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Original Studies |
Surgery (E.Y.S., Q-Y.D., S.A.B., D.M.Y., M.G.W., Y.D.M., R.F.G., A.E.S., O.H.C.) and Pathology (H.D.E.) Service, University of California San Francisco/Mount Zion Medical Center and Veterans Affairs Medical Center (Q-Y.D.), San Francisco, California, 94115; Genentech (K.G.), South San Francisco, California 94080-4990
Address all correspondence and requests for reprints to: Quan-Yang Duh, Surgery Service, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, California 94121–11598.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Vascular endothelial growth factor (VEGF) is unique among angiogenic factors because it is both mitogenic for vascular endothelial cells and is secreted by the cancer cells (7, 8, 9, 10). The cancer cells, therefore, can stimulate the development of host blood vessels to bring more nutrients to support growth. VEGF is a 34- to 42-kilodalton heat- and acid-stable, dimeric, heparin-binding glycoprotein (11, 12). VEGF binds to membrane receptor tyrosine kinase (flt-1, KDR), and increases the proliferation of endothelial cells (13). VEGF also increases vascular permeability, causing extravasation of plasma proteins and deposition of fibrin. This extravascular fibrin gel matrix then supports the ingrowth of new blood vessels (11, 14). Systemic administration of anti-VEGF antibody has been found to inhibit the growth of xenografted tumor cells in nude mice (15, 16).
While investigating an in vivo invasion model, we observed extensive angiogenesis surrounding the human thyroid cancer cells xenografted in nude mice. Because thyroid cancers are vascular tumors, and follicular cancer metastasizes via the blood vessel, we postulated that thyroid cancer cells produce and secrete VEGF, and that VEGF may stimulate thyroid cancer growth, invasion, and metastasis.
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Material and Methods |
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We used human thyroid cell lines derived from follicular cancers (FTC-133, FTC-236, and FTC-238), kindly provided by Peter Goretzki, M.D., Düsseldorf, Germany, papillary cancer (TPC-1), kindly provided by Nobuo Satoh, Japan, Hürthle cell cancer (XTC-1) developed in our laboratory, primary cultures of medullary thyroid cancer (MTC-1.1 and MTC-2.2), and normal thyroid tissue (NT 1.0). FTC-133, FTC-236, and FTC-238 were derived from primary tumor, metastatic lymph node, and pulmonary metastases from the same patient. MTC-1.1 was derived from the thyroid tumor and MTC-2.2 from a lymph node metastasis. The cells were obtained in accordance with approved human experimentation protocols and maintained in standard incubating conditions (5% CO2, 95% humidity, 37 C) in DMEM/F12 (Mediatech, Herndon, VA) supplemented with 10% FCS (Irvine Scientific, Santa Ana, CA), insulin (0.25 IU/mL; Sigma, St. Louis, MO), TSH (10 mIU/mL, Sigma), and antibiotics. A cell line from a patient with colon cancer (Colo-201 cell line), was obtained from the American Type Culture Collection (Rockville, MD) and grown in RPMI 1640 containing 20% FCS.
RNA extraction and Northern analysis
Total RNA was prepared from cultured cells using the RNA stat-60
(Tel-Test B, Friendswood, TX). For Northern analysis, RNA
samples (15 µg) were size fractionated on 1% agarose gels containing
6% formaldehyde and transferred to nitrocellulose membranes (Hybond-N;
Amersham Life Sciences, Arlington Heights, IL) with low-vacuum (785
vacuum blotter; Bio-Rad, Richmond, CA), cross-linked with ultraviolet
(UV) (UV cross-linker; Fisher Scientific) and then dried in a vacuum
oven (80 C) for 2 h. The RNA was hybridized for 12–36 h at 42 C
in 50% deionized formamide, 4.7x SSPE, 0.47x Denhart’s solution,
0.1% SDS, and 10% dextran sulfate (17). The DNA probes (kindly
provided by Dr. Robert B. Jaffe, University of California, San
Francisco, CA) were labeled using random primers labeling method with
[
-32P]deoxycytidine triphosphate (Amersham) to a SA of
2–3 x 108 cpm/mg DNA. Typically, 2–3 x
107 cpm of 32P-labeled probe was used for 70
cm2 filter in 10 mL hybridization room temperature, 0.1x
SSC/0.1% SDS for 20 min at 50–55 C, and exposed to Kodak XAR-5 film
(Eastman Kodak, Rochester, NY) at -80 C with intensifying screens for
2–3 days. To control for the integrity and amount of messenger RNA
(mRNA), the membranes were stripped in 0.1% SDS/0.01x SSC for 10 min
and reprobed for ß-actin mRNA.
Immunohistochemistry
Immunohistochemistry studies were done both in cell lines and in the paraffin-embedded tissues. The thyroid cell lines were cultured on chamber slides (Nunc, Naperville, IL) for 2–3 days and fixed with cold acetone. The human thyroid tissues used for immunohistochemistry were initially fixed by 10% buffered formalin phosphate and embedded in paraffin. Tissues studied included Graves’ thyroid tissue, follicular adenomas, papillary cancers, follicular cancers, medullary cancers, and anaplastic cancers. Five-micrometer thick tissue sections were deparafinized and hydrated in PBS, incubated for 30 min in 1% hydrogen peroxide to inhibit endogenous peroxidase, and incubated in 2% goat serum in PBS for 20 min to block nonspecific binding. The sections were then incubated for 30 min at room temperature with a rabbit anti-VEGF polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200 dilution (0.5 µg/mL) using PBS and 1% BSA, and washed three times in PBS. The secondary antibody was biotinylated goat antirabbit IgG (1:300 dilution; Vector Labs., Burlingame, CA). The secondary antibody was then detected using the avidin-peroxidase complex (Vector Labs.) for 30 min. The sections were stained by diaminobenzidine solution (Santa Cruz Biotechnology) for 5 min and counterstained with hematoxylene. For negative controls, we used all reagents except the primary anti-VEGF antibody.
Western blot
Cell lysates from the cell lines [XTC-1, TPC-1, FTC-133, FTC-236, FTC-238, and control human umbilical cord cells (HUV-EC-C cell lines)] were prepared by washing cells once with PBS. Cell pellet(s) were obtained by centrifugation. PBS buffer was aspirated off, and 1 mL 6 M Urea + ß-mercaptoethanol was added. The cell lysate was sonicated for 1 min. Proteins were quantified using the Lowry protein assay method. Two SDS-PAGE Gels (Biorad Ready-made 4–15% gel) were prepared (one gel for transfer and the other for Coomassie blue staining). Ten to fifty micrograms of proteins were loaded onto each well using a 1:1 mix of protein volume and a 2x loading buffer (Novex, San Diego, CA). Samples were denatured at 90 C for 5 min and were quickly cooled on ice. Electrophoresis was carried out at 70 V for 3 h. One gel was stained with Coomassie brilliant solution (50% methanol, 0.05% Coomassie brilliant blue R, 10% acetic acid, 40% water) for 4 h. It was destained with destaining solution (5% methanol, 7% acetic acid, 88% water) overnight. The second gel was transferred onto a Hybond-N membrane (Amersham) for 3 h at 50 V using a Biorad transblot apparatus containing transfer buffer (39 mM glycine, 50 mM Tris base, 20% methanol). After the overnight transfer, the membrane was dried for 5 min. The membrane was blocked using 3% BSA/TBST for 1 h. The blocking reagent was replaced with the primary antibody (Santa Cruz Biotechnology, 1:100 in TBST buffer) and incubated for 2 h. The membrane was washed three times with TBST buffer for 5 min/wash. The TBST buffer was removed, and the membrane was incubated with secondary antibody (biotinylated antibody and/or HRP-labeled second antibody diluted in 3% goat serum/TBST) for 1 h. The membrane was then washed three times with TBST for 5 min/wash. The buffer was removed, and the membrane was incubated with streptavidin/HRP complex diluted in TBST for 1 h. The membrane was washed three times with TBST. The last TBST buffer wash was removed, and the membrane was incubated for 1 min in detection solution (1:1 vol of detection reagent 1 and detection reagent 2 from Amersham, Arlington Heights, IL). The detection solution was removed, and the membrane was wrapped in plastic wrap. The membrane was exposed for 15–60 sec on Hyperfilm-ECL and developed.
Enzyme-linked immunosorbent assay (ELISA)
Cells (105) were cultured in 200 µL MEM + 10% FCS
for 7 days. Aliquots of culture media were collected and assayed in
ELISA. ELISA plates were coated with 2.5 µg/mL monoclonal antibody
(mAb) to VEGF (mAb 3.5F8; Genentech, South San Francisco, CA) in 50
mM carbonate buffer, pH 9.6, at 4 C overnight and blocked
with 0.5% BSA in PBS. Standards (0.03–2 ng/mL recombinant
V165, Genentech) and 3-fold serially diluted samples
(initial dilution 1:5) in PBS contain-ing 0.5% BSA, 0.05%
polysorbate 20, 0.25% 3-[(3-cholamidopropyl)-dimethyl
ammoniol]-1-propane sulfate (Sigma), 0.2% bovine 
-globulin
(Sigma), 5 mM EDTA, and additional 0.35 N NaCl
were incubated on the plate for 2 h. Bound VEGF was detected using
biotinylated mAb to VEGF (mAb 4.6.1, Genentech), followed by
streptavidin peroxidase (Sigma) and 3,3',5'5'-tetramethyl benzidine
(Kirgaard & Perry Labs.) as the substrate. Plates were washed between
steps. Absorbance was read at 450 nm on Vmax plate reader
(Molecular Devices, Menlo Park, CA). The standard curve was fitted
using a four-parameter nonlinear regression curve-fitting program
(developed at Genentech). Data points that fell in the linear range of
the standard curve were used for calculating the VEGF concentration in
samples.
Statistical method
Statistical significance of ELISA data was determined by Student’s t test.
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Results |
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Cultured human thyroid cell lines expressed a 4.2-kilobase VEGF
mRNA. The VEGF mRNA levels were higher in thyroid cancer cell lines
than in normal thyroid cells. Thyroid cancer cell lines of follicular
cell origin (FTC-133, TPC-1, and XTC-1) expressed more VEGF mRNA than
did thyroid cell lines of parafollicular cell line (MTC-1.1) (Fig. 1A
). There were no differences in the
VEGF mRNA signals between the cells derived from the primary thyroid
tumors and metastases (FTC-133 vs. FTC-236 or FTC-238,
MTC-1.1 vs. MTC-2.2, Fig. 1B).
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Western blots showed production of VEGF121,
VEGF165, and VEGF189 splice variants by the
XTC-1 (Hürthle cell) and TPC-1 (papillary cancer) cell lines
(Fig. 1C
).
Immunohistochemical studies in thyroid cell lines and human thyroid tissue
The findings of immunohistochemical staining of the cell lines
parallels the findings of the Northern blots (Fig. 2). Thyroid cancer cell lines stained
more positively than normal thyroid cells, and thyroid cancer cells of
follicular cell origin stained stronger than thyroid cancer cells of
parafollicular cell origin. Similar VEGF staining was again observed in
primary and metastatic thyroid cancer cell lines. The deparafinized
sections of human papillary thyroid cancer, follicular thyroid cancer,
and Hürthle cell thyroid cancer also stained strongly. Adjacent
normal thyroid tissues stained only weakly compared with the cancers
(Fig. 3). Benign thyroid tumors
(follicular adenoma), hyperplastic thyroid tissue (Graves’ thyroid),
and medullary thyroid cancer also stained less strongly than did
thyroid cancers of follicular cell origin.
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All thyroid cells secreted VEGF in the conditioned medium as
determined by ELISA (Fig. 4A). Thyroid
cancer cell lines secreted more VEGF protein than did normal thyroid
cells in primary culture. Among the cancer cell lines, XTC-1 secreted
the most (39.1 ng/mL) and MTC-1.1 secreted the least (15.0 ng/mL) VEGF.
There was no difference in VEGF secretion between the cell lines
derived from the primary thyroid tumors and metastases (FTC-133
vs. FTC-236 or FTC-238, MTC-1.1, vs. MTC-2.2,
Fig. 4B). Under the conditions in which these cells were grown, there
was no appreciable cell proliferation during this period as measured by
the MTT assay. Correction for cell proliferation was therefore not
necessary.
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Immunohistochemical studies also demonstrated that cancer tissues stained more intensely than benign thyroid tumors and more intensely than adjacent normal tissues. The patterns of mRNA expression as well as immunohistochemical staining were not different between the primary thyroid tumors (FTC-133 and MTC-1.1) and respective metastases in lymph nodes (FTC-236 and MTC-2.2) or lung (FTC-238).
Our findings are consistent with other studies that found higher levels of VEGF mRNA in colon cancer and renal cell carcinoma than adjacent normal tissues and higher levels in tumorigenic cell lines (HT 1080 fibrosarcoma, MNNG HOS osteosarcoma) than in nontumorigenic cell lines.
Viglietto et al. (20) showed that thyroid tumors expressed more VEGF mRNA and stained stronger with anti-VEGF antibody than normal thyroid tissue. Ours is the first study, however, to show that Hürthle cell thyroid cancer and medullary thyroid cancer cells also express VEGF mRNA. Viglietto and co-workers found that the expression of VEGF seems to correlate with the aggressiveness of the tumors in vivo and their tumorigenic ability, and they showed that TPC-1 did not express VEGF. In contrast, we found TPC-1 cell line to express VEGF mRNA, to stain strongly with anti-VEGF antibody, and to secrete more VEGF into the condition media than the follicular thyroid cell lines FTC-133, FTC-236, and FTC-238. One may also have expected that the cell lines derived from metastases (FTC-236 and FTC-238) would have expressed more VEGF than the cell line derived from the primary thyroid cancer (FTC-133) if VEGF expression correlates with the ability of the cells to metastasize, but we did not find any difference among these three cell lines derived from the same patient.
The concentrations of VEGF in the conditioned media from the thyroid cell lines were significantly higher (15.8–39.1 ng/mL) than the concentration in the ocular fluid (3.6 ng/mL) obtained from patients with active proliferative diabetic retinopathy (21) and similar to the concentration in the conditioned media of G55 glioblastoma multiforme (41 ng/mL) and SK-LMS-1 leiomyosarcoma (14 ng/mL) cell lines (15). It should be pointed out, however, that our results were obtained from a closed culture system in which the VEGF can accumulate. The ocular fluid samples, in contrast, were isolated and quantitated in vivo from a open system with associated transport, clearance, metabolism, etc., which may account for lower values of VEGF. The high concentration of VEGF in the conditioned media suggests that VEGF may be more important for the growth and invasion of thyroid cancers than in other cancers.
Follicular thyroid cancers are vascular tumors and metastasize hematogenously. The effect of VEGF in increasing vascular permeability may facilitate this tendency for hematological dissemination (23, 24). Small papillary thyroid cancers are relatively common tumors but are rarely clinically significant because they usually do not grow appreciably or metastasize (25). Although it is possible that VEGF expression may be increased in the more metastatic phenotype (20, 26), we observed no appreciable differences among cell lines derived from primary tumors and cell lines from metastatic sites from the same patient, or among tumors of differentiated histological types.
It is intriguing, however, that the only medullary cancer we studied produced less VEGF than the other thyroid cancers. Medullary thyroid cancers are derived from the parafollicular cells and are generally more aggressive than cancers derived from the follicular thyroid cells. It appears that other angiogenic factors are more important in supporting the growth and invasion of medullary thyroid cancers.
We have also recently found that VEGF expression is stimulated by TSH in thyroid cancer cell lines (27). In this study, the cell line that had the highest basal secretion of VEGF (XTC-1, Hürthle cell cancer) was least stimulated by TSH, whereas the FTC lines (follicular cancer) and the TPC-1 (papillary cancer) were significantly stimulated by TSH and increased their VEGF production by 2- to 5-fold.
In conclusion, we demonstrated increased VEGF expression and secretion in differentiated thyroid cancers of follicular cell origin. It is likely that VEGF is important for thyroid tumor growth and invasion. Antiangiogenic agents may thus be useful in treating patients with papillary and follicular thyroid cancers.
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Acknowledgments |
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Footnotes |
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Received March 22, 1996.
Revised January 8, 1997.
Revised May 18, 1997.
Accepted July 10, 1997.
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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