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Molecular Oncology, Markers, Clinical Correlates |
Center for Research on Reproduction and Women’s Health [L. Z., J-R. C-G., A. M-H., G. C.], Abramson Family Cancer Research Institute [K. S., A. T., B. L., G. C.], Division of Gynecologic Oncology, Department of Obstetrics and Gynecology [S. C. R., G. C.], and Cell and Molecular Biology Program [N. Y.], University of Pennsylvania, Philadelphia, Pennsylvania 19104, and Department of Obstetrics and Gynecology, University of Turin, Turin, Italy [D. K., S. F.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EG-VEGF, the first tissue-specific angiogenic molecule, was identified recently in steroidogenic endocrine organs, including the ovary, testis, adrenal, and placenta (11 , 15) . Although EG-VEGF does not show any homology to the VEGF family (11) , it possibly induces activation of mitogen-activated protein kinase p44/42 and phosphatidylinositol-3 kinase pathways and leads to proliferation, migration, and survival of endothelial cells (15) , a role reminiscent of VEGF. EG-VEGF selectively induces proliferation, migration, and fenestration (the formation of membrane discontinuities) in capillary endothelial cells specifically derived from endocrine glands, suggesting the existence of tissue-restricted specific receptors (11) . Importantly, EG-VEGF contributes to the endocrine function of these organs, as endothelial cell fenestration may accelerate the entry of hormones in the bloodstream (10 , 11) . Because angiogenic factors play a critical role in tumorigenesis, the discovery of tissue-specific angiogenic mechanisms raises hopes of developing tumor type-specific angiogenesis inhibitors (10 , 11) .
Angiogenesis mechanisms play a critical role in ovarian carcinogenesis. VEGF is overexpressed in ovarian carcinoma (16, 17, 18, 19) , where it exerts important biological functions, such as promoting tumor growth and the formation of ovarian cysts and ascites (20, 21, 22, 23) . Importantly, increased serum and/or tumor levels of VEGF have been associated with poor clinical outcome (24, 25, 26, 27, 28) . To date, it is unknown whether EG-VEGF is expressed in ovarian carcinoma and whether it plays a role in supporting angiogenesis and tumor growth. In the present study, we analyzed the expression of EG-VEGF and VEGF mRNA in human ovarian neoplasms. We report for the first time that EG-VEGF is expressed in human ovarian carcinoma but most likely derives from nonepithelial components of the tumor, including stroma cells and tumor-infiltrating leukocytes.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines and Cell Culture.
A total of 17 human ovarian cancer cell lines (A2780, A2780/C30, A2780/CP70, A2008, A1847, SKOV-3, PE01, PE04, OVCAR2, OVCAR3, OVCAR4, OVCAR5, OVCAR7, OVCAR8, OVCAR10, UPN251, and OAW42) was generously provided by Drs. Steven Johnson and Kang-Sheng Yao, Department of Cancer Pharmacology, University of Pennsylvania. All cell lines were cultured in 5% CO2 atmosphere, at 37°C, and in DMEM medium supplemented with 10% fetal bovine serum and 100 units/ml penicillin. HOSE cells were isolated intraoperatively from normal ovaries by gently scraping the ovarian surface with a wooden spatula. Cells were collected in ice-cold PBS and immediately transported to the laboratory. Primary HOSE cultures were established in RPMI 1640 supplemented with 10% fetal bovine serum and 100 units/ml penicillin. All reagents for cell culture were purchased from Invitrogen (Carlsbad, CA), unless stated otherwise.
PBLs were isolated by elutriation from the apheresis product of normal healthy donors and cultured at 1 x 106 cells/ml in RPMI 1640 containing 10% human AB sera (BioWhittaker, Walkersville, MD) at 37°C and 5% CO2. Cells stimulated by anti-CD3/anti-CD28-coated beads (29) were cultured at a 3:1 bead:cell ratio at a final concentration of 1 x 106/ml for 48 h. Cells were then harvested, beads were removed by magnetic separation, and cells were subjected to RNA isolation.
Flow Cytometry.
Single-cell suspension from fresh tumor nodules were generated as described previously (30)
. Briefly, fresh tumor specimens were mechanically dissected and enzymatically digested overnight at room temperature with type IV collagenase (0.1%; Sigma Chemical Co., St. Louis, MO), type IV DNase (30 units/ml; Sigma), and type V hyaluronidase (0.01%; Sigma). The tumor suspension was then filtered through a wire grid to yield a single-cell suspension. Cells were separated on a percoll (Pharmacia Biotech AB, Uppsala, Sweden) density gradient for 30 min at 1,500 x g at room temperature and subjected to four-color flow cytometry on a FACSCalibur flow cytometer using CellQuest 3.2.1f1 software (Becton Dickinson, San Jose, CA), using monoclonal antibodies against CD45 (HI30), CD3 (UCHT-1), CD4 (RPA-T4), CD8 (RPA-T8), CD19 (HIB19), CD57 (NK-1), IgG1, and IgG2a (BD PharMingen, San Diego, CA). Data representing 10,000–30,000 events were recorded and analyzed with CellQuest software (Becton Dickinson).
Isolation of Tumor-infiltrating Lymphocytes.
Tumor-infiltrating lymphocytes were isolated as described previously (30)
. Briefly, single-cell suspensions obtained from dispersed tumor specimens, as described above, were separated on a percoll density gradient. The dense layer, enriched for lymphocytes, was collected. Tumor-infiltrating T lymphocytes were selected by fluorescence-activated cell sorting using monoclonal antibodies against CD4 and CD8 (BD PharMingen) and a MoFlo cell sorter (Cytomation, Fort Collins, CO) equipped with an argon laser beam.
Immunohistochemistry and Immunofluorescent Staining.
Immunohistochemical staining was performed using the avidin-biotin-peroxidase method. Sections were fixed in cold acetone for 10 min, pretreated with 3% H2O2 for 20 min to block endogenous peroxidase activity, and incubated in normal serum (Vector, Burlingame, CA) for 30 min. After incubation with the primary antibody (Table 1)
in a humid chamber for 1 h, the VECTASTAIN avidin-biotin complex method kit was applied as described by the manufacturer (Vector). Briefly, sections were incubated in biotin-labeled secondary antibody for 30 min, followed by incubation in biotin-peroxidase/avidin complex for 30 min. The immunoreactive sites were visualized with freshly prepared 3,3'-diaminobenzidine (Vector) containing H2O2. All staining steps were performed at room temperature. Sections were counterstained lightly with Gill’s hematoxylin (Vector). Images were acquired through Cool SNAP Pro color digital camera (Media Cybernetics, Carlsbad, CA). For immunofluorescent staining, sections were sequentially incubated in 5% normal serum, antihuman cytokeratin antibody (DAKO, Carpinteria, CA) diluted at 1:500 for 1 h, and FITC-labeled antirabbit IgG (Vector) for 2 h.
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. LCM was performed as described by others (31
, 32)
, implementing minor modifications as suggested by the manufacturer of the LCM used. Briefly, cryosections (6 µm) from selected tumors were mounted on a polyethylene foil slide (SL Microtest, Jena, Germany) according to the manufacturer’s instructions. After rapid H&E staining, sections were subjected to LCM using the µCUT Laser-MicroBeam System (SL Microtest), which uses a fine UV laser that performs circumferential dissection of selected tissue areas following precisely a drawn incision path. By this cold ablation, the material to be extracted is never directly exposed to the laser. This LCM system also enables the contact-free isolation of single cells or groups of cells (Fig. 2B)
. Microdissected cells were catapulted into the lid of a 0.5-ml reaction tube containing RNA isolation buffer. RNA was isolated by Micro RNA isolation kit (Ref. 32
; Stratagene, La Jolla, CA).
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were designed based on published sequences (11
, 33) . To avoid false positive results caused by amplification of contaminated genomic DNA in the cDNA preparation, all primers were designed to reside on exons separated by an intron; the product of human EG-VEGF spanned the boundary of exons 2 and 3, whereas the product of human VEGF spanned the boundary of exons 1 and 2. Because all five VEGF isoforms share the same exons 1–5, the VEGF primers were able to amplify all human VEGF isoforms.
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Real-time RT-PCR.
The mRNA expression of EG-VEGF and VEGF was quantified by real-time RT-PCR on the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). PCR was performed using SYBR Green PCR Core Reagents (Applied Biosystems) according to the manufacturer’s instructions. PCR cycles consisted of an initial denaturation step at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and at 60°C for 60 s. A standard curve was constructed with PCR products containing the same fragments amplified by the Taqman system. The relative expression in each sample was calculated with respect to the standard calibration curve, as described previously (34
, 35)
. Normalization of cDNA load was performed against housekeeping GAPDH. Each sample was analyzed twice, and each PCR experiment included two nontemplate control wells. PCR products were confirmed as single bands using gel electrophoresis and by DNA sequencing.
Nested PCR.
To detect the expression of EG-VEGF mRNA in ovarian cancer cell lines, a two-step nested PCR was used. Briefly, after a first round of PCR performed with the outer primers (Table 2)
, a second round of PCR was carried out using 0.5 or 3 µl of the first round PCR product and inner primers (real-time PCR primers). Conditions for both the first and second round of PCR reactions were the same as with standard RT-PCR mentioned above. PCR products were run on a 2.5% agarose gel.
Statistical Analysis.
Data statistical analysis was performed using the SPSS statistics software package (SPSS, Chicago, IL). All results were expressed as mean ± SE, and P < 0.05 was used for significance. Survival distributions were depicted with Kaplan-Meier curves, and Log-rank statistics were used to compare curves.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20-fold higher in normal human testis and premenopausal ovary than in other organs. EG-VEGF mRNA could be detected at low levels in brain, colon, skeletal muscle, small intestine, spleen, thymus, uterus, and liver, whereas it was undetectable in nonlactating breast and skin (Fig. 1A)
. In contrast, VEGF mRNA was detected in all specimens, and no tissue-specific expression pattern was seen (data not shown). EG-VEGF mRNA was detected by RT-PCR both in resting as well as in PBLs activated ex vivo by CD3 cross-linking and CD28 costimulation, with slightly increased levels after costimulatory activation (Fig. 1B)
. The identity of the PCR product isolated from PBLs with EG-VEGF primers was confirmed by sequencing.
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). In contrast to EG-VEGF, malignant ovarian tumors significantly overexpressed VEGF mRNA compared with benign and low malignant potential neoplasms (P < 0.05; Fig. 1D
). No significant difference was identified between stage I and III carcinomas (P = 0.866). Because EG-VEGF can be up-regulated by hypoxia via the same mechanism as VEGF (11)
, we analyzed the correlation between the EG-VEGF and VEGF mRNA in stage III tumors. No correlation was found between the mRNA levels of these two angiogenic molecules (P = 0.462; Fig. 1E
).
EG-VEGF Expression in Stroma and Tumor Islets.
EG-VEGF mRNA was quantified comparatively in tumor islets and peritumoral stroma. First, stage III tumor specimens were screened by immunohistochemistry using cytokeratin, an epithelial tumor marker, to identify tumors with clear architectural distinction between tumor islets and stroma. Only specimens with such distinction (Fig. 2A)
were selected to perform LCM (Fig. 2B)
. By immunohistochemistry, we determined that as many as 70–80% of cells within tumor islets were cytokeratin-positive tumor cells, whereas no cytokeratin-positive cells could be seen in peritumoral stroma areas. EG-VEGF mRNA was detected in both stroma and tumor islets. Remarkably, mRNA levels were consistently higher in peritumoral stroma compared with tumor islets, with a ratio of stroma:islets ranging between 2 and 7 (Fig. 2C
shows two representative cases).
EG-VEGF Expression in Established Ovarian Cancer Cell Lines.
We investigated the constitutive expression of EG-VEGF by ovarian carcinoma cells in vitro. A total of 17 epithelial ovarian cancer cell lines was analyzed by RT-PCR. EG-VEGF was undetectable in all cell lines (Fig. 3A)
. The quality of cDNA was tested by analysis of housekeeping gene GAPDH, which was detected in all cell lines (data not shown). For comparison, VEGF mRNA expression was also tested in the same specimens. High levels of VEGF mRNA were found in all 17 ovarian cancer cell lines (Fig. 3A)
. To enhance the sensitivity of RT-PCR, we used nested RT-PCR. In 15 of 17 cell lines, a detectable band was observed at the expected molecular weight, the intensity of which depended on the amount of the first-round PCR product (Fig. 3B)
. Finally, no EG-VEGF could be detected by RT-PCR in cultured HOSE cells (data not shown). The above data indicate that EG-VEGF mRNA is present at extremely low levels in ovarian cancer cell lines in vitro.
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, including CD4+ and CD8+ subsets (data not shown) in >50% of patients. By contrast, other types of lymphocytes, such as CD19+ B cells (Fig. 4B)
and CD57+ NK cells (Fig. 4C)
, were scarcely represented. By flow cytometry, we confirmed that CD45+ leukocytes represent 
20–35% of cells isolated from mechanically dispersed and enzymatically digested ovarian carcinomas, whereas T cells were the main immune cell population encountered in ovarian carcinoma (Fig. 4D)
, representing 35.1 ± 3.3% of tumor-infiltrating leukocytes (Table 3)
. EG-VEGF expression was assessed in tumor-infiltrating T cells by RT-PCR. In all cases, EG-VEGF mRNA was expressed in tumor-infiltrating T lymphocytes (Fig. 4E)
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), but no difference in outcome was observed between patients with relatively high and low levels of EG-VEGF (P = 0.491; Fig. 5A
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The normal ovary is formed by cells belonging to three distinct lineages: (a) the germ cell lineage, which comprises all eggs; (b) the sex cord-stroma lineage, which comprises the cortical stroma, the cumulus oophorus surrounding the eggs, the granulosa, and theca layers of follicles and interstitial stroma; and (c) the epithelial lineage, which comprises the surface epithelium (36, 37, 38) . In the normal ovary, EG-VEGF mRNA is highly expressed in elements of the sex cord-stroma lineage, including the cortical stroma, the cumulus oophorus, as well as the granulosa and theca layers of developing follicles (11) . Epithelial ovarian carcinomas, which represent >90% of ovarian cancers, likely derive from the epithelial lineage of the ovary (39, 40, 41) . EG-VEGF was found to be expressed at the mRNA level in all human epithelial ovarian neoplasms, including benign cystadenomas, tumors of low malignant potential, and invasive carcinomas. However, the present data indicate that tumor cells are not an important source of EG-VEGF in ovarian cancer. In fact, EG-VEGF expression was lower in cystadenomas, tumors of low malignant potential, and early stage carcinomas to normal premenopausal ovary. In addition, through LCM and RT-PCR, we showed that EG-VEGF levels were consistently higher in tumor stroma compared with tumor islets. Finally, EG-VEGF mRNA was not detected in primary ovarian surface epithelial cells, the purported precursors of epithelial ovarian carcinomas (39, 40, 41) , or 17 established cell lines by RT-PCR.
Despite the above evidence, a proangiogenic role of EG-VEGF in ovarian cancer may not be a priori excluded. In fact, tumor-infiltrating leukocytes, such as macrophages, are known to support angiogenesis by releasing VEGF in response to hypoxia (42 , 43) . EG-VEGF was also shown to be up-regulated by hypoxia via hypoxia-inducible factor, indirectly suggesting functional implication in angiogenesis (11) . To assess the role of EG-VEGF in the biology of ovarian cancer, we sought to establish associations of EG-VEGF with stage, tumor outcome, or VEGF levels. Although an association of VEGF overexpression could be readily established with advanced stage or poor outcome in stage III ovarian carcinoma, such association was not evident for EG-VEGF. In fact, EG-VEGF expression declined in late stage ovarian cancer compared with stage I tumors. Furthermore, no correlation could be found between VEGF and EG-VEGF levels, suggesting different mechanisms of regulation. Taken together, these data suggest that EG-VEGF likely plays a marginal role in promoting angiogenesis in advanced stage disease. Ovarian carcinomas may exhibit an abundant rich stroma component, which may be highly cellular. It has been postulated that stroma-epithelial cell interactions may inhibit the growth of tumorigenic surface epithelium in early stages of ovarian oncogenesis (44 , 45) . In a xenograft model, it was shown that normal ovarian stromal cells, when admixed with tumor cells, had the ability to inhibit the growth of ovarian carcinoma transplants but were gradually substituted by host (murine) stromal cells, which were recruited to the tumor and supported its growth (46) . These experimental data are supported by histopathologic data; only 13% of ovarian cancer specimens examined in one series displayed evidence of stromal luteinization (47) , whereas in another series, steroid cells were identified in <3% of advanced ovarian cancers, most of which were of the relatively uncommon mucinous histotype (48) . It is possible that the overall influence of ovarian steroidogenic stroma on tumor development might be inhibitory, resulting in its progressive loss during malignant progression, which might provide an explanation for the decline of EG-VEGF expression.
In the present study, we found that EG-VEGF mRNA is dramatically overexpressed in two steroidogenic organs, ovary and testis, but is also expressed in other normal tissues at markedly lower levels. The difference in our results is likely attributable to the use of RT-PCR, which is more sensitive than Northern blot, and the tissue RNA array used in LeCoutor’s study (11) . Additional studies need to be performed to assess whether EG-VEGF expression in nonsteroidogenic tissues has any physiological function. The most interesting finding in our present study was that EG-VEGF mRNA is also expressed in resting as well as activated PBLs, which is consistent with the detection of EG-VEGF mRNA in lymphoid tissues, such as spleen and thymus. Tumor-infiltrating T cells were also found to express EG-VEGF. This is the first evidence that peripheral and tumor-infiltrating T cells secrete EG-VEGF. T cells are known to secrete VEGF (49) . Furthermore, tumor-infiltrating T cells may secrete VEGF and promote angiogenesis (50) . The potential roles of EG-VEGF in tumor-infiltrating T cells remain to be elucidated.
In summary, in this study, we provide evidence indicating that EG-VEGF, a purported tissue-specific angiogenic factor, is expressed in ovarian carcinoma but is most likely derived from nontumor cells of the sex cord-stromal lineage, as well as tumor-infiltrating T lymphocytes. Furthermore, in contrast to VEGF, the expression of EG-VEGF in advanced stage tumors significantly declines compared with early stage cancers, low malignant potential tumors, or benign tumors and does not correlate with outcome in advanced stage disease. On the basis of these findings, we postulate that EG-VEGF-targeted antiangiogenesis therapy may prove useful in early stage but not in advanced stage ovarian carcinoma.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the American Association of Obstetricians and Gynecologists Foundations, the Gynecologic Cancer Foundation, the Abramson Family Cancer Research Institute, and National Cancer Institute Specialized Programs of Research Excellence CA-98-008. Select normal human tissues were provided by the Cooperative Human Tissue Network, which is supported by the National Cancer Institute. 
2 To whom requests for reprints should be addressed, at Center for Research on Reproduction and Women’s Health, University of Pennsylvania, 421 Curie Boulevard, 1355 Biomedical Research Building II/III, Philadelphia, PA 19104. Phone: (215) 746-5136; Fax: (215) 573-7627; E-mail: gcoukos{at}obgyn.upenn.edu
3 The abbreviaitons used are: VEGF, vascular endothelial growth factor; HOSE, human ovarian surface epithelial; EG-VEGF, endocrine gland-derived vascular endothelial growth factor; LCM, laser capture microdissection; PBL, peripheral blood lymphocyte; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription-PCR; NK, natural killer; DFI, disease-free interval.
Received 5/16/02; revised 8/ 9/02; accepted 8/ 9/02.
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