[CANCER RESEARCH 49, 1758-1762, April 1, 1989) Antiestrogenic Action of Toremifene on Hormone-dependent, -independent, and Heterogeneous Tumor Growth in the Athymic Mouse1

Simon P. Robinson and V. Craig Jordan2

Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, Wisconsin 53792

ABSTRACT where tumor remission is initially observed, therapy ultimately fails. The toremifene has been used to study the growth control Immunocytochemical techniques with monoclonal antibodies of hormone-dependent (MCF-7), -independent (MDA-MB-231), or to the ER and PgR have been used to stain receptor-containing mixed tumor cell populations in athymic mice. Maximal MCF-7 tumor cells in frozen breast tumor sections. These studies indicate that growth was produced in ovariectomized athymic mice by circulating a heterogeneous distribution of both ER (3, 4)- and PgR- levels of approximately 200 pg/ml (produced by 0.5-cm silastic capsules implanted s.c.). The antiestrogen toremifene (77 ±4 Mg/day containing cells (5) is a frequent occurrence and support the from a 2-cm silastic capsule) inhibited estradiol (0.5-cm capsule)-stimu- findings that repeat microsamples from the same tumor can lated growth by more than 70%. No tumor growth was observed in mice contain markedly differing receptor levels (6). An antiestrogen treated with toremifene alone, although toremifene acted as a weak partial would be expected to control the ER-positive cell population. on the mouse . However, if the drug cannot exert control over the ER-negative The growth of hormone-independent MDA-MB-231 breast tumors cells, this population might be expected to continue to prolif implanted in athymic mice was not influenced by either estradiol (0.5-cm erate so that the antiestrogen would eventually fail. The timing capsule) or toremifene (2-cm capsule) when administered alone or in of this failure would depend upon the relative proportion of combination. Furthermore, even very large doses of toremifene (S mg/ cell types. The control of both hormone-dependent and -inde day p.o.) did not alter the rate of MDA-MB-231 tumor growth. Mixtures of MCF-7 and MDA-MB-231 cells in 9:1 and 99:1 ratios pendent tumor cells in heterogeneous breast tumors would be inoculated into athymic mice produced tumors which grew in the absence a major advantage in breast cancer therapy. Recently, a new of estradiol but responded to estradiol supplementation (0.5-cm capsule) antiestrogen, toremifene, has been developed and is currently with a more rapid rate of tumor growth. Tumors grown from inoculated being studied in clinical trials (7). Interestingly, toremifene has MCF-7:MDA-MB-231 cells (99:1 ratio) in the presence of estradiol had been reported to have some activity against a hormone-inde receptor levels of 33.2 ±9.2 fmol/mg of protein at Day 44 pendent mouse uterine sarcoma (8). compared to 84.8 ±4.8 fmol/mg of protein in pure MCF-7 tumors. Recent laboratory studies suggest that ER-positive breast Toremifene (2-cm capsule) treatment inhibited the estrogen stimulation cancer may be capable of influencing the proliferation of ER- of these mixed tumors (99:1 starting ratio) to that of toremifene alone. negative breast cancer cells (9, 10). can cause an However, toremifene-alone treatment produced a more rapid rate of increased secretion of transforming growth factor ßfrom ER- tumor growth than control or tumors grown from irradiated MCF-7 cells mixed with viable MDA-MB-231 cells. Increasing the ratio of MCF- positive cells (9), and this factor is inhibitory to the proliferation of the ER-negative breast cancer cell line, MDA-MB-231. 7:MDA-MB-231 cells (999:1) initially inoculated resulted in tumors which developed less rapidly than the lower ratio (99:1). Toremifene (2- The aim of this study is to examine the activity of toremifene cm capsule) again produced partial inhibition of 17/J-estradiol-stimulated on hormone-dependent, -independent, and mixed tumors using tumor growth while increasing tumor growth above control when the breast cancer cell lines grown into solid tumors in the athymic antiestrogen was administered alone. mouse (11-13). These results demonstrate that toremifene is effective in inhibiting estrogen stimulation of hormone-dependent tumors and partially success ful at controlling mixed hormone-dependent/independent tumors; how MATERIALS AND METHODS ever, the antiestrogen cannot control the growth of a hormone-independ ent tumor in this model. Tumors. MCF-7 cells (American Type Culture Collection passage 144) were karyotyped and maintained in minimal essential medium (Gibco, Grand Island, NY) with phenol red, containing nonessential amino acids, 10 IHM. 4-(2-hydroxyethyl)-l-piperazineethanesulfonic INTRODUCTION acid, insulin (6 ng/ml), penicillin (100 units/ml), and 10% complete The hormone receptor content of breast tumor biopsies is calf serum (Gibco, Grand Island, NY). MDA-MB-231 cells (American currently used as a predictive marker; the presence of both ER3 Type Culture Collection passage 25) were maintained in the same medium with 5% calf serum that was charcoal stripped. and PgR indicates about a 77% chance of a tumor responding Cells were grown in 150- x 25-mm dishes (Falcon, Cockeysville, to endocrine therapy, whereas a tumor containing neither recep MD), harvested at log phase with a disposable cell scraper (Costar, tor type has only about an 11% chance of a response to similar Cambridge, MA), and diluted in serum-free medium. When required, therapy (1). The antiestrogen, , has now become the cell mixtures were made, and 10 cells in 0.1 ml of medium were endocrine therapy of choice in the postmenopausal breast can inoculated into the axillary mammary fat pads of 4- to 5-wk-old cer patient. Tamoxifen therapy produces objective remission in ovariectomized BALB/c athymic mice (HaríanSprague-Dawley, Indi about 30% of unselected cases (2). However, even in cases anapolis, IN). Transplantation procedures for solid tumors, animal housing conditions, and tumor size calculations were all as previously Received 8/10/88; revised 11/17/88; accepted 1/3/89. described (11). The costs of publication of this article were defrayed in part by the payment Drug Administration. Estradiol administration was by silastic cap of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sules implanted s.c. on the back of the animals using a trochar. Torem 1Supported by Adria Laboratories, Columbus, OH. ifene was administered either in a similar manner or, when very large 2To whom requests for reprints should be addressed, at the Department of doses were required, by intubation (p.o.) of a crystalline suspension in Human Oncology, University of Wisconsin Clinical Cancer Center, 600 Highland 0.1 ml of peanut oil. Avenue, Madison, WI 53792. 3The abbreviations used are: ER, , PgR, progesterone recep Silastic capsules were formed by plugging one end of lengths of tor; EIA, immunoassay. medical grade silastic tubing (0.078 in inner diameter by 0.125 in outer 1758

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. ANTIESTROGENIC ACTIONS OF TOREMIFENE diameter; Dow Corning, Midland, MI) with silastic 382 medical grade dependent MCF-7 tumors, the 0.5-cm 17/3-estradiol capsule elastomer (Dow Corning) and then filling with either crystalline torem- was used to stimulate tumor growth. These capsules released ifene-free base (a gift from Adria Laboratories, Columbus, OH) or 17/3- an average (±SEM)4 ±0.4Mg/day of 17/3-estradiol. Toremifene estradiol (Sigma Chemical Company, St. Louis, MO) mixed 1:3 (w/w) produced more than a 70% reduction in the 17/3-estradiol- with elastomer (without catalyst). Capsules were completed by Tilling stimulated growth regardless of whether 0.5-, 1-, or 2-cm torem the open end with elastomer and sterilizing with radiation (20,000 rads). Toremifene and 17/3-estradiol release was calculated as preim- ifene capsules were used [silastic capsules released on average plantation weight minus dry weight after removal. (±SEM)28 ±0.2 jig, 45 ±1.0 ^g, or 77 ±4.0 ^g/day over the Hormone Levels and Tumor Receptor Measurements. Circulating treatment period, respectively]. However, even with 77 jig/day 17/3-estradiol levels were measured in serial serum samples taken from of toremifene, the tumor growth was not inhibited to control tumor-bearing mice. Blood samples were obtained by light ether anes or toremifene-treated alone (Fig. 2). thesia and bleeding from the eye orbit. After clotting overnight at 4°C, Long-term (27 days) treatment of athymic mice with torem samples were centrifuged at 2000 x g, and serum was removed and ifene silastic capsules (2 cm) produced some uterine stimula stored at -20°Cuntil analysis. 17/3-Estradiol measurements were made tion. However, a marked inhibition of 17/3-estradiol (0.5-cm on 50 //I samples using the commercially available estradiol coat-a- silastic capsule)-stimulated uterine weight was produced by count radioimmunoassay (Diagnostic Products, Los Angeles, CA). toremifene (Fig. 3). Ovariectomized mouse serum was used as a test control, and spiked samples were used to validate the assay (data not shown). The lowest The influence of estrogen and toremifene on the hormone receptor-negative breast cancer cell line, MDA-MB-231 (15), standard used for the standard curve was 20 pg/ml. ER was determined by homogenization of tumors in buffer contain was determined using the 0.5-cm 170-estradiol capsule and 2- ing 0.4 KC1 to extract nuclear ER and assayed using the commercially available ER-EIA kit (ER-EIA Monoclonal; Abbott Laboratories, Chi cago, IL) as previously described (11). 2uJ 0.6 -i W •¿0.5H RESULTS 0.4 H The sensitivity of MCF-7 tumor pieces implanted in the athy mie mouse to 170-estradiol-stimulated growth was exam 0.3 - ined using different sized 17/3-estradiol capsules (Fig. 1). The capsules produced a constant release of 17/3-estradiol in the 0.2 H tumor-bearing mice (Table 1). The smallest capsule capable of U) 0.1 - producing maximal tumor growth was 0.5 cm in length and W produced circulating levels of approximately 200 pg/ml of 17/3- l o.oJ estradiol. ER tended to be higher in tumors grown with lower 20 40 60 80 17/3-estradiol levels (Table 1). Days To examine the antitumor action of toremifene in hormone- Fig. 2. The growth rates of MCF-7 tumors (n = 10) transplanted in athymic 2 0.5-, mice treated with: estradiol, 0.5-cm (•)silastic capsule alone; toremifene, 0.5 cm (A) or 2 cm (D). Silastic capsule alone or estradiol, 0.5-cm silastic capsule, bl combined with toremifene, 0.5-cm (O), 1-cm (A), or 2-cm (•)silastic capsule. M *' 0.4 H Control animals (O) received no treatment. O m rv - 0.3 H

0.2 - 60-0»E

» O.I - ^^50-cnSo> o E .= 0.0 J £UJ CO40-<- „¿_

IO 20 30 40 50 60 +10) c£ Days o30-E~ Fig. 1. The effect of estradiol stimulation on the growth rate of MCF-7 tumors implanted in athymic mice. Estradiol administration was achieved with 2-cm (O, T-T11 n = 8), 1-cm (•,n= 7), 0.5-cm (•,n= 7), and 0.25-cm (O, n = 6) silastic capsules 20-a510 implanted under the skin on the back. Control animals (A, n = 8) received no treatment. Circulating estradiol levels from these mice are shown in Table 1. -0-I1T Table 1 Sensitivity of MCF-7 tumor to ¡7ß-estradiol 17/3-estradiol Silastic (Pg/ml)4wk794 content of 1° 1 capsule MCF-7 tumors £TJ c ±S (cm)2 (fmol/mgprotein)89.2 •¿Â»-o O O » c1 «^ •¿â€”»^ ±50°(7)* ±34 (8) ±6.6 (10) 10.5 363 ±32 (7) 330 ±26 (7) 84.7 ±6.1' (10) 1 11 2 198 ±8(8) 206 ±23 (5) 92.7 ±8.0 (10) 0.25 74 ±10(6) 104 ±8(6) 106.2 ±6.7 (10) 0Circulating 0(7)9wk671 0(7)ER Fig. 3. The effect of long-term (27 days) toremifene treatment (2-cm silastic " Mean ±SEM. capsule) or estradiol treatment (0.5-cm silastic capsule) on the uterine wet weight * Numbers in parentheses, number. of ovariectomized athymic mice when given either alone or in combination (n = c Significantly differed (P < 0.05) from tumors grown with a 0.25-cm capsule. 7). Control animals received no treatment (n = 6). 1759

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. ANTIESTROGENIC ACTIONS OF TOREM1FENE cm toremifene capsule. The growth rate of MDA-MB-231 MCF-7/MDA ER 2 (ratio) (tmol/mg protien) tumors was not changed by 17/S-estradiol or toremifene either U to 0.8- (9:I) +E2 6.2+1.5 alone or in combination (Fig. 4). Furthermore, the p.o. admin +i istration of 5 mg of toremifene per day for 10 days did not 2 33.219.2 change the rate of MDA-MB-231 tumor growth compared to 0.6 -{ controls (Fig. 5). This large dose of toremifene did, however, 0.4- produce a loss in body weight and 50% fatality. (I00:0) + Et 84.814.8 Having established the influence of estrogen and toremifene (9:1) 1.6+0.3 on MCF-7 and MDA-MB-231 cells when grown alone as solid OT0.2 - (99:1) 2.010.4 w [UIO6 MDA-MB-2311 tumors, the effect of these agents was examined on tumors O grown from mixtures of these cell lines. 0.0 Mixing MCF-7 cells with MDA-MB-231 cells in 9:1 and 10 20 30 40 50 Days 99:1 ratios and inoculating 10 million cells produced tumors which grew in the absence of 170-estradiol but responded to Fig. 6. The growth rate of MCF-7:MDA-MB-231 mixed tumors. Mixtures of tumor cells (total of 10 x IO6 cells) were inoculated into athymic mice with or 17/3-estradiol (0.5-cm capsule) with a more rapid rate of growth without estradici supplementation (0.5-cm capsule). After 44 days tumors were (Fig. 6). When 1 million MDA-MB-231 cells were inoculated dissected, and ER levels were measured by ER-EIA (see "Materials and Meth ods"). As a control, 1 X 10' MDA-MB-231 cells alone were inoculated. At 44 alone, tumor developed less rapidly than the mixed inoculates days, insufficient tumor was available for ER-EIA, so continued growth was containing the same number of MDA-MB-231 cells (9:1 ratio). followed and ER measured at Day 70. ER were not detected in MDA-MB-231 tumors, whereas MCF-

7 tumors contained on average 84.8 ±4.8 fmol/mg of protein O.?-, after 44 days of growth. At 44 days, little ER was present in V) mixed tumors not treated with estrogen or tumors starting with 0.6- a 9:1 ratio of MCF-7 cells to MDA-MB-231 and supplemented 00.5 H with 17/3-estradiol. However, tumors grown in the presence of S 170-estradiol for 44 days from a 99:1 ratio of MCF-7:MDA- —¿0.4- CVJeo. •¿S 0.2- tiJ 1.5- CO CO +1 c o = 0.0 J 25 l.O- 10 20 30 E Days u Fig. 7. The growth rate of a transplanted mixed tumor (n = 14) in athymic a0.5H mice treated with: estradiol, 0.5-cm silastic capsule (•)alone; toremifene, 2-cm co silastic capsule alone (D); or estradiol, 0.5-cm silastic capsule, and toremifene, 2- o cm silastic capsule (•).Control animals (O) received no treatment. The trans E planted tumor was grown from a 99:1 ratio of MCF-7:MDA-MB-231 in the 0.0 J presence of estradiol (as shown in Fig. 6).

IO 20 30 40 MB-231 cells were still ER positive with more than a third of Days the receptor content measured in pure MCF-7 tumors (Fig. 6). Fig. 4. The effect of toremifene on the growth rate of MDA-MB-231 tumors (n = 12) transplanted in athymic mice treated with: estradici, 0.5-cm (•)silastic On the basis of the tumor responses and ER content, a tumor capsule alone; toremifene, 2-cm (D) silastic capsule alone; or estradici, 0.5-cm grown from a 99:1 ratio of MCF-7:MDA-MB-231 cells was silastic capsule, and toremifene, 2-cm silastic capsule, combined (•).Control chosen to examine the action of antiestrogens on a heteroge animals (O) received no treatment. neous tumor. At Day 44, the tumor was retransplanted into athymic mice, and the effect of 17/3-estradiol (0.5-cm capsule) or toremifene (2-cm capsule) either alone or in combination on the growth was examined (Fig. 7). Tumors on all animals grew rapidly and were not influenced by the treatments. These results were similar to the findings for the pure MDA-MB-231 tumors and show that the initially heterogeneous tumor was now pre dominantly hormone independent. ER measurements on the transplanted mixed tumors 27 days after implantation now showed no difference between the treatment groups with all tumors having <3 fmol/mg of protein. The action of toremifene on a mixed cell population was studied further using the 99:1 ratio of MCF-7:MDA-MB-231 cells. Cell mixtures were inoculated into athymic mice bearing 10 20 30 40 17-/3-estradiol (0.5 cm) capsules and/or toremifene (2-cm cap Days sules). This experiment supports the earlier result that 170- Fig. 5. The effect of high-dose toremifene treatment on the growth rate of estradiol treatment produced a more rapid tumor growth than MDA-MB-231 tumors transplanted in athymic mice. Mice were treated daily p.o. the control. The combination of toremifene with 17(8-estradiol with either 5 mg of toremifene suspended ¡n0.1 ml of peanut oil (D) or peanut oil alone (O) for 10 days. Tumors on mice surviving treatment (toremifene, n = produced a partial inhibition of the estrogen-stimulated growth. 5; control, n = 10) were measured with calipers at weekly intervals. However, toremifene alone caused a more rapid rate of tumor 1760

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. ANTIESTROGENIC ACTIONS OF TOREMIFENE growth than control (Fig. 8). The combination of irradiated DISCUSSION MCF-7 (10,000 rads) cells with viable MDA-MB-231 cells at The growth of MCF-7 tumors in the athymic mouse has 99:1 ratio produced a tumor growth pattern very similar to previously been demonstrated to require estrogen stimulation control. This suggests the control tumor is resulting from (11-13). Our present Findings indicate that maximal MCF-7 predominantly MDA-MB-231 cell growth. Increasing further tumor growth is produced by circulating levels of 17/3-estradiol the ratio of MCF-7:MDA-MB-231 cells (999:1) initially inoc in the athymic mouse comparable to the levels observed in the ulated resulted in tumors which took approximately 10 to 15 days longer to reach a size comparable to tumors inoculated at premenopausal woman (14) and suggest that estrogen levels in a 99:1 cell ratio. This difference was seen whether 17/3-estradiol- the postmenopausal woman (29 to 60 pg/ml of , 10 to 21 pg/ml of 170-estradiol; Ref. 14) are sufficient to cause partial treated or control tumors were compared (compare Figs. 8 and 9). Toremifene (2-cm capsule) produced a marked inhibition of stimulation. The trend of an increased ER content of MCF-7 the 170-estradiol-stimulated portion of the growth of these tumors grown with low circulating levels of 170-estradiol is tumors which started with 999 hormone-dependent cells to consistent with findings in culture where estrogen deprivation every hormone-independent cell. However, even with this large results in a marked rise in the ER content of MCF-7 cells (15, starting ratio of MCF-7:MDA-MB-231 cells, toremifene still 16). stimulated tumor growth above control when administered The present finding that toremifene can inhibit the growth of MCF-7 tumors in vivo is in keeping with the report that alone (see Fig. 9). The combination of irradiated (10,000 rads) MCF-7 cells with viable MDA-MB-231 cells at a 999:1 ratio toremifene is inhibitory to this cell line in culture (8) and is resulted in a long period to tumor development. However, as consistent with the antiestrogenic action reported for this agent in the study with 99:1 ratio of MCF-7:MDA-MB-231 cells, the (8, 17). Inhibition of MCF-7 and other estrogen-dependent breast tumors has been described for tamoxifen (11-13) and growth of tumors from these cells is similar although slightly slower than control. metabolites (18). Tamoxifen has been reported to be ineffective in preventing the growth of hormone-independent tumors such as MDA-MB-231 (13, 18) or T60 (19) in the athymic mouse. 1.4- LÃœ Our findings for toremifene on the MDA-MB-231 tumor are 1.2- consistent with the hypothesis that antiestrogens are ineffective in preventing the growth of hormone-independent tumors. The I .0- lack of antitumor action of massive doses of toremifene in the 0.8 - MDA-MB-231 tumor contrasts with the reported reduced

o 0.6- growth rate produced by similar toremifene treatment of a mouse uterine sarcoma shown to be estrogen receptor negative .~ 04- (8). This difference may, however, be related to the difference W in tumor type or species origin. 5 0.2H As described in the "Introduction," many breast tumors E = 0.0 J contain both hormone-dependent and hormone-independent elements. Our studies on MCF-7:MDA-MB-231 mixed tumors io 20 30 40 50 60 70 demonstrate, when sufficient hormone-dependent cells are pre Days sent, that estrogen can stimulate heterogeneous tumors to grow Fig. 8. The growthrate of MCF-7:MDA-MB-231mixedtumors.Tumor cells (9.9 x 10' MCF-7 cellsand 0.1 x 10' MDA-MB-231cells)were inoculatedinto more rapidly [i.e., inoculated cells in the ratio of 9:1, 99:1, or athymic mice treated with: estradici, 0.5-cm silasticcapsules(•;n= 10)alone; 999:1 MCF-7:MDA-MB-231 were stimulated by 170-estradiol, toremifene,2-cm silasticcapsules(D;n = 10)alone; or estradiol, 0.5-cm silastic capsules,and toremifene,2-cmsilasticcapsules(•;n= 10).Control animals(O; whereas the transplanted initially heterogeneous tumor (ulti n = 7) receivedno treatment. A further group of animals were inoculatedwith mately ER negative) was not]. The inhibitory action of toremi irradiated (10,000 rads) MCF-7 cells mixedwith viableMDA-MB-231cells (A; fene on estrogen-stimulated growth may be analogous to the n = 8). inhibition of endogenous and environmental by anti- estrogens in the postmenopausal breast cancer patient. Since 1.4-1 only partial inhibition of the estrogen-stimulated effect was produced by toremifene, this may be a consequence of incom plete suppression of 170-estradiol-stimulated MCF-7 growth as seen in pure MCF-7 tumors. However, the increased rate of tumor growth produced by toremifene alone over control in heterogeneous tumors is inconsistent with a partial inhibition of estrogen stimulation alone. Tamoxifen has estrogenic actions in the mouse (20) and has been reported to stimulate the growth of the EnCalOl endometrial tumor (11,21) when implanted in this species. Similarly, toremifene has an estrogen-like action on the normal mouse uterus (17), and we have demonstrated a action on the uterus of the athymic mouse uterus. The stimulatory action of toremifene in the heterogeneous Fig. 9. The growthrate of MCF-7:MDA-MB-231mixedtumors.Tumor cells tumor may be related to these effects. Whether this represents (9.99 x 10' MCF-7 cells and 0.01 x 10' MDA-MB-231cells) were inoculated partial stimulation of MCF-7 cells by toremifene, as observed into athymic mice treated with: estradiol, 0.5-cm silastic capsules (•;n= 16) alone; toremifene,2-cm silasticcapsules(D; n = 10) alone; or estradiol, 0.5-cm for tamoxifen in culture (22), or a host-mediated action such as silastic capsules, and toremifene, 2-cm silastic capsules (•;n= 10). Control natural killer cell suppression (23) is unclear. There have been animals (O; «= 16) receivedno treatment. A further group of animals were inoculatedwith irradiated (10,000 rads) MCF-7 cells mixed with viable MDA- some reports that antiestrogens are ineffective in preventing MB-231cells(A;n = 14). growth factor-stimulated T47D (24) and MCF-7 (25, 26) cell 1761

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. ANTIESTROGENIC ACTIONS OF TOREMIFENE growth in culture. If toremifene promotes increased growth T. V., Pallud, C., Spyratos, F., and Milgrom, E. Immunocytochemical study with monoclonal antibodies to progesterone receptor in human breast tumors. factor production in the mammary fat pads of the athymic Cancer Res., 47: 2652-2661, 1987. mouse, then this may influence the growth of both the im 6. Van Netten, J. P., Algard, F. T., Coy, P., Carlyle, S. J., Brigden, M. L., planted hormone-dependent and -independent breast cancer Thornton, K. R., Peter, S., Fraser, T., and To, M. P. Heterogeneous estrogen receptor levels detected via multiple microsamples from individual breast cells. cancers. Cancer (Phila.), 56: 2019-2024, 1985. The principle of inoculating the same number of cells when 7. Valvarra, R., Pyrhönen,S., Heikkinen, M., Rissanen, P., Blanco, G., Thölix, making comparisons of tumor growth is obviously important. E., Nordman, E., Taskinen, P., Holsti, L., and Hajba, A. Toremifene, a new antiestrogenic compound, for treatment of advanced breast cancer. Phase II This is seen in the present study where inoculated MDA-MB- study. Eur. J. Cancer Clin. Oncol., 24:785-790, 1988. 231 cells alone grew into tumors less rapidly than when mixed 8. Rangas. L., Nieminen, A.-L., Blanco, G., Gronroos, M., Kallio, S., Karjalai- nen, A., Perila, M., Sodervall, M., and Toivola, R. A new with viable MCF-7 cells. The mixtures of MDA-MB-231 cells compound, Fc-1157a. II. Antitumor effects. Cancer Chemother. Pharmacol., with irradiated MCF-7 cells formed tumor at a similar rate to 77:109-113, 1986. mixtures with viable MCF-7 tumors. Clearly the MCF-7 cells 9. Knabbe, C., Lippman, M. E., Wakefield, L. M., Flanders, K. C., Kusid, A., act as either a "feeder layer" or buffer the MDA-MB-231 cells Derynck, R., and Dickson, R. B. Evidence that transforming growth factor- la is a hormonallv regulated negative growth factor in human breast cancer against the host immune system. cells. Cell, «.-417-428, 1987. The increased time to tumor development observed when the 10. Lippman, M. E., Dickson, R. B., Bates, S., Knabbe, C., Huff, K., Swain, S., McManaway, M., Bronzert, D., Kusid, A., and Gelmann, E. P. Autocrine ratio of MCF-7:MDA-MB-231 cells was increased (and con and paracrine growth regulation of human breast cancer. Breast Cancer Res. sequently the number of MDA-MB-231 cells in each inocula Treat., 7: 59-70, 1986. tion reduced) is consistent with the MDA-MB-231 tumor cells 11. Gottardis, M. M., Robinson, S. P., Satyaswaroop, P. G., and Jordan, V. C. Contrasting actions of tamoxifen on endometrial and breast tumor growth being the predominant factor in mixed tumor growth. The in the athymic mouse. Cancer Res., 48:812-815, 1988. dominance of the hormone-independent cells was indicated by 12. Shane. S. M., and Granthan, F. II. Role of hormones in the growth and regression of human breast cancer cells (MCF-7) transplanted into athymic the ER differences seen between pure MCF-7 tumors and nude mice. J. Nati. Cancer Inst., 67: 51-56, 1981. heterogeneous tumors when measured at 44 days by ER-EIA. 13. Osborne, C. K., Hobbs, K., and Clark, G. M. Effect of estrogens and antiestrogens on growth of human breast cancer cells in athymic nude mice. Since the completion of these studies, a report using DNA flow Cancer Res., 45: 584-590,1985. cytometry has indicated that the MDA-MB-231 cell is the 14. Vorherr, H. Sex in relation to breast cancer. In: Breast Cancer predominant cell type at 3 wk after inoculation of either a 2:1 Epidemiology, Endocrinology, Biochemistry, and Pathology, Chap. 10, pp. or 10:1 mixture of MCF-7:MDA-MB-231 cells into the athymic 136-200. Baltimore: Urban and Schwarzenberg, 1980. 15. Katzenellenbogen, B. S., Kendra, K. L., Norman, J. M., and Berthois, Y. mouse. MDA-MB-231 cells are not promoting MCF-7 cell Proliferation, hormone responsiveness, and estrogen receptor content of growth through a paracrine mechanism, and MDA-MB-231 MCF-7 human breast cancer cells grown in the short-term absence of estrogen. Cancer Res., 47:4355-4360,1987. cells do not substantially stimulate MCF-7 cell growth (27). 16. Welshons, W. V., and Jordan, V. C. Adaptation of estrogen-dependent MCF- The present study now indicates that the control of hormone- 7 cells to low estrogen (phenol-red-free) culture. Eur. J. Cancer Clin. Oncol., independent elements of heterogeneous tumors by hormone- 25:1935-1939,1987. 17. Kallio, S., Kangas, L., Blanco, G., Johansson, R., Karjalainen, Perila, M., dependent cells is not achieved in this model even with a 999:1 Pippo. I., Sundquist, H., Sodervall, M., and Toivola, T. A new triphenyl ratio of MCF-7:MDA-MB-231 cells, as untreated or antiestro- ethylene compound, Fc-1157a. I. Hormonal effects. Cancer Chemother. Pharmacol., 17:103-108,1986. gen-treated heterogeneous tumors grew at a similar rate or 18. Gottardis, M. M., Robinson, S. P., and Jordan, V. C. Estradiol-stimulated more rapidly than tumors grown from inactivated (by radiation) growth of MCF-7 tumors in athymic mice: a model to study the tumoristatic MCF-7 cells mixed with viable MDA-MB-231 cells. This is in action of tamoxifen. J. . Biochem., 20: 311-314, 1988. 19. Brunner, N., Spang-Thomsen, M., Vindelov, L., Wolff, J., and Engelholm, contrast to findings made in culture at a similar ratio (9) but S. A. Effect of tamoxifen on the receptor-positive T61 and the receptor- consistent with clinical findings that tamoxifen therapy cannot negative T60 human breast carcinoma growth in nude mice. Eur. J. Cancer control the growth of all receptor-positive tumors, and even Clin. Oncol., 21:1349-1354,1985. 20. Terenius, L. Structure-activity relationships of antiestrogens with regard to when this is achieved, the therapy ultimately fails (25). Never interaction with 17/3-estradiol in the mouse uterus and . Acta Endo theless, questions regarding the suitability of the athymic mouse crino!., 66:431-447,1971. 21. Clarke, C. L., and Satyaswaroop, P. G. Photo affinity labeling of the proges to study the therapy of breast cancer must be considered, and terone receptor from human endometrial carcinoma. Cancer Res., 45:5417- the limitations of the model need to be further characterized 5420,1985. and defined. 22. Reddel, R. R., and Sutherland, R. L. Tamoxifen stimulation of human breast cancer cell proliferation in vitro: a possible model for tamoxifen tumour flare. Eur. J. Cancer Clin. Oncol., 20:1419-1424,1984. 23. Screpanti, J., Santoni, A., Gulino, A., Herberman, R. B., and Frati, L. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. Antiestrogenic Action of Toremifene on Hormone-dependent, -independent, and Heterogeneous Breast Tumor Growth in the Athymic Mouse

Simon P. Robinson and V. Craig Jordan

Cancer Res 1989;49:1758-1762.

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