher

Scientific.

Mice

C57BL/6 129SvJ SPARC-null (SP/) and wild-type

(SP+/+) mice (6-8 weeks old) were a kind gift of Dr. Helene

Sage (Hope Heart Program, Benaroya Research Institute at

Virginia Mason, Seattle, WA). Mice were backcrossed against

wild-type C57BL/6 mice for at least six generations beforeuse in tumor studies. All experimental procedures were

approved by the Laboratory Animal Services of the Medical

College of Georgia and were done in specific pathogen-free

facilities.

In vivo Tumor Generation

Intraperitoneal and subcutaneous tumors were generated by

injection of ID8 and ID8-VEGF cells into SP+/+ and SP/

mice (n = 10 per group) as previously described (25, 39).

Animal survival and tumor development were monitored twice

weekly. At the end of the experimental period, animals were

sacrificed and ascitic fluid and tumor tissues from the animals

were collected and processed as previously reported (25, 39).

RNA Isolation and Reverse Transcription-PCR

RNA was isolated from tissues and cells with Trizol reagent

(Invitrogen) and was purified with RNeasy isolation kit

(Qiagen). Total RNA (2 Ag) was reverse transcribed using

Improm II RT enzyme kit (Promega) and cDNA was amplified

using Jumpstart Taq polymerase (Sigma) as recommended by

the manufacturers. Specific oligonucleotide primers were

synthesized according to published sequences (Table 1). PCR

products were resolved in 2% agarose gels and were visualized

with ethidium bromide staining under UV light. Gel imaging,

documentation, and analysis were done with a Kodak Gel Logic

100 imaging system equipped with Kodak D 3.6 software

(Eastman-Kodak).

Immunohistochemistry

The expression of MMP-2, MMP-9, TIMP-1, TIMP-2, and

F4/80 was determined in deparaffinized tumor sections after

antigen retrieval with AutoZyme (BioMeda Corp.). Sections

were incubated with specific primary antibodies overnight at

4jC, washed with PBS-0.2% Tween 20, and incubated for

1 h with peroxidase-labeled appropriate secondary antibodies

(Jackson ImmunoResearch laboratories, Inc.). Sections were

then developed with Vectastain ABC Elite kit (Vector

Laboratories) and stable 3,3-diaminobenzidene (ResGen),

counterstained with hematoxylin, and mounted in Permount

(Fisher Scientific). Images were acquired with Leica micro-

scope (DM5000) equipped with a Q-Imaging digital camera.

The mean vascular density in tumor sections was determined

by immunostaining with anti-CD31 antibody and averaging the

TABLE 1. PCR Primers Used in Semiquantitative ReverseTranscription-PCR

Primer Sequence Cycles

rpS6

F: 5-AAGCTCCGCACCTTCTATGAGA-3 24R: 5-TGACTGGACTCAGACTTAGAAGTAGAAGC-3

MMP-2F: 5-TTGGATATTTGCAATGCAGCC-3 35R: 5-AAGGTTGAAGGAAACGAGCGA-3

MMP-9

F: 5-CCATTTCGACGACGAGT-3 35R: 5-CCAAATTTGCCGTCCTTATCGTA-3

TIMP-1

F: 5-CTGTGGGAAATGCCGATA-3 30R: 5-TGAGCAGGGCTCAGAGTACGC-3

TIMP-2F: 5-TCATCAGGGCCAAGTTCGTG-3 30

R: 5-GGATCATGGGACAGCGAGTGA-3 h3 integrin

F: 5-CTGGTAAAAACGCCGTGAAT-3 35R: 5-GAGTAGCAAGGCCAATGAGC-3

h1 integrin

F: 5-GCCAGGGCTGGTTATACAGA-3 35R: 5-TCACAATGGCACACAGGTTT-3

h5 integrinF: 5-TAGCTAGGCACGCACGGATAAT-3 35

R: 5-TTTGTACACACAGACTCTTCCGGATA-3 av integrin

F: 5-AAACGTGTCCGACCACCTC-3 35

R: 5-CTTTACAATCCTAATTGCCCTAACG-3a5 integrin

5-TCCTATCCAGTGCACCACCATT-3 355-GCAACAAAGTCTTCTGTGTCATCAC-3



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number of positively stained vessels in five microscopic

fields per section of tumors from at least four different animals

(n = 4).

Adhesion AssaysAdhesion assays were done as previously described (25).

Harvested cells were preincubated with and without MMP

inhibitors (40 Amol/L), VEGF inhibitors (40 Ag/mL), integrin

blocking antibodies (10 Ag/mL), SPARC (10 Ag/mL), or RGD

peptides (20 Amol/L) followed by stimulation with VEGF164(40 ng/mL), integrin activating antibody (5-10 Ag/mL), MnCl2(1-5 mmol/L), IL-6 (5-50 ng/mL), LPA (5-50 Amol/L), or

ascitic fluid. Cell suspensions were then added to ligand-coated

plates. After a 4-h incubation at 37jC/5% CO2, nonadherent

cells were washed out and adherent cells were stained with

Hemacolor 3 stain (Fisher Scientific) as recommended by the

manufacturer. Cell adhesion was determined by measuring the

absorbance at 590 nm.

Isolation of Murine Peritoneal Mesothelial Cells

Murine peritoneal mesothelial cells were isolated from SP+/+

and SP/ mice by peritoneal lavage and from collagenase/

dispase-digested explants from the mesentery, as previously

described (84). The digested tissue was placed into collagen

Icoated plates and cultured for 2 days in DMEM containing

12% fetal bovine serum and EGF (20 ng/mL). Mesothelial cells

used for these studies (passages 1-2) were confirmed to be

positive for the expression of cytokeratin 5, cytokeratin 18, and

vimentin, as determined by immunocytochemical staining.

Adhesion of ID8-GFP Cells to SP+/+ and SP/ Peritoneal

Explants In vitro

Fresh peritoneal tissues were excised and prepared as

previously described (85). Briefly, 6 6-mm2 peritoneal pieces

were dissected from the diaphragmatic peritoneum and the

peritoneum lining the anterior abdominal wall and were placed

in 24-well plates with the mesothelium facing upward. ID8-

GFP cells (5 105/500 AL of DMEM-0.4% bovine serum

albumin) were added onto peritoneal pieces in each well. Plates

were then incubated for 72 h in a humidified incubator at 37jC/

5% CO2. At the end of the incubation period, peritoneal pieces

were rinsed thrice in PBS to remove nonadherent cells. Adherent

cells were visualized under a fluorescent stereomicroscope,

photographed, and the mean green fluorescence intensity in six

fields per experimental condition was determined.

Invasion Assays

Invasion assays were done as previously described (25).

Harvested cells were treated exactly as described in adhesion

assays and cell suspensions were added to the upper chamber of

polycarbonate inserts (8-Am pore size, Corning Costar) pre-

coated with fibronectin, collagen I, or vitronectin. After a

5-h incubation at 37jC/5% CO2, cells in the upper chamber

were removed by Q-tips (Fisher Scientific). Cells attached to

the underside of the inserts (the invasive cells) were stained

with Hemacolor 3 stain and counted under an inverted

microscope equipped with DFC 320 digital camera (Leica)

under200 magnification.

ELISA

Mouse IL-6 ELISA kit (RayBiotech), MCP-1 ELISA kit

(BD Biosciences), and 8-isoprostane EIA kits (Cayman

Chemical) were used to determine IL-6, MCP-1, and 8-

isoprostane levels in ascitic fluid samples, respectively,

according to the manufacturers instructions.

Immunoblotting

Tumor tissues from SP+/+ and SP

/ animals (n = 4) were

homogenized in lysis buffer [20 mmol/L Tris (pH 7.4),

150 mmol/L NaCl, 1 mmol/L EDTA, 50 mmol/L NaF, 0.5%

sodium deoxycholate, 1% NP40, 1 mmol/L Na3VO4, and

1 protease inhibitor cocktail mixture] as previously described

(25). One hundred micrograms of cell lysates were resolved

by 10% SDS-PAGE under nonreducing conditions and

transferred onto polyvinylidene difluoride membranes (Bio-

Rad). The membranes were then incubated overnight at 4jC

with antimouse specific integrin antibodies (Santa Cruz Bio-

technology) and monoclonal antiglyceraldehyde-3-phosphate

dehydrogenase (Ambion) as loading control. Protein detection

was carried out with horseradish peroxidase conjugated

secondary antibodies and a SuperSignal West Dura Chemilu-

minescence kit (Pierce).

Nonradioactive Cell Proliferation Assay

CellTiter96 kit (Promega) was used according to the

manufacturers instructions. The number of proliferating cells

was determined colorimetrically by measuring the absorbance

at 590 nm (A590) of the dissolved formazan product after the

addition of 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxy-

phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) for 3 h.

Statistical Analysis

Statistical analysis was done using GraphPad Prism version

3.1 for Windows (GraphPad Software). Survival curves were

generated using the Kaplan-Meier method followed by log-rank

test for comparison of curves. The concentration-dependent

studies were done by one-way ANOVA followed by Newman-

Keuls multiple comparison test. All other statistical analyses

were determined by Students t test. Differences were

considered significant at P < 0.05.

AcknowledgmentsWe thank Dr. George Coukos for providing ID8-GFP and ID8-VEGF-GFP cells

and Dr. E. Helene Sage for providing SP/ and SP+/+ mice.

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