TO explore the expression and function of epidermal growth factor receptor (EGFR) ... The correlation between EGFR expression and metastasis of uveal ...
Role of Epidermal Growth Factor Receptor in the Metastasis of Intraocular Melanomas Ding Ma and Jerry Y. Niederkorn PURPOSE. TO explore the expression and function of epidermal growth factor receptor (EGFR) expression on human uveal melanoma cells.
Five human uveal melanoma cell lines were examined by flow cytometry for the expression of EGFR. The correlation between EGFR expression and metastasis of uveal melanoma cells was tested in a nude mouse model of intraocular melanoma. The effect of EGFR on liver homing of blood-borne uveal melanoma cells was tested by tracing the fate of radiolabeled cells treated with anti-EGFR monoclonal antibody. The capacity of EGFR to inhibit the cytotoxic effects of tumor necrosis factor-a (TNF-a) was determined in vitro. The role of EGFR in promoting metastatic disease was studied by infusing intraocular melanoma- bearing mice using a neutralizing antibody against EGFR.
METHODS.
EGFR was expressed to varying degrees on all eight human uveal melanoma cell lines. Expression of EGFR correlated with metastatic potential and capacity of blood-borne uveal melanoma cells to localize in the liver. EGFR rendered uveal melanoma cells resistant to the cytolytic effects of TNF-a. Blocking EGFR with a neutralizing monoclonal antibody increased the susceptibility of uveal melanoma cells to TNF-mediated cytolysis, inhibited metastases, and prolonged host survival.
RESULTS.
CONCLUSIONS. The expression of EGFR on five human uveal melanoma cell lines is correlated with an increased capacity to localize in the liver, an increased resistance to TNF-mediated lysis, and decreased survival. Targeting EGFR expression and function may be a fruitful strategy for managing patients with uveal melanoma. (Invest Ophthalmol Vis Set. 1998;39:1067-1075)
U
veal melanoma is the most common intraocular malignancy in adults.1 The 5-year survival rate for uveal melanoma is 75% and is comparable to cutaneous melanoma.2 Unlike cutaneous melanoma, which can metastasize by lymphatic and hematogenous routes to multiple organs, including the lungs and lymph nodes, uveal melanoma spreads by the hematogenous route and preferentially localizes in the liver.3 Metastatic disease of the liver remains the leading cause of death in patients with uveal melanoma, with up to 95% of patients with uveal melanoma having liver metastases at the time of death.4 Metastasis is a highly selective, nonrandom process that follows a distinct series of steps, which include the detachment of cells from the primary tumor, invasion of surrounding tissues, intravasation of the blood vessels, implantation into the capillary bed of the target organ, extravasation, invasion of the
From the Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas. Supported in part by National Institutes of Health, Bethesda, Maryland (CA 30276) and an unrestricted grant from Research to Prevent Blindness, New York, NY. Submitted for publication January 9, 1998; accepted February 24, 1998. Proprietary interest category: N. Reprint requests: J. Y. Niederkorn, Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Investigative Ophthalmology & Visual Science, June 1998, Vol. 39, No. 7 Copyright © Association for Research in Vision and Ophthalmology
parenchyma of the target organ, and formation of new blood vessels in the secondary organ.5'6 The importance of cell surface molecules in determining the organ-specific metastasis of tumors has been well documented since the seminal study of Fidler.7 We have previously shown that radiolabeled human uveal melanoma cells, but not human cutaneous melanoma cells, display a strong predilection to localize in the liver following intravenous injection.8 However, organ-specific homing alone cannot account for the propensity of blood-borne tumor cells to colonize specific organs. Experiments in different animal models suggest that paracrine stimulation of tumor cells by organ-derived growth factors contribute to organ-specific metastasis.9 Colon carcinoma, like uveal melanoma, displays a predilection to metastasize to the liver.9 The capacity of colon carcinomas to form progressive liver metastases in nude mice is dependent on the ability of colon carcinoma cells to proliferate in the liver, especially in response to hepatocyte growth factor and transforming growth factor-a (TGF-a).9 TGF-a and hepatocyte growth factor are produced in the liver and stimulate the proliferation of various tumor cells by interacting with epidermal growth factor receptor (EGFR), which is expressed on a wide variety of normal and malignant cells.910 Importantly, EGFR expression has been correlated with the growth and metastasis of various neoplasms, including hepatic carcinoma,'' renal carcinoma,'2 laryngeal cancer,13 esophageal tumors,14 colon carcinoma,9 and lung cancer.15 However, we are unaware of any studies 1067
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that have examined the expression of EGFR on uveal melanomas. The predilection of uveal melanomas to form liver metastases, the importance of EGFR as a receptor for using liverderived growth factors (e.g., TGF-a and hepatocyte growth factor), and the wealth of data indicating a role for EGFR in the metastasis of other neoplasms prompted us to examine the expression and function of EGFR on human uveal melanoma cells. METHODS Mice d
Athymic nude balb/c (H-2 ) and C57BL/6 mice were purchased from The Jackson laboratories (Bar Harbor, ME). The use of animals conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Uveal Melanoma Cells OCMl, OCM3, and OCM8 human uveal melanoma cell lines were generously provided by June Kan-Mitchell (University of California, San Diego, CA). OCMl has a predominantly spindle morphology, whereas OCM3 and OCM8 have epithelioid and mixed morphologies, respectively.1617 The MEL202 human uveal melanoma cell line was kindly provided by Bruce Ksander (Schepens Eye Institute, Boston, MA). MEL202 is comprised predominantly of spindle cell elements.18 OM431 was generously provided by Daniel Albert (University of Wisconsin, Madison) and is predominantly epithelioid.19 Cell line 92.1 is comprised of epithelioid and spindle elements and was generously provided by Martine Jager (Leiden University Medical Center, The Netherlands). Its origin and characterization have been described previously.20 OMM1 was isolated from a subcutaneous metastasis in a patient with uveal melanoma and displays a mixed cell morphology.21 OCMl, OCM3, OCM8, OMM1, and OM431 cells were cultured in Ham's F-12 medium containing 10% heat-inactivated fetal calf serum, 1% L-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, 1% HEPES buffer, and 1% antibiotic-antimycotic solution. Cell lines 92.1 and MEL202 were cultured in complete RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 1% L-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, 1% HEPES buffer, and 1% antibiotic-antimycotic solution.
Intracameral Transplantation and Hepatic Metastasis Determination A modified quantitative technique for the orthotopic intracameral (IC) transplantation of precise numbers of tumor cells into the mouse eye has been described previously.22 Mice were deeply anesthetized with 0.66 mg ketamine hydrochloride (Vetalar; Parke-Davis, Detroit, MI) given intramuscularly. Tumor cells (105/5 /xl) were inoculated IC using a 1.0-ml syringe (Hamilton) fitted with a 35-gauge glass needle. Fifty days later metastatic hepatic tumor foci were readily demonstrable by histopathologic examination of the liver and were scored as previously described.23"25 Severity of metastases was scored by three independent observers and reported as: clear (0 = no discernible foci); minimal involvement ( 1 + = metastatic tumors involved less than 10% of the liver); moderate (2 + = metastatic tumors involved 10% to 25% of the liver); or extensive (3 + = metastatic tumor mass involved > 25% of the liver).
IOVS, June 1998, Vol. 39, No. 7
Liver metastases cultures The 92.1 human uveal melanoma cells were transplanted IC into nude mice. Fifty days later, the mice were euthanatized and macroscopic metastases were microdissected from the liver under a dissecting microscope and the first generation metastases (Fl) were cultured in vitro. Liver metastases cultures were expanded and transplanted IC into nude mice, and the process of isolating and culturing liver metastases was repeated to generate second (F2) and third (F3) generation metastases cultures.
In Vitro Selection of Epidermal Growth Factor Receptor—Positive Melanoma Cells An EGFR+ cell population of the original 92.1 human uveal melanoma cell culture was isolated and enriched using goat anti-mouse IgG-coated magnetic beads (Dynabeads, catalog number M-450; Dynal, Lake Success, NY) according to the manufacturer's instructions. A single-cell suspension of 92.1 melanoma cells was prepared and adjusted to 1 X 107 cells/ml and incvibated in a 1:10 dilution of mouse anti-human EGFR monoclonal antibody (mouse IgG,, hybridoma 225; American Type Culture Collection [ATCC], Rockville, MD) for 30 minutes at 4°C. Cells were resuspended, washed three times in Hanks' balanced salt solution (HBSS), and incubated with antimouse IgG-coated magnetic beads (Dynabeads; 4 beads/melanoma cell) for 30 minutes at 4°C on a rocking platform. Cells adhering to the magnetic beads were immobilized with a magnet (MPC; Dynal). The rosetted beads were resuspended in HBSS, and the procedure was repeated four additional times. Cells were washed in HBSS, and the surface phenotype was determined by flow cytometry.
Flow Cytometry The expression of EGFR on uveal melanoma cell lines was quantified by flow cytometry as previously described.26 Melanoma cells were incubated on ice with a 1:40 dilution of mouse monoclonal antibody specific for human EGFR (IgG,; ATCC hybridoma 225). Cells were then washed three times in phosphate-buffered saline (pH 7.2) containing 15% fetal calf serum and incubated with fluorescein isothiocyanate-labeled, goat IgG F(ab')2, anti-mouse IgG secondary antibody (Organon Teknika, Durham, NC) diluted 1:100 in HBSS. Cells were washed three times in HBSS (15% fetal calf serum) and resuspended in 1.0% paraformaldehyde in phosphate-buffered saline. Fluorescence was quantitated in an analyzer (Epics Profile Analyzer; Coulter Electronics, Hialeah, FL). In other experiments, liver metastases were confirmed by flow cytometry using a melanoma-specific mouse monoclonal antibody, designated MAb8.1H, which was generously provided by Larry Donoso (Wills Eye Hospital, Philadelphia, PA) and which has been described in detail previously.27
In Vivo Melanoma Cell Localization Assay Melanoma cells were labeled in vitro with 0.1 mCi Na251CrO4 (New England Nuclear, Boston, MA) for 1 hour (37°C), washed three times with HBSS, and resuspended in HBSS at a concentration of 1 X 107 cells/ml. 51Cr-labeled melanoma cells (1 X 106 in 0.1 ml) were incubated in either anti-EGFR monoclonal antibody (IgG2b, catalog number 14891 A; Pharmingen, San Diego, CA), normal mouse IgG (Pharmingen), or HBSS for 1 hour at 37°C. Mice received intravenous (TV) injections of 250
EGFR and Uveal Melanoma Metastases
IOVS, June 1998, Vol. 39, No. 7 TABLE 1. Epidermal Growth Factor Receptor
Expression on Human Uveal Melanomas Cell Line OCM1 OCM3 OCM8 92.1 OM431 OMM1* MEL202
Morphology
EGFR Expression (% Positive)
Spindle Epithelioid Mixed Mixed Epithelioid Mixed Spindle
20.27 2.52 4.41 31.49 1.73 12.38
5.34
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ventional in vitro 51Cr-release assay. Briefly, melanoma cells were radiolabeled with 5 l Cr as described above. Cells were then added to 96-weU microtiter plates at a concentration of 1 X 104 melanoma cells per well in the presence of 1 /xg/ml actinomycin D (Sigma Chemical, St. Louis, MO) and varying concentrations of TNF-a (10 ng, 100 ng, or 1 jag/well). Plates were incubated in 5% CO 2 for 18 hours at 37°C. In other experiments, melanoma cells were incubated with anti-EGFR (1 ng, 10 ng, 100 ng, and 1 jag/well; IgG,) before exposing the cells to TNF-a. Percentage lysis was calculated using the same formula used for measuring antibody-mediated lysis (see above).
' Cell line established from a skin metastasis of a uveal melano-
RESULTS jxg mouse anti-human EGFR monoclonal antibody, 250 jag normal mouse IgG (Pharmingen), or HBSS 1 hour before IV injection of radiolabeled melanoma cells. Six hours later, lungs, liver, spleen, kidneys, and 0.5 ml blood were removed from each euthanatized animal, and the radioactivity of each tissue was determined by counting in a gamma counter. The total counts per minute (CPM) for the lungs, liver, spleen, kidneys, and 0.5 ml blood were determined for each animal and the total CPM was considered the total isotope recovered for each animal. The CPM for each organ and blood sample were divided by the CPM for the total isotope recovered to give "percentage CPM recovered" for each sample. Results were expressed as the mean percentage CPM recovered for each organ or blood sample in the respective groups.
Antibody-Mediated Cytolysis of Epidermal Growth Factor Receptor—Positive Human Uveal Melanoma Cells Human uveal melanoma cells were labeled in vitro with 0.1 mCi Na 2 5l CrO 4 (New England Nuclear) for 1 hour (37°C), washed three times with HBSS, and resuspended in HBSS at a concentration of 1 X 10 7 cells/ml. Cells were washed 3X in HBSS and resuspended in HBSS at a concentration of 5 X 10 6 cells/ml. Radiolabeled cells were added to 96-well microtiter plates (5 X 104 cells/well) with either anti-EGFR monoclonal antibody (IgG2b) diluted 1:50, 1:100, or 1:200 or HBSS. Plates were incubated for 45 minutes at 4°C, the wells were washed with HBSS containing 0.1% bovine serum albumin, centrifuged, and the cell pellet was resuspended in 0.2 ml of a 1:10 dilution of low-tox rabbit complement (Accurate Chemical, San Diego, CA). Plates were incubated for 45 minutes at 37°C and centrifuged, and the CPM of the supernatant were counted in a gamma counter. Percentage lysis was calculated based on the formula: Percentage of lysis experimental CPM — spontaneous release CPM X 100. maximum release CPM — spontaneous release CPM
Tumor Necrosis Factor—Mediated Lysis of Melanoma Cells The susceptibility of human uveal melanoma cells to lysis by tumor necrosis factor-a (TNF-a) was determined using a con-
Epidermal Growth Factor Receptor Expression on Human Melanoma Cell Lines Flow cytometric analysis of eight human uveal melanoma cell lines and one uveal melanoma metastasis cell line did not reveal a clear dichotomy between spindle and epithelioid cell types in their expression of EGFR (Table 1). To determine whether EGFR expression influenced metastatic behavior of human uveal melanomas, prospective studies were performed using a nude mouse model of intraocular melanoma. For these studies, two spindle cell melanomas, two epithelioid cell melanomas, and two mixed uveal melanoma cell lines were compared. Melanoma cells were transplanted intracamerally in nude mice, and liver metastases were assessed 50 days later as previously described. 23 " 25 The results revealed a close correlation between the expression of EGFR and the severity of liver metastases (Table 2). Uveal melanoma cell lines in which >20% of the cells expressed EGFR produced extensive metastases in >75% of the hosts. By contrast, metastases were mild or absent in hosts bearing melanomas originating from in vitro cultures that contained 60% (data not shown). Over one hundred years ago, Paget33 proposed the "seed and soil" hypothesis to explain the propensity of certain tumors to form metastases in specific organs. The "soil" of certain organs was believed to provide the proper nutrients for nurturing the growth of disseminated tumor cells. Although surface molecules clearly play a role in organ-specific homing of blood-borne tumor cells, the parenchyma of the secondary organ also contributes to the survival and growth of metastatic tumor cells. The ability of EGFR to serve as a ligand for capturing and using hepatic growth factors such as TGF-a and hepatocyte growth factor may help to explain the predilection of uveal melanomas to form hepatic metastases. Targeting this phase of the life history of the tumor may be a fruitful strategy for reducing morbidity and mortality in patients with uveal melanoma.
Acknowledgments The authors thank Daniel Albert, Martine Jager, June Kan-Mitchell, Bruce Ksander, Theo Luider, and Gregorius Luyten for generously providing the various human melanoma cell lines.
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