Inhibition of Tumor Growth, Vascularization, and Collagenolysis

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

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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 containing 450 jug of medroxyprogesterone. Note absence of vascularization 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 implants 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 corneas. In all 94 control eyes capillary growth from the limbal region 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 tumor 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 vascularized 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 tumor 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, stroma infiltrated with V2 tumor cells (V), bloodvessels (Bl), and small unidentified cells (C). (Phase-contrast optics, epoxy section, x210.) (b) large defects in the basement membrane were observed in the Electron micrograph showing disruption and apparent fragmentation presence of intact epithelium. Where cells were plentiful, the (arrow) ofnormal collagenous structure ofstroma near corneal epithe- intracellular spaces often had lost their collagen fibrils, which lium(E). (x 11, 400.) Downloaded by guest on September 30, 2021 1178 Medical Sciences: Gross et al. Proc. Nad Acad. Sci. USA 78 (1981) were replaced by loose amorphous and filamentous material (Fig. 2b). The densely packed layers of collagen below the tumor, including Decemet's membrane, remained uninvaded. In the presence of medroxyprogesterone, either as Depo- provera or in the crystalline acetate form, there was little or no vascularization and no three-dimensional tumor growth (Fig. 'lb). The tumor cells grew as a thin plate extending laterally 6- E ,E6 |1 64

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0 ~~~~ 0 2 6 10 14 18 22 26 30 34 38 Time, days ;.X. -?. '. 'a) "., FIG. 4. Vascular growth in V2 carcinoma implanted in cornea. I -l-' Vessel length refers to mean length of longest vessels. o, Untreated I controls, 94 eyes; *, medroxyprogesterone-treated, 40 eyes; A, medroxyprogesterone pellets removed after 26 days, 9 eyes. All values repre- sent mean and SEM forall eyes in series.

in the region of the preformed pocket (Fig. 3a), with little D evidence of infiltration perpendicular to the tightly packed D a laminated bundles of collagen (Fig. 3b). Few blood vessels were seen. In 40corneas that were implanted simultaneously with tumor fragments and medroxyprogesterone pellets, neovascularization was negligible during the first 10 days after implantation and was very limited thereafter (Fig. 4). Inhibition of neovascularization and tumor growth was promptly reversed in nine eyes in which the polymer was removed at 26 days (Fig. 4). Conversely, hormone implants inserted 5!A days after the tumor also inhibited further growth and vessel proliferation (Fig. 5). Medroxyprogesterone reduced neovascularization and tumor growth even when the polymer was placed 8-10 mm away from the tumor on the other side of

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FIG. 3. V2 tumor implanted with medroxyprogesterone pellet, at 0- 17 days. (a) Photomicrograph ofsection cut perpendicular to surface of 14 1 28 corneal epithelium (E) showing V2 tumor cells (V) implanted in cor- Time, days neal pocket near polymer containing 450 ug ofmedroxyprogesterone. D, Descemet's membrane. (x220.) (b) Electron micrograph showing or- FIG. 5. Blood vessel growth in vascurarizing tumor. Measure- deredcollagenous stroma (S) nearepithelium (E) in-area similarto that ments as in Fig. 4. Q, Untreated controls, 8 eyes; e, medroxyprogester- shown in a. Section prepared as in Fig. 2b. (x 13,100.) one pellets implanted 5 days after tumor implant, 8 eyes. Downloaded by guest on September 30, 2021 Medical Sciences: Gross et aL Proc. NatL Acad. Sci. USA 78 (1981) 1179

Table 2. Collagenase activity of mouse melanoma and tumor angiogenic extract in rabbit cornea 6- No. of Collagenase, Experiment eyes units/ml B-16 control tumor 6 0.4 ± 0.04 B-16 and medroxyprogesterone 5 0.05 ± 0.03 Angiogenic extract implant 7 0.09 ± 0.01 Angiogenic extract and medroxyprogesterone 8 0.03 ±0.005 Results are mean ± SEM. for 11 eyes implanted with extract and medroxyprogesterone. Dexamethasone was equally effective.

9 11 13 15 17 19 21 23 Collagenase Activity. As reported in Table 1, about 4 times Time, days more enzyme activity was released from the untreated tumor tissue than from explants of the hormone-treated tissue. The FIG. 6. Blood vessel growth in vascularizing tumor: effect of other "halo" region, which contained a thin layer of tumor cells be- hormones. Measurements as in Fig. 4. o, Untreated tumor, 94 eyes; yond the highly vascularized three-dimensional control tumor *, 1 mg of estradiol, 4 eyes; A, 1 mg of testosterone, 5 eyes; *, 450 ug outgrowth, produced about V3 the amount of enzyme that the of medroxyprogesterone, 40 eyes; A, 1 mg of cortisone, 5 eyes; o, 1 mg vascular tumor proper did. Uninvolved regions of the cornea of dexamethasone, 7 eyes. containing tumor alone were less than ¼io as active as tumor tissue, and the analogous regions ofhormone-treated preparations the cornea (data not shown). Ofother steroids tested, dexameth- were even less active. asone and, to a lesser degree, cortisone also delayed vasculariza- In tumor-containing corneas from which medroxyprogester- tion and tumor growth. Testosterone and estradiol were ineffec- one had been removed 8 days before harvest, enzyme activity tive (Fig. 6). was only slightly lower than that of the untreated tumor-con- Mouse B-16 melanoma in the presence ofmedroxyprogester- taining eyes. When the hormone was inserted 6 days after the one failed to vascularize or spread beyond the implant (Fig. ld), tumor had been implanted, mean collagenase activity at the whereas rapid neovascularization was observed with some cor- time ofharvest, approximately 20 days later, was about Y2 that neal cloudiness and flat tumor spread within the pocket (Fig. found in controls; however, variability was so great, determined Ic) within 48 hr in all five control eyes. Mean (+SEM) vascular by T test, as to make this difference uncertain. Hormone im- growth at 11 days for the five control eyes measured 2.08 ± 0.35 plants placed 1 cm away from the tumor were partially effective mm as compared with 0.34 ± 0.26 mm for five corneas carrying in blocking collagenase production. tumor plus medroxyprogesterone. The melanoma never be- In experiments designed to assess the response ofthe cornea came three-dimensional and by 9 or 10 days the corneas became to a different tumor, explants ofthe mouse B-16 melanoma from quite cloudy in three ofthe five control eyes, possibly heralding the rabbit cornea produced significant amounts of collagenase the onset ofa rejection response. in culture, whereas those previously exposed to medroxypro- In the absence of tumor, pellets containing angiogenic hepa- gesterone released about /io that ofthe control tumor explants toma extract implanted along with medroxyprogesterone (Fig. (but more than that ofuninvolved cornea ofthe same eyes) (Ta- if) or dexamethasone-containing pellets showed little or no ves- ble 2). sel growth, whereas implants oftumor extract alone, with blank Explants from the region of rapid vascular ingrowth in re- polymer, elicited a luxuriant vascular response (Fig. le). Mean sponse to tumor extract released low levels of collagenase vascular growth for hepatoma extract implants alone at 14 days and, again, this activity was blocked by medroxyprogesterone in 10 eyes was 2.01 ± 0.27 mm compared with 0.21 0.12 mm (Table 2). Dexamethasone, which proved to be a potent inhibitor ofvas- cularization and tumor growth, also blocked collagenase pro- Table 1. Collagenase activity ofV2 carcinoma implants in rabbit duction as extensively as did medroxyprogesterone (Table 1). cornea: Effect oflocal hormones Cortisone was less effective and neither testosterone nor estra- No. of Collagenase,* diol had any inhibitory effect, also in keeping with their failure Experiment* eyes units/ml to inhibit tumor growth or vascularization. V2 tumor proper (control) 35 1.2 ± 0.12 The proportions of spontaneously active to inactive (latent) V2 and medroxyprogesterone (A) 22 0.3 ± 0.06 enzyme varied from 0% to 50% with no obvious correlation with V2 and medroxyprogesterone (B) 4 0.5 ± 0.34 any of the in vivo experimental conditions. V2 and medroxyprogesterone (C) 4 0.9 ± 0.24 V2 and medroxyprogesterone (D) 5 0.6 ± 0.23 V2 and dexamethasone 4 0.2 ± 0.09 DISCUSSION V2 and cortisone 4 0.4 ± 0.20 The most of this was the V2 and testosterone 4 1.4 ± 0.10 significant finding study complete tumor V2 and estradiol 4 1.6 ± 0.40 block of neovascularization and three-dimensional growth Polymer alone in cornea 8 0.15 ± 0.03 by local implants ofmedroxyprogesterone and dexamethasone. Cortisone had similar but reduced action; testosterone and es- * Assay 4 hr at 37°C, 200-,ul sample; collagen substrate specific activity tradiol had none. The first two agents also effectively blocked was 67 cpm/4g; 1 unit = 1 ag ofcollagen degraded per min. Results neovascularization induced by tumor extract, an action that may are mean ± SEM. (A) Tumor and hormone pellet implanted simulta- be for tumor to neously. (B) Hormone implanted 6 days after tumor. (C) Hormone re- responsible preventing growth. Subsequent moved 8 days before harvest. (D) Hormone implanted 8 mm away completion ofthis study, we became aware ofa report by Shubik from tumor. et aL (15) that methylprednisolone given parenterally blocked Downloaded by guest on September 30, 2021 1180 Medical Sciences: Gross et al. Proc. Natl. Acad. Sci. USA 78 (1981) angiogenesis in the hamster's cheek pouch induced by locally comitantly with three-dimensional tumor growth, which de- implanted neoplasms. stroys surrounding connective tissue. Prevention ofthese struc- Although a briefly delayed (6 days) implantation ofhormone tural alterations in the cornea by hormones further supports the resulted in considerable reduction in further vessel and tumor proposed association ofcollagenolysis with the invasive process. growth and collagenase production, none-ofthe three measured However, other effects such as increased hyaluronate produc- properties proved reversible. Hormone implantation in a sin- tion may also contribute to the observed changes (19). gle pellet after 16 days of tumor growth blocked extension of Using these observations to construct a working hypothesis, the vascular bed only in the immediate vicinity of the polymer we suggest the following scheme. V2 carcinoma cells (and prob- implant. ably other tumor types) induce a blood supply that they need The action ofthe hormones is not limited to one tumor type, for their further growth. Tumor cells have the ability to stim- because similar effects were observed with mouse melanoma ulate collagenase production by normal host cells. Increased implants in the rabbit cornea. Medroxyprogesterone is also numbers of tumor cells provide increased stimulation of more effective in blocking V2 carcinoma growth in the rabbit ear host cells, leading to collagen destruction and greater freedom (unpublished data), indicating no unique property of the cor- for tumor cell spread. Several steroid hormones can block the neal site. process at least at two points, neovascularization and collagen- Collagenolytic activity released by rapidly growing tumor ase production by host cells, and perhaps also can depress the masses was considerably greater than that ofexplants from me- postulated stimulatory potential of the tumor cells. No doubt, droxyprogesterone-treated tumors, although activity in the lat- multiple properties of the malignant tumor, working in concert, ter was higher than that obtained from uninvolved regions of are required for invasiveness. cornea containing untreated tumors. Because untreated tumor- bearingcorneais much more cellular and thicker than hormone- The authors thank Mr. Clement Darling for his excellent technical treated preparations, one might conclude that enzyme activity assistance. This is publication 827 of the Robert W. Lovett Memorial is correlated with gross cell number. However, we do not yet Group for the Study of Diseases Causing Deformities, of the Medical various cell types, are Services andthe Department ofMedicine, Massachusetts General Hos- knowwhat proportion of, and which ofthe pital, and the Harvard Medical School, Boston. These studies were sup- "turned on" to make the enzyme. ported by research grants from the National Institutes of Health The possible role ofcollagenase in facilitating vascularization (CA19158, AM03564, and EY02252) to the Massachusetts General Hos- of normal tissues by elaboration or activation of the enzyme at pital and CA 14019 to the Childrens Hospital Medical Center and a the growing tips ofnew blood vessels is suggested by our exper- grant from the Monsanto Company to Harvard University. iments with inserts oftumor extract alone. Simultaneous inhibition of vascularization, collagenase pro- 1. Jeffrey, J. J. & Koob, T. J. (1973) in Proceedings of the Excerpta duction, and tumor growth by local implants ofmedroxyproges- Medica Foundation International Congress, (Excerpta Medica terone and dexamethasone poses an interesting problem in Foundation, Amsterdam), No. 273, pp. 1115-1123. causal relationships. These three properties responded in par- 2. Newsome, D. A. & Gross, J. (1977) Invest. Ophthalmol Vision Sci. allel to the relative inhibitory effectiveness ofthe five different 16, 21-31. 3. Folkman, J. (1971) N. EngL J. Med. 285, 1182-1186. steroid hormones tested. The effective hormones actively 4. Eisenstein, R., Kuettner, K., Soble, L. W. & Sorgente, N. (1975) blocked vascularization ofthe cornea, elicited by hepatoma ex- Am. J. Path. 81, 337-347. tract and also independently, are potent inhibitors ofcollagen- 5. Brem, H. & Folkman, J. (1975) J. Exp. Med. 141, 427-439. ase production. The small increase in collagenolytic activity of 6. Eisenstein, R., Goven, S. B., Schumacher, B. & Choromokos, E. tissue vascularized by tumor extract induction could not account (1979) Am. J. Ophthalmol 88, 1005-1012. for the much greater enzymatic activity of the vascularized 7. Brem, S., Preis, I., Langer, R., Brem, H.-, Folkman, J. & Platz, A. (1977) Am. J. Ophthalmol 24, 323-328. three-dimensional tumor tissue. Whether or not V2 cells can 8. Kidd, J. G. & Rous, R. J. (1940)J. Exp. Med. 71, 813-837. make collagenase awaits success in culturing pure lines. Rabbit 9. Gimbrone, M. A., Jr., Cotran, R. S., Leapman, S. B. & Folkman, corneal stromal cells, in contrast, will produce this enzyme J. (1974) J. Natl Cancer Inst. 52, 413-427. when stimulated by secreted products ofcirculating monocytes 10. Langer, R. & Folkman, J. (1976) Nature (London) 263, 797-800. (16) and epithelial cells (17). We also know (unpublished) that 11. Rhine, W., Hsieh, D. S. T. & Langer, R. (1980)J. Pharm. Sci. 69, medroxyprogesterone and dexamethasone in concentrations 265-270. 12. Zetter, B. R. (1980) Nature (London) 285, 41-43. below 10 and 0.1 nM, respectively, will inhibit such stimulation 13. Nagai, Y., Gross, J. & Piez, K. A. (1964) Ann. N.Y. Acad. Sci. 121, and also block production of stimulating factors by epithelial 494-500. cells. These observations suggest that interactions between tu- 14. Johnson-Wint, B. (1980) Anal. Biochem. 104, 175-181. mor and host cells, probably through products secreted by the 15. Shubik, P., Feldman, R., Garcia, H. & Warren, B. A. (1976)J. Natl. former, may turn on collagenase production by the latter. Cancer Inst. 57, 769-774. IV that 16. Newsome, D. A. & Gross,J. (1979) Cell 16, 895-900. We did not assay for the collagenase (type specific) 17. Johnson-Wint, B. (1980) Proc. NatL Acad. Sci. USA 77, 5331-5335. degrades basement membrane collagen (18). This enzyme may 18. Liotta, L. A., Abe, S., Robey, P. G., & Martin, G. R. (1979) Proc. well be required to release tumor cells from blood vessels, but Natl. Acad. Sci. USA 76, 2268-2272. not for destruction of interstitial collagen described here. 19. Toole, B. P., Biswas, C. & Gross, J. (1979) Proc. Natl Acad. Sci. The morphologic data suggest that collagenolysis occurs con- USA 76, 6299-6303. Downloaded by guest on September 30, 2021