extending to within 1 mm of the limbus (9) was used to house small tumor ..... Langer, R. & Folkman, J. (1976) Nature (London) 263, 797-800. 11. Rhine, W.
Proc. Nati Acad. Sci. USA Vol. 78, No. 2, pp. 1176-1180, February 1981
Medical Sciences
Inhibition of tumor growth, vascularization, and collagenolysis in the rabbit cornea by medroxyprogesterone (invasion/angiogenesis/collagenase/steroid hormones)
JEROME GROSS*, RICHARD G. AZIZKHANt, CHITRA BIswAs*, ROMAINE R. BRUNS*, DEAN S. T. HSIEHt, AND JUDAH FOLKMANt *Developmental Biology Laboratory of the Medical Services of the Massachusetts General Hospital, tthe Department of Surgery of the Childrens Hospital Medical Center, the Departments of Medicine and Surgery, Harvard Medical School, Boston, Massachusetts 02114
Contributed by Jerome Gross, October10, 1980
ABSTRACT Medroxyprogesterone, dexamethasone, or cortisone, locally applied in sustained release polymer to rabbit V2 carcinoma implanted in the rabbit cornea, blocked neovascularization and three-dimensional growth of the tumor. These hormones similarly prevented the vascular proliferative response to implants in the rabbit cornea of mouse B-16 melanoma and also the response to implants of polymer containing tumor extract with angiogenesis activity. The inhibitory responses were accompanied by considerable reduction in collagenolytic activity released into culture medium by explants of the two tumors and of the corneal region containing angiogenic hepatoma extract. Morphologic studies revealed extensive three-dimensional disruption of the compact laminated collagenous structure ofthe cornea by untreated V2 carcinoma. In the presence of hormone the tumor grew slowly as a noninvasive two-dimensional plaque limited to the narrow region of the insertion pocket in the cornea, with no obvious disturbance of structure elsewhere. Cortisone was much less effective than medroxyprogesterone or dexamethasone. Testosterone and estradiol had no effect on the three measured properties. The data suggest that local hormonal interference with neovascularization, collagenase production, and tumor growth can prevent neoplastic invasion and destruction of a dense collagenous connective tissue.
MATERIALS AND METHODS Young male and female New Zealand White rabbits were used for propagating V2 carcinoma stocks originally derived from a Shope virus-induced papilloma (8), which had been carried for many years as an intramuscular tumor. An intra-corneal pocket extending to within 1 mm of the limbus (9) was used to house small tumor fragments and a 1-mm3 block of ethylene vinyl acetate polymer (10, 11) containing the hormone. In most experiments the polymer pellet containing either the test substance or buffer as blank control was positioned at the base of the pocket, followed by a 1-mm3 piece of tumor. One eye was used as control and the other as experimental. In experiments in which the hormone-containing polymer was inserted 6 days after the tumor, the pocket was reopened and the pellet was placed adjacent to but behind the tumor. In another series, the tumor and polymer containing the test substance were inserted about 10 mm apart. Medroxyprogesterone (Depo-provera) and crystalline medroxyprogesterone acetate were obtained from Upjohn and the other steroid hormones from Sigma. In a separate group of rabbits 1-mm3 fragments of B-16 mouse melanoma (Arthur D. Little, Cambridge, MA), which invaded and killed C57 black mice, were substituted for the V2 carcinoma. In other experiments polymer pellets containing 5 mg of angiogenic human hepatoma extract (12) were substituted for tumor. Microscopic Observations. Corneas were examined every 2 days with a Zeiss slit lamp stereomicroscope, and growth rate of new vessels and tumor size were measured with an ocular micrometer at X 10 magnification (accuracy ± 0.1 mm). For morphologic studies, the animals were killed with intravenous pentobarbitol. The corneas were fixed in situ with Karnovsky's fixative injected into the anterior chamber and by dripping fixative onto the outer surface. The excised corneas were postfixed with osmium tetroxide, embedded in epoxy resin, and sectioned for both light and electron microscopy. Collagenase Determination. Intact eyes were enucleated post mortem and, sterilized in Betadine solution for 2 min, and the antiseptic was carefully washed away with phosphate-buffered saline. Whole corneas were excised to include about 1 mm of the sclera. Uniform, coherent discs 4 mm in diameter were punched from the center of tumor growth, from a cloudy region containing plaque-like extensions of tumor cells just outside the vascularized three-dimensional tumor, and from distant uninvolved cornea. These discs were placed individually in Costar dishes having 24 16-mm wells, were covered with 1 ml of Dulbecco's modified Eagle's medium containing antibiotics, and were incubated at 37°C in a moist 5% CO2 atmosphere for 9 days. Culture medium was harvested at 3, 6, and occasionally 9 days and assayed. Reaction products were examined by polyacrylamide gel electrophoresis (13).
These studies were designed to determine whether the pattern of tumor growth in a highly ordered and densely packed collagenous matrix, the corneal stroma, when correlated with collagenase production, might furnish more concrete evidence for a significant participation of collagenolysis in invasion. Penetration of tumor cells perpendicular to the densely packed collagen layers with destruction of fibrils would indicate collagenolytic activity. Because medroxyprogesterone blocks collagenase production by explants of postpartum rat uterus (1) and alkaliburned rabbit cornea, preventing perforation ofthe latter (2), it seemed appropriate to use this hormone to test the proposed relationship between collagenolytic activity and tumor invasion. V2 carcinoma growing in the rabbit cornea, a system developed by Folkman and associates, has been used extensively by this group for studies on the role of vascularization in tumor growth. Folkman (3) proposed that inhibition of angiogenesis could be a significant deterrent to neoplastic growth and invasiveness. Inhibitory substances isolated from cartilage (4, 5) aorta (6), and vitreous body (7) blocked both angiogenesis and growth of the V2 carcinoma in the rabbit cornea. However, these substances are available in small amounts, require purification, and are variable in effect. Experiments reported here led to an unexpected observation of almost complete interference with vascularization and subsequent tumor growth by low concentrations of medroxyprogesterone and dexamethasone implanted locally in sustained-release polymer depots. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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Proc. Natd Acad. Sci. USA 78 (1981)
Medical Sciences: Gross et al.
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