(CANCER RESEARCH 50, 6959-6965. November I. 1990]

Constitutive Production of Interleukin 6 by Ovarian Cancer Cell Lines and byPrimary Ovarian Tumor Cultures1

Joanna M. Watson, John L. Sensintaffar, Jonathan S. Berek, and Otoniel Martinez-Maza2

Departments of Microbiology and Immunology [J. M. W., O. M-M.J and Obstetrics and Gynecology [J. L. S., J. S. B., O. M-M.¡,and Jonsson Comprehensive CancerCenter, University of California-Los Angeles School of Medicine, Los Angeles, California 90024

ABSTRACT

We examined the production and utilization of interleukin 6 (11-(>),amultifunctional cytokine with diverse biological effects, by both ovariancancer cell lines and primary ovarian tumor cultures. \Ve have found thatepithelial ovarian cancer cell lines (CAOV-3, OVCAR-3, and SKOV-3)constitutive!) produce varying amounts of IL-6. This molecule is biologically active as determined by the proliferation of an IL-6-dependenthybridoma cell line, MH60.BSF-2, and is detectable by an IL-6 enzyme-linked immunosorbent assay. By cytoplasmic immunoperoxidase staining,>98% of the cells produce at least some IL-6, with variation in thestaining intensity between individual cells. The ovarian cancer cell-produced protein has a molecular »eightof approximately 24,000, andexhibits some molecular weight heterogeneity, with M, 27,000 and 28,000minor forms of IL-6. The levels of IL-6 produced by ovarian cancer cellscan be modulated by other inflammatory cytokines, such as tumor necrosis factor-a, interleukin-1/3. and interferon-7. Our results suggest thatIL-6 is not an autocrine growth factor for these established ovarian tumorcell lines, because the addition of either exogenous IL-6 or antibodies toIL-6 did not affect the cellular proliferation of the cell lines. We alsofound significant levels (>3 ng/ml) of IL-6 in ascitic fluids of ovariancancer patients and in the supernatants of primary cultures from freshlyexcised ovarian tumors. The production of IL-6 by epithelial ovariancancer cells may prove to be a useful diagnostic tool and aid in investigation of the host immune response to ovarian cancer.

INTRODUCTION

Of all gynecological cancers, ovarian carcinoma has the highest rate of mortality. At the time of diagnosis most patientsalready present with advanced disease (1). Failure to detect thedisease in the early stages results in part from the lack ofsuitable secreted markers, of which the ovarian tumor-specificmarker CA 125 has been the most widely pursued (2). Also,epithelial ovarian cancers are insidious and have a high frequency of subclinical metastasis that frequently remains localized within the peritoneal cavity.

Tumor development has often been associated with eitheroncogene expression and amplification or inappropriate growthfactor regulation. With the exception of the recently identifiedHER-2/neu oncogene (3), which was found to be overexpressedin approximately 30% of ovarian cancers and was usuallyindicative of poor clinical prognosis, there has been little successin correlating significant oncogene expression and amplification with ovarian tumors (4). The HER-2/neu oncogene encodes an epidermal growth factor-like receptor (5). Therefore,these findings support the concept that ovarian tumor development and progression are more likely related to inappropriategrowth factor regulation. One such factor that offers potential

Received 4/2/90; accepted 7/30/90.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This material is based upon work supported by grants from the University ofCalifornia Cancer Research Coordinating Committee, the California Institute forCancer Research (M900419). the NIH (CA12800 and NIH Tumor ImmunologyInstitutional Training Grant CA09120), and the American Cancer Society (JFRA-165).

2To whom requests for reprints should be addressed.

relevance to the progression of ovarian carcinomas is IL-6.'

Also known as BSF-2, IFN-/32, hybridoma/plasmacytomagrowth factor, and M, 26,000 protein (6-9), IL-6 is a pleiotropiccytokine involved in numerous diverse biological functions (10-12). IL-6 has been shown to be produced by tumor cells suchas cardiac myxoma cells (13) and myeloma cells and will in factaugment growth of myeloma cells in an autocrine loop asdemonstrated by the growth inhibition of myeloma cells byanti-IL-6 antibodies (14). Recent evidence has shown IL-6 tobe produced by both human epidermal cells and epidermoidcarcinoma cell lines (15), as well as other epithelium such asbladder carcinoma cells (16) and renal cell carcinoma (17). Asthe majority of ovarian carcinomas often arise from the coe-lomic epithelium that covers the ovarian surface (1), we examined the production of IL-6 from several established ovariancarcinoma cell lines (3 of epithelial origin and 1 of germ cellorigin) and from freshly excised epithelial ovarian tumors.

In this study we show that 3 of 4 ovarian carcinoma cell lines,as well as primary cultures taken from ovarian tumors, secreteIL-6. By all criteria examined, this IL-6 is indistinguishablefrom IL-6 produced by other epithelial, lymphoid, or monocy-toid cell lines. These epithelial ovarian tumor cell lines do notappear to produce IL-6 as an autocrine growth factor. Theresults of this study will allow further characterization of therole of cytokine production by ovarian tumor cells and provideinformation on the role of IL-6 as a growth factor for ovariancancer cells, as well as provide a new potential marker usefulin the diagnosis and treatment of ovarian carcinoma.

MATERIALS AND METHODS

Cells. CAOV-3, NIH-OVCAR-3, SKOV-3, and PA-1 are epithelialovarian cancer cell lines obtained from and maintained according todirections from American Type Culture Collection. Small portions offresh primary ovarian tumors were minced with sterile scissors in RPMI1640, the resultant supernatant was seeded into 25cm2 tissue culture

flasks (Corning Glass Works, Corning, NY); fed with complete media(RPMI 1640; Whittaker Bioproducts, Inc., Walkersville, MD). 10%PCS (Irvine Scientific, Irvine, CA), 1% L-glutamine, and 1% penicillin/streptomycin (Gibco, BRL, Bethesda, MD); and grown at 37°Cwith

5% CO2. The cells were washed in PBS and fed prior to reachingconfluency (2-3 weeks), and the supernatant was collected 14 dayslater. Ascitic fluid from ovarian cancer patients was clarified by low-speed centrifugation for 20 min. An aliquot of the supernatant wasstored at 4°Cuntil tested for IL-6 activity as described below.

IL-6 Assays. Supernatants were determined to have IL-6 biologicalactivity by bioassay using the IL-6-dependent murine hybridomacell line MH60.BSF-2 (kindly provided by Drs. T. Kishimoto andT. Mirano, Osaka, Japan) as described previously (18). Briefly,MH60.BSF-2 cells were maintained in RPMI 1640 supplemented with10% PCS, 1% antibiotics, 1% glutamine, 2% 2-mercaptoethanol, and5 units/ml rhIL-6 (provided by Dr. T. Kishimoto). Duplicate supernatants were serially diluted in 96-well flat-bottomed plates (Nunc, Inc.,Roskilde, Denmark). A total of 1 x IO4 washed MH60.BSF-2 cells

3The abbreviations used are: IL-6, interleukin 6; IFN. interferon; PCS, fetalcalf serum: PBS, phosphate-buffered saline; rhIL-6, recombinant human interleukin 6; ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor;IL. interlcukin: SDS, sodium dodecyl sulfate: BSF-2. B-cell stimulatory factor-2.

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were added per well and the plates were incubated at 37°Cfor 42 h. Atthis time, the cells were pulsed with 1 ¿/Ciof ['H]thymidine (ICN

Biochemicals, Irvine, CA) for 6 h and harvested onto glass membranefilters, and the radioactivity was counted. The concentration of IL-6that gave 50% maximal proliferation was assigned the value of 0.03unit/ml. Relative concentration of IL-6 in the culture supernatants wasalso determined using an IL-6-specific sandwich ELISA (18), whichdiffers from the bioassay by the failure to detect complexed forms ofIL-6 (19). This ELISA used a monoclonal anti-human IL-6 primaryantibody (kindly provided by Dr. F. Takatsuki, Ajinomoto Co. Inc.,Kawasaki. Japan) coated onto plastic 96-well flat-bottomed microplatesovernight. Supernatants or rhIL-6 (provided by Dr. T. Kishimoto)standards were added to coated wells, incubated overnight, and thenincubated further in the presence of a rabbit anti-IL-6 polyclonalimmunoglobulin fraction (Genzyme Corp.). The plates were then incubated with an anti-rabbit horseradish peroxidase-coupled goat serum(Tago, Inc., Burlingame, CA) prior to development with the o-pheny-lenediamine substrate (Sigma Chemical Co., St. Louis. MO).

Immunoperoxidase Staining for Cytoplasmic IL-6. Ovarian cancercells cultured on slides were washed in PBS. fixed in precooled acetonefor 10 min. and air dried. Prior to staining, cells were washed in PBSfor 10 min and incubated with blocking reagent (mouse serum) for 20min at room temperature. Primary antibody (murine monoclonal anti-IL-6; 80 fjg/ml) or isotype control (IgGl) was then added, incubatedfor 90 min, and washed with PBS for 10 min. After this, thebiotinylatedantibody (horse anti-mouse IgG) was added, incubated for 30 min, andwashed with PBS for 10 min. Following this, the ABC kit (VectorLaboratories, Burlingame. CA) reagents were added according to themanufacturer's instructions, incubated for 30 min, and washed with

PBS for 10 min, and 3-amino-9-ethyl-carbazole substrate solution

(freshly prepared) was added and incubated for 10 min, with colordevelopment checked by light microscopy. The enzyme reaction wasstopped with tap water wash to remove substrate.

Neutralization of IL-6 Activity. Fifty ¿ilof culture supernatant collected 5 days postpassage were incubated for 2 h at 37°Cin the presence

of anti-IL-6 polyclonal antisera (Genzyme) at a final antibody concentration of 0.2. 0.1, or 0.05 ng/nl. The supernatants were further incubated at 4°Cuntil tested in MH60.BSF-2 bioassay as described.

Modulation of IL-6 Secretion by Cytokines. Cells were plated in 96-well flat-bottomed plates at 1-2 x IO4cells/well to which either TNF-a (Amgen, Thousand Oaks, CA), IL-Iff (Amgen), or IFN--y (Amgen)was added in various concentrations (1-10.000 units/ml final concentration). The final volume was 200 /jl/wrell. The plates were incubated

for 48 h at which time the supernatants were removed and tested forIL-6 concentration by ELISA.

IL-6mRNA Analysis. Isolation and slot-blot analysis of mRNA fromthe ovarian cancer cell lines were performed as previously described(20) using the guanidinium isothiocyanate/cesium chloride ultracen-trifugation method (21). The blot was probed with the "P-labeled 440-base pair Taq\-Ban\\ fragment of BSF-2/IL-6 complementary DNA(pBSF-2.38, kindly provided by Dr. T. Mirano) (22).

Immunoprecipitation and Gel Electrophoresis. To determine the molecular weight of the IL-6 produced by the ovarian cancer cell lines, 10x IO5 cells were plated in the presence of 100 units/ml IL-lrf andincubated for 48 h prior to pulsing the cells with 200 ^Ci of "S-Trans(ICN Biochemicals) in methionine-deficient RPMI 1640 (ICN Bio-chemicals), supplemented with 5% FCS for 24 h. The supernatant wasincubated overnight at 4°Cwith anti-IL-6 polyclonal antisera (kindly

provided by Dr. F. Takatsuki, Ajinomoto Co. Inc., Kawasaki, Japan)at an antibody dilution of 1:100, followed by a 3-h incubation at roomtemperature after the addition of 150 p\ of a 10% solution of proteinA-Sepharose (Pharmacia, Inc., Piscataway. NJ). The beads were washed5 times in radioimmunoprecipitation assay buffer (0.05 M Tris-HCl,pH 8, 0.1% SDS, 1.0% Triton X-100, 2.0 m.M phenylmethylsulfonylfluoride, 0.15 M NaCl) once in 0.0625 M Tris, pH 6.8. and resuspendedin sample buffer (0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol, 10%2-mercaptoethanol). Antigen-antibody complexes were released fromprotein A-Sepharose by boiling for 5 min. then incubated on ice for 10min prior to separation on a 12.5% SDS-polyacrylamide gel. The gelwas fixed overnight in 50% ethanol and 7% glacial acetic acid, washed

twice in distilled water, incubated for 20 min in l M sodium salicylate,and dried prior to autoradiography.

Statistics. Statistical analysis was done by using Student's t test.

RESULTS

Epithelial Ovarian Cancer Cell Lines Produce IL-6. IL-6activity in culture supernatants from several well-characterizedestablished ovarian tumor cell lines and supernatants fromprimary ovarian tumor cells grown in vitro was examined.Culture supernatants from human ovarian cancer cell linesCAOV-3, NIH-OVCAR-3, and SK-OV-3 were found to support the growth of the IL-6-dependent murine hybridoma cellline MH60.BSF-2, indicating the presence of biologically activeIL-6 in these supernatants (Fig. 1). This MH60.BSF-2 cell line,with a lower sensitivity limit of 0.03 unit/ml, has been shownto be solely responsive to IL-6; other cytokines, including IL-1«,IL-1 /3, IL-2-5, IFN-jtf, IFN--y, and granulocyte-colony stimulatory factor, do not induce cell growth (18). Only PA-1supernatant failed to produce any detectable IL-6 activity.Preincubation of these culture supernatants with a polyclonalanti-IL-6 antibody significantly reduced the proliferation of theMH60.BSF-2 cells (data not shown). As shown in Table 1 andFigure 1, these supernatants not only produce a molecule thathas IL-6 biological activity, but also contain a molecule that

100

ES

10 -

1.0 -

=! 0.1 -

0.01 J< 0.01-1 en

3

Time

4 5

Culture (days)

100

10 -

ES

0.01 J

< o.on CD ca D :

1 23 4 5 67891011121314

Time in Culture (days)

Kig. 1. Epithelial ovarian cancer cell lines produce IL-6. Supernatants fromovarian cancer cell lines collected 5 days after passage al IO x IO4cells/ml weretested for IL-6 biological activity by the proliferative response of IL-6-dependentMH60.BSF-2 hybridoma cells (.4). and for the presence of antigenically relatedIL-6 by an IL-6-specific ELISA (A). Points, mean value of duplicate supernatants.•.CAOV-3; •SKOV-3; O. OVCAR-3; D, PA-1 culture supernatants.

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Table 1 Ovarian cancer cell lines produce IL-6

CelllineCAOV-3OVCAR-3PA-lSKOV-3Culture

mediumIL-6

activity(units/ml)"8.4

±0.9C0.2

±0.02«0.032.5

±0.3<0.03IL-6

concentration(ng/ml)*9.1

±0.40.2±0.1£0.052.7

±0.5S0.05

°Results determined by MH60.BSF-2 bioassay.* Results determined by ELISA.c Mean ±SEM from triplicate experiments using 5-day supernatants harvested

from cells plated at 10 x IO4cells/ml.

Table 2 Oraríancancer cell lines and primary tumor cells stain positive forcytoplasmic IL-6 by immunoperoxidase staining

EstablishedlinesCAOV-3OVCAR-3PA-lSKOV-3Primary

tumorcells449s451453455cr

of positive

cells>98>98>75>98>75>80>80>80Stainingintensity0++/++++++±++±±±±

°±.faint, cytoplasmic staining: +. majority of cells lightly stained: ++. areas

of dark staining: +++. areas of intense staining.

shares antigenic reactivity with human IL-6 as detected in anIL-6 ELISA (sensitive to 0.05 ng/ml). The media preparationalone failed to support the growth of the MH60.BSF-2 hybrid-

oma cells (Table 1). indicating that the media itself did notcontain any IL-6 activity.

Figure 1 shows that 3 of 4 of these ovarian cancer cell linesconstitutively exhibit IL-6 activity. The production of this activity is dependent on cell concentration and is linear with time(data not shown). It is interesting to note that the levels of IL-6 produced by each of the cell lines are dramatically different:CAOV-3 produces significantly greater quantities than doesSKOV-3, whereas OVCAR-3 produces several-fold less thandoes SKOV-3 at all times and cell concentrations tested. Increases in the IL-6 levels were noted to coincide with the timethe cultures reached confluency, after 72 h for SKOV-3, 5 daysfor OVCAR-3, and between days 5 and 7 for CAOV-3.

In light of the differences obtained in IL-6 production fromplating varying cell concentrations, we sought to determine therelative IL-6 contribution on a per-cell basis using cytoplasmicimmunoperoxidase staining. We found that 100% of CAOV-3,SKOV-3, and OVCAR-3 cells are immunoperoxidase-positiveusing a monoclonal anti-IL-6 antibody (Table 2). There wasslight heterogeneity in the intensity of the staining for cytoplasmic IL-6. However, this staining heterogeneity was alsoseen when the cells were stained for major histocompatibilityclass I (data not shown). The PA-l cell line, which failed tosecrete IL-6 as determined by bioassay and ELISA of culturesupernatants, appeared to be lightly positive for cytoplasmicIL-6. Both ovarian cancer cells isolated from a random selectionof solid tumors and ascitic fluids also stained lightly positivefor IL-6 in a majority of the cells examined (Table 2).

To confirm that the ovarian cancer cell lines were producingIL-6 without exogenous stimulation, such as endotoxin contamination of the fetal calf serum used to culture the cell lines, wevaried the culture conditions and again measured the IL-6concentration in the supernatants. CAOV-3 produced biologically active IL-6 detectable in all supernatants, including bothlow serum conditions and Iscoves medium supplemented withalbumin and transferrin (Fig. 2), although the level of IL-6

120 -

100 -

80 -

10* FCS 5* FCS 2* FCS Iscoves-AT

Fig. 2. Constitutive production of IL-6 by ovarian cancer cell lines. Ovariancancer cells were grown in the presence of 10% FCS, 5% FCS, 2% FCS-supplcmented RPMI. or transferrin-albumin-supplemented Iscoves medium for5 days. The IL-6 concentration was measured by bioassay. •CAOV-3; D,OVCAR-3; D, SKOV-3. Points, mean of triplicate samples.

decreased by approximately 75% from 158 units/ml to 39 units/ml. Like CAOV-3, SKOV-3 appears to exhibit a relative orpartial dependence upon serum for the production of biologically active IL-6; IL-6 decreased in culture supernatants grownin either 2% FCS by 30% (from 0.57 to 0.41 unit/ml) or by60% when grown in Iscoves medium supplemented with albumin and transferrin (0.22 unit/ml) as compared with the control. OVCAR-3 produced IL-6 in all media tested, which wasat least 90% that of the control (0.49 unit/ml at 5 days). Whenthe cells were also grown in screened FCS-containing mediumknown not to induce IL-6 from unstimulated peripheral bloodmononuclear cells (lipopolysaccharide concentration of thismedium was 0.16 ng/ml as determined by the Limulus assay),comparable relative concentrations of IL-6 were detected fromconfluent cultures of each of the cell lines grown in such FCS-supplemented medium as compared with cells grown in mediumcontaining untested FCS: CAOV-3, 6.31 and 7.54 units/ml;OVCAR-3 0.36. 0.48 unit/ml; and SKOV-3 1.38, 2.18 units/ml, respectively.

As the production of IL-6 can be induced in different celltypes by various different agents, we examined the effects onproduction of IL-6 by the ovarian cancer cells grown in thepresence of other inflammatory cytokines, TNF-«, IL-10, andIFN--y. IL-6 production by CAOV-3 was found to be enhanced(P < 0.01) in the presence of either TNF-« (all concentrations)or IFN-7 (>100 units/ml), while being unaffected by the presence of IL-10 (Table 3). OVCAR-3 modulated IL-6 productionin a vigorous dose-dependent fashion to IL-1/3 (P < 0.005 at>10 units/ml), with a significant response to TNF-a (P< 0.01)and a negligible response to IFN-7 (Table 2). SKOV-3 showeddose-dependent modulation of IL-6 production by all 3 cytokines tested with significant increases in IL-6 production seenat the highest doses used (P < 0.01). The most dramaticenhancement of IL-6 secretion by SKOV-3 was induced by 100units/ml of IL-10 where the IL-6 level increased 10-fold overthe control level (Table 3). Neither TNF-n, IL-l/S, nor IFN-7was capable of stimulating production of IL-6 from PA-l (datanot shown).

Fibroblasts and monocytes have been shown to produce multiple forms of IL-6 (23). To determine what molecular weightform(s) of IL-6 were being synthesized and secreted by theseovarian cancer cell lines, we immunoprecipitated IL-6 from theculture supernatants of metabolically labeled cells. It appearsthat the IL-6-producing lines all secrete a major species of IL-

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Table i Modulation of IL-6 secretion from ovarian cancer cells by TNF-u, IL-lß,ami /F.V--> 1 234 56 78 9 10 1112 13 14

IL-6 concentration(ng/ml)°ControlTNF-n10

units/ml100 units/ml1,000units/mlIL-

If)1unit/ml

10 units/ml100units/mlIFN-71

unit/ml100 units/ml10.000 units/mlCAOV-3

cellline35.3

±0.751.4

±0.154.9 ±3.052.1 ±1.935.0

±0.337.2 ±9.333.4 ±1.245.6

±10.650.6 ±6.752.8 ±1.7OVCAR-3

cellline0.1

±0.030.3

±0.090.9 ±0.20.8 ±0.30.04

±0.01.1 ±0.07

10.3±1.30.1

±0.020.1 ±0.050. 1 ±0.03SKOV-3

cellline0.6

±0.41

.00 ±0.001.7 ±0.22.8 ±0.20.5

±0.41.7 ±0.56.2 ±1.80.9

±0.041.4 ±0.051.5 ±0.06

' Mean ±SD of triplicate samples from one representative experiment (n =

4).

6 that has a molecular weight of 24,000 (Fig. 1A). CAOV-3also secretes isoforms of IL-6 at M, 27,000 and 28,000. Thisheterogeneity has been seen with other epidermal cells andepidermoid carcinoma cell lines (15). It is possible that OVCAR-3 and SKOV-3 also secrete other forms of IL-6 but inconcentrations too low to be detected by immunoprecipitation.As a control, IL-6 was immunoprecipitated from the superna-tants both from a human monocytic cell line (THP-1) and fromlipopolysaccharide-activated peripheral blood mononuclearcells (Fig. 3Ä).The major species immunoprecipitated with thisantibody is M, 24,00, with minor species of protein detectableat slightly higher molecular weights.

Slot-blot RNA analysis revealed the presence of IL-6-specificmRNA in SKOV-3 and CAOV-3 cell lines but failed to detectOVCAR-3 and PA-1 mRNA specific for IL-6 (Fig. 4). Thelevels of mRNA do not appear to directly correlate with therelative protein activity detectable in the culture supernatantsbecause CAOV-3, which secretes more IL-6 protein thanSKOV-3, has less specific mRNA than SKOV-3. The cause ofthis variation is unknown but could be the result of eithergreater mRNA stability or translation efficiency in the CAOV-3 cell line as opposed to the SKOV-3 line. Precedent fordetection of a biologically active protein and subsequent failureto detect associated mRNA, as in the case of OVCAR-3-produced IL-6, has been demonstrated. Ramakrishnan et al.(24) have shown detectable macrophage-colony stimulating factor activity in culture supernatants of ovarian cancer cell lines,including OVCAR-3, while failing to detect corresponding specific cytokine mRNA.

IL-6 Production by Freshly Isolated Ovarian Cancer Cells.Since ovarian cancer cell lines represent fully transformed cellsthat have been selected for their ability to grow in vitro, theunstimulated constitutive production of IL-6: by these cell linesmay be a consequence of such selection. To determine therelevance of ovarian cancer cell line-produced IL-6 to an in vivo

condition, we examined freshly isolated ovarian tumors for theproduction of IL-6. In this study, all primary culture supernatants (n = 19) from freshly excised ovarian tumors and allovarian cancer patient ascitic fluids (n = 24) were found to havedetectable IL-6 levels (Table 4). The concentration ranged from3 to 40 ng/ml and was detectable in both the IL-6 ELISA andMH60.BSF-2 bioassay (data not shown). The high (>3 ng/ml)IL-6 activity in all ascitic fluids (n = 24) tested here cannot bedirectly linked to IL-6 produced by ovarian tumor cells. Ascitic

2OO-

30

14

46

30-

14-

B

Fig. 3. Immunoprecipitation of IL-6 from culture supernatants of epithelialovarian cancer cell lines reveals a M, 24.000 protein. Supernatants from cellsincubated in the presence of IOOunits/ml II.-U «eremelabolically labeled with•"S-Transfor 24 h and immunoprecipitated with a polyclonal antiserum for IL-6(l:l()0 final dilution). Immunoprecipitates were separated on a I2.5rr SDS-

polyacrylamide gel cleclrophoresis. dried, and autoradiographed for 3 days. .4.Lanes I and 2. immunoprecipitatc from OAOV-3 supernatant; Lanes 3 and 4.from OVCAR-3; Lanes 5 and 6, from PA-1; Lanes 7 and fi. from SKOV-3; LanesV and IH. from primary culture 436; Lanes 11-14. from the monocytic line THP-I, representing supernatants from unstimulated cells. I.PS- and IKN--)-stimulatcdcells, and stimulated cells plus excess of cold IL-6. respectively. Lanes 2. 4. 6. 8,HI. and 14 are immunoprecipitates from supernatants treated with pre-immunerabbit antiscra. /?, Lanes 1-5. immunoprecipitates from peripheral blood mono-nuclear cells. Lane I from unstimulated cells; Lanes 2-5. peripheral bloodmononuclear cells were stimulated with 1 ^g/ml lipopolysaccharide and immunoprecipitated with Ajinomoto antisera (Lanes 2 and 4) or Genzyme polyclonalanii-IL-6 (Lane .?): Lane 4 supernatant was pretreated with cold IL-6 prior toimmunoprecipitation; Lane 5 immunoprecipitate with preimmune rabbit antisera.Molecular weight markers are indicated in thousands.

fluid, obtained from one patient with a gynecological conditionother than ovarian cancer, was found to have an IL-6 level of<3 ng/ml (1.8 ng/ml). Peritoneal fluid obtained from healthywomen undergoing voluntary tubai ligations (n = 7) was foundto contain IL-6 levels comparable with that of the non-cancerpatient (2.1 ng/ml).

IL-6 is Not an Autocrine Growth Factor for Kpithclial OvarianCancer Cells. To determine whether IL-6 is an autocrine growthfactor for ovarian cancer cells, we examined the cellular prolif-erative response of the ovarian cancer cells in the presence ofeither exogenous rhIL-6 over a range of concentrations (0.01

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OVARIAN CANCER-PRODUCED II.-6

CAOV-3

OVCAR-3

PA-1

SKOV-3 I t

Fig. 4. Epithelial ovarian cancer cell lines contain IL-6-specific mRNA. Equivalent amounts (5, 2.5, 1.25. and 0.75 ME)of total RNA from each cell line wereblotted, probed with "P-labeled IL-6 complementary DNA, and autoradi-

ographed. Blots »ereexposed for 48 h.

Table 4 Primary culture supernatants from freshly excised ovarian tumors anaascitic fluid from oraríancancer patients contain high levels of IL-6

IL-6 concentration (ng/ml)

Range- Mean ±SEM

Ovarian cancer patientsAscitic fluids (n = 23) 3.2-42.0 20.0 ±2.6Primarv culture supernatants (n = 11.6-35.9 24.4 ±1.819) "

Non-cancer patientsPeritoneal fluids (n = 8) 1.8-2.6 2.1 ±0.1

to 100 units/ml) or polyclonal anti-IL-6 antibodies (0.05 to 5Mg/ml) (from Dr. F. Takatsuki, Ajinomoto). Each of the variouscell lines was titrated at low seeding densities to increase thepossible dependence on exogenous factors and to dilute out anyautocrine factors that are produced by culturing the cells athigh densities. None of the IL-6-producing lines, CAOV-3,SKOV-3, or OVCAR-3, appeared to have enhanced prolifera-tive responses in the presence of exogenous IL-6 or decreasedproliferative responses in the presence of polyclonal anti-IL-6(data not shown). We conclude from these results that IL-6 isnot a positive autocrine growth factor for these epithelial ovarian cancer cells.

DISCUSSION

Here we report that 3 well-characterized human ovariancarcinoma cell lines, CAOV-3, NIH-OVCAR-3, and SKOV-3,

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constitutively produce a molecule with the biological and anti-genie characteristics of IL-6. SKOV-3 and CAOV-3 are celllines that constitutively produce moderate to high levels of IL-6 and detectable IL-6 mRNA. OVCAR-3 produces low amountsof IL-6 (<0.5 unit/ml) even at culture confluency and has nodetectable IL-6 mRNA. The IL-6 produced by these ovariancancer cell lines has a molecular weight of approximately24,000, and exhibits some of the molecular weight heterogeneity often associated with IL-6 produced by epithelial cells(15).

Significant levels (>3 ng/ml) of IL-6 activity were also detected in the supernatants of primary ovarian tumor culturesand in ovarian cancer patient ascitic fluid. Although every effortwas made to ensure that there were no contaminating peritonealmonocyte/macrophage cells in the cultures, the possibility ofsuch cells contributing to IL-6 production cannot be excluded.However, by nonspecific esterase staining of a random selectionof the primary cultures, we found that there was <10% macrophage contamination in these cultures (data not shown),whereas the cultures stained for cytoplasmic IL-6 by the im-munoperoxidase technique expressed >75-80% IL-6 reactivity.The levels of IL-6 in the primary tumor cultures are significantin that tumor cells are exposed to no additional stimulus otherthan being placed in culture and go through changes in culturemedium prior to testing the supernatant, removing any IL-6secreted by contaminating monocytes upon adherence to thetissue culture flask.

In this study, we emphasize the production of IL-6 from thetumor cells themselves, unlike Erroi et al. (25), who havereported that IL-6 is constitutively produced by tumor-associated macrophages in ovarian carcinomas, without significantcontribution from the neoplastic elements. Differences in isolation procedures and cell line selections probably account forthe variation between their reported results and ours. However,the original source from which their cell lines were derived isnot stated, and as shown here, cell lines from germ cell tissue(PA-1) did not secrete IL-6, whereas those of epithelial origin(CAOV-3, OVCAR-3, and SKOV-3) were seen to produce andsecrete IL-6. Our results support those of Tabibzadeh et al.(26), who examined IL-6 immunoreactivity in human tumorsby immunohistological staining and found that the majority ofhuman tumors of epithelial or mesenchymal origin stain positive for IL-6. From such immunohistological procedures it wasnot apparent whether the tumors actually produced IL-6 orsimply utilized and stored IL-6 produced by other cells. Ourresults suggest that the neoplastic elements themselves produceIL-6.

The source of the activity detected in the ascites fluids cannotbe ascribed to IL-6 solely produced by the ovarian tumors.Despite the uncertain source of the ascitic fluid IL-6, it isapparent that there are extremely high levels of this cytokinewithin the peritoneal cavity of patients with ovarian cancer.Because the accumulation of ascitic fluid is an abnormal condition per se, the appropriate control for examining ascitic fluidIL-6 levels would be ascitic fluid from women with gynecological conditions other than ovarian carcinoma. Interestingly, theone such fluid tested contained only low levels (<3 ng/ml) ofdetectable IL-6, comparable with that found in peritoneal fluidfrom healthy women undergoing voluntary tubai ligations. Thehigh concentrations of IL-6 within a confined and localizedregion reported here are not unique because high levels (>1000units) of IL-6 have been reported to be present in amniotic fluid(27), in the synovial fluid of rheumatoid arthritis patients (28),and in localized regions of acute bacterial infections (29). These

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OVARIAN CANCER-PRODUCED IL 6

high levels of IL-6 do not appear to directly contribute to theimmunosuppressive effects of ascitic fluid (30) because neutralization of the IL-6 in the ascitic fluid was not capable ofrestoring proliferation to phytohemagglutinin-stimulated peripheral blood mononuclear cells (data not shown). It remainsto be determined what immune function IL-6 plays in suchlocalized regions, and whether in high, sustained concentrationsIL-6 actively suppresses or enhances other immune functionsor induces a state of nonresponsiveness through indirect means.

Although IL-6 has been implicated as an autocrine growthfactor in other tumors (14), the results presented here showingthat the levels of IL-6 continue to rise even after the cultureshave reached confluency, the lack of proliferative inhibition inthe presence of antibodies, and the lack of proliferative responseto exogenous IL-6 suggest that these ovarian carcinoma celllines are not producing this cytokine as an autocrine growthfactor. Recent evidence from Klein et al. (31) suggested thatIL-6 regulated myeloma cell growth by a paracrine (as opposedto an autocrine) mechanism (14). The possible role of ovariancell-produced IL-6 as an exocrine or paracrine growth factorfor other cells of the peritoneal cavity is intriguing, especiallyif considered in light of the high levels of the cytokine that areproduced and retained within the peritoneal cavity and the lackof metastasis outside of the cavity. It has been proposed byTamm et al. (32) that IL-6 may aid in tumor metastasis andinvasiveness, since exogenous IL-6 was shown to increase mo-tility and decrease adhering-type junctions in the breast carcinoma cell lines T47D and Zr-75-1. It is not known whetherautonomously produced IL-6 would affect the motility andadherence of the ovarian cancer cell lines, but this could providean explanation for the high degree of local seeding that occursthroughout the peritoneal cavity in patients with ovarian carcinoma.

All the primary ovarian tumors and ovarian cell lines thatproduced IL-6 activity were classified as adenocarcinoma tumors. The single ovarian carcinoma cell line that failed toproduce any IL-6, PA-1, was originally derived from a terato-carcinoma (33). This germ cell tumor is derived from undiffer-entiated cells exhibiting characteristics of embryonal carcinomacells, as opposed to the adenocarcinomas, which characteristically contain more differentiated cells. The relevance of thestate of differentiation to IL-6 production cannot be drawnfrom this study. However, it would be interesting to examinethis further to determine if such a correlation exists, since thismay provide a useful diagnostic tool. The ovarian carcinomamarker CA 125 has been demonstrated to be associated withapproximately 85% of serous ovarian tumors and 70% ofundifferentiated tumors, and only rarely with mucinous tumors(2). Likewise there has been some association between othermarkers such as CEA and CA 19-9 and other types of ovariancarcinoma (mucinous and endometroid). However, none ofthese particular tumor markers shows specificity for cancer orcorrelates with their respective secreted marker serum values.Studies to examine circulating serum IL-6 levels in ovariancancer patients are currently underway. If a correlation is foundbetween circulating IL-6 levels and ovarian tumor status, IL-6may prove useful as another marker for ovarian cancer.

Evidence from Boltz et al. (4), regarding the lack of anysignificant amplification of the c-ras-Ki oncogene in ovariantumors, and the results of this study suggest that ovarian tumorprogression may be related to abnormal growth factor regulation as opposed to inappropriate oncogene expression andamplification. Evidence relating abnormal growth factor regulation to ovarian tumor progression has been demonstrated by

the amplification and overexpression (3) of an epidermalgrowth factor-like receptor (5) encoded by the HER-2/neuoncogene. In further support of this idea, we have shown herethat 2 of the IL-6-producing cell lines (CAOV-3 and SKOV-3)constitutive!}- express IL-6 mRNA. Although the overexpression of IL-6 has been implicated in the pathogenesis of numerous diseases, including some lymphoid cancers (34), it remainsto be determined what role such deregulated expression of IL-6 has in the development and progression of ovarian cancer.

ACKNOWLEDGMENTS

The authors would like to thank Reba Knox, Meta Vander Meyden.and Ahmad Rezai for their assistance with the IL-6 ELISA, ¡mmuno-peroxidase staining, and RNA analysis; Drs. Tadamitsu Kishimotoand Toshio Mirano of Osaka University, Osaka, Japan, for providingIL-6, IL-6 complementary DNA, and the IL-6-responsive cell line,MH60.BSF-2; Dr. Fumihiko Takatsuki of Ajinomoto Co., Inc., Kawasaki, Japan, for providing polyclonal and monoclonal antibodies toIL-6; and Dr. Donna Vredevoe for the use of laboratory facilities.

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1990;50:6959-6965. Cancer Res   Joanna M. Watson, John L. Sensintaffar, Jonathan S. Berek, et al.   Lines and by Primary Ovarian Tumor CulturesConstitutive Production of Interleukin 6 by Ovarian Cancer Cell

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