Inhibition of Tumor Growth, Vascularization, and Collagenolysis
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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 byJerome Gross, October10, 1980 ABSTRACT Medroxyprogesterone, dexamethasone, or cor- MATERIALS AND METHODS tisone, locally applied in sustained release polymer to rabbit V2 carcinoma implanted in the rabbit cornea, blocked neovascular- Young male and female New Zealand White rabbits were used ization and three-dimensional growth of the tumor. These hor- for propagating V2 carcinoma stocks originally derived from a mones similarly prevented the vascular proliferative response to implants in the rabbit cornea ofmouse B-16 melanoma and also the Shope virus-induced papilloma (8), which had been carried for response to implants ofpolymer containing tumor extract with an- many years as an intramuscular tumor. An intra-corneal pocket giogenesis activity. The inhibitory responses were accompanied by extending to within 1 mm of the limbus (9) was used to house considerable reduction in collagenolytic activity released into cul- small tumor fragments and a 1-mm3 block ofethylene vinyl ace- ture medium by explants of the two tumors and of the corneal re- tate polymer (10, 11) containing the hormone. In most experi- gion containing angiogenic hepatoma extract. Morphologic studies ments the polymer pellet containing either the test substance or revealed extensive three-dimensional disruption of the compact laminated collagenous structure ofthe cornea by untreated V2 car- buffer as blank control was positioned at the base ofthe pocket, cinoma. In the presence of hormone the tumor grew slowly as a followed by a 1-mm3 piece of tumor. One eye was used as con- noninvasive two-dimensional plaque limited to the narrow region trol and the other as experimental. In experiments in which the of the insertion pocket in the cornea, with no obvious disturbance hormone-containing polymer was inserted 6 days after the tu- ofstructure elsewhere. Cortisone was much less effective than me- mor, the pocket was reopened and the pellet was placed adja- droxyprogesterone or dexamethasone. Testosterone and estradiol cent to but behind the tumor. In another series, the tumor and had no effect on the three measured properties. The data suggest that local hormonal interference with neovascularization, colla- polymer containing the test substance were inserted about 10 genase production, and tumor growth can prevent neoplastic in- mm apart. Medroxyprogesterone (Depo-provera) and crystal- vasion and destruction ofa dense collagenous connective tissue. line medroxyprogesterone acetate were obtained from Upjohn and the other steroid hormones from Sigma. These studies were designed to determine whether the pattern In a separate group ofrabbits 1-mm3 fragments ofB-16 mouse of tumor growth in a highly ordered and densely packed colla- melanoma (Arthur D. Little, Cambridge, MA), which invaded genous matrix, the corneal stroma, when correlated with colla- and killed C57 black mice, were substituted for the V2 carci- genase production, might furnish more concrete evidence for a noma. In other experiments polymer pellets containing 5 mg significant participation of collagenolysis in invasion. Penetra- of angiogenic human hepatoma extract (12) were substituted for tion oftumor cells perpendicular to the densely packed collagen tumor. layers with destruction of fibrils would indicate collagenolytic Microscopic Observations. Corneas were examined every 2 activity. Because medroxyprogesterone blocks collagenase pro- days with a Zeiss slit lamp stereomicroscope, and growth rate of duction by explants of postpartum rat uterus (1) and alkali- new vessels and tumor size were measured with an ocular mi- burned rabbit cornea, preventing perforation ofthe latter (2), it crometer at X 10 magnification (accuracy ± 0.1 mm). For mor- seemed appropriate to use this hormone to test the proposed re- phologic studies, the animals were killed with intravenous pen- lationship between collagenolytic activity and tumor invasion. tobarbitol. The corneas were fixed in situ with Karnovsky's V2 carcinoma growing in the rabbit cornea, a system devel- fixative injected into the anterior chamber and by dripping fix- oped by Folkman and associates, has been used extensively by ative onto the outer surface. The excised corneas were postfixed this group for studies on the role of vascularization in tumor with osmium tetroxide, embedded in epoxy resin, and sec- growth. Folkman (3) proposed that inhibition of angiogenesis tioned for both light and electron microscopy. could be a significant deterrent to neoplastic growth and inva- Collagenase Determination. Intact eyes were enucleated siveness. Inhibitory substances isolated from cartilage (4, 5) post mortem and, sterilized in Betadine solution for 2 min, and aorta (6), and vitreous body (7) blocked both angiogenesis and the antiseptic was carefully washed away with phosphate-buff- growth of the V2 carcinoma in the rabbit cornea. However, ered saline. Whole corneas were excised to include about 1 mm these substances are available in small amounts, require purifi- of the sclera. Uniform, coherent discs 4 mm in diameter were cation, and are variable in effect. punched from the center oftumor growth, from a cloudy region Experiments reported here led to an unexpected observation containing plaque-like extensions oftumor cellsjust outside the of almost complete interference with vascularization and sub- vascularized three-dimensional tumor, and from distant unin- sequent tumor growth by lowconcentrations ofmedroxyproges- volved cornea. These discs were placed individually in Costar terone and dexamethasone implanted locally in sustained-re- dishes having 24 16-mm wells, were covered with 1 ml of Dul- lease polymer depots. becco's modified Eagle's medium containing antibiotics, and were incubated at 37°C in a moist 5% CO2 atmosphere for 9 The publication costs ofthis article were defrayed in part by page charge days. Culture medium was harvested at 3, 6, and occasionally 9 payment. This article must therefore be hereby marked "advertise- days and assayed. Reaction products were examined by poly- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. acrylamide gel electrophoresis (13). 1176 Downloaded by guest on September 30, 2021 Medical Sciences: Gross et al. Proc. Natd Acad. Sci. USA 78 (1981) 1177 E - 4 *- ,_.,..'m,'.<>*_<'**'*. ej _ S<f i n- 0 v -~~~~~r . M art~~~~Op v1v-_ _ -dp - ~ ~ d , - Un@. ,. ', , ?> ,.'-"_ -f8e. ' e-_- w s 'F..4 AP FIG. 1. Vascular response and tumor growth in the rabbit cornea. C.',,0~~~~~~~~~~~~~~. (a) Three-dimensional vascularized tumor 22 days after implantation of V2 carcinoma and blank polymer. Polymer is obscured by tumor #~~ mass. (b) Contralateral eye implanted with tumor and polymer con- taining 450 jug of medroxyprogesterone. Note absence of vasculariza- tion and tumor growth. (c and d) B-16 mouse melanoma implant with- s~~~~t out (c) and with (d) polymer containing medroxyprogesterone. Note ~ ~ ~ absence of tumor spread in d. (e) Polymer implants containing 5 mg ," of crude tumor angiogenesis extract (T) and a second blank implant. (f) Same as e, but with medroxyprogesterone. Arrows indicate mass of tumor cells; L, limbus; N, nictitating membrane; *, polymer im- plants with or without medroxyprogesterone. Irregular white spots (unlabeled) are light reflections from the moist eye. '4~~~~~~~~~~~~~~~~~~4 Both latent and active collagenase were assayed, the former after activation with 90 tkg of 1-tosylamido-2-phenylethyl chlo- romethyl ketone-treated trypsin per ml ofsample fluid for 5 min at 370C, followed by a 5-fold excess ofsoybean trypsin inhibitor. Collagenase activity was measured by the assay procedure of Johnson-Wint (14). RESULTS Neovascularization and Tumor Growth. V2 carcinoma was implanted with and without blank copolymer in 94 rabbit cor- neas. In all 94 control eyes capillary growth from the limbal re- gion closest to the implant had begun at 7 days. By 10-14 days a ribbon ofcapillary sprouts extended across the tumor surface, which bulged outward as a three-dimensional mass. Rapid tu- mor growth was paralleled by the density and extension of new blood vessels. The length of the longest vessels was a direct measure ofgrowth ofthe three-dimensional region ofthe tumor. Ai' In nearly all cases tumor cells also extended beyond the vascu- larized mass as a thin flat plate seen as a cloudy "halo. " By 4-5 weeks each tumor was a large exophytic mass often greater than 1 cm thick (Fig. la).The fully developed three-dimensional tu- mor showed extensive disruption ofthe collageneous layers with FIG. 2. V2 tumor implanted without medroxyprogesterone, at 22 numerous blood vessels and infiltration by tumor cells, princi- days, similar to tumor in Fig. la. (a) Photomicrograph of section cut fibrous pally in the anterior region ofthe cornea, often leaving intact the perpendicular to surfaceofthe corneal epithelium (E), showing basement membrane and epithelium (Fig. 2a). Occasionally,