Tumor Types Derived from Epithelial and Myoepithelial Cell Lines of R3230AC Rat Mammary Carcinoma1

(CANCER RESEARCH 52, 1553-1560, March 15, 1992] Tumor Types Derived from Epithelial and Myoepithelial Cell Lines of R3230AC Rat Mammary Carcinoma1

Anna Sapino, Mauro Papotti, Brunella SanfÃ¬lippo,Patrizia Gugliotta, and Gianni Bussola!i Department of Biomedicai Sciences and Human Oncology, University of Turin, Via Santena 7, Turin, 1-10126, Italy

ABSTRACT MATERIALS AND METHODS

Epithelial and myoepithelial cells coexist in the rat R3230AC mam Tumor. R3230AC transplantable mammary tumor was obtained mary tumor. To test the hypothesis that these two cell types constitute from Biomeasure (Hopkinton, MA) and maintained by serial s.c. trans interactive but independent neoplastic populations, we obtained in vitro plantations into the dorsal region of female Fischer 344 inbred rats (100-120 g body weight; Nossan, Correzzana, Milan, Italy). cell lines with epithelial or myoepithelial patterns and transplanted them in syngeneic animals. One stabilized line (EPI) and four cloned lines (A, Tumor fragments were obtained from nonnecrotic areas of the donor rat, mechanically dispersed in a Potter homogenizer and suspended in C, D, E) with epithelial characteristics, confirmed by positive reactions RPMI. Approximately 1 mm3 of tumor (or 1-3 x IO6neoplastic cells) for keratins in immunocytochemical and immunoblot tests, constantly gave rise in vivo to carcinomas, which, however, lacked structural and was used for monthly transplantations. The tumor consistently grew to a 2-em mass within 20 days. Animals were killed by decapitation every functional patterns typical of the original tumor. A fusiform shape and 25-35 days; tumor tissue was in part inoculated in recipient rats as immunocytochemical characteristics of myoepithelial cells were observed described above, in part fixed in absolute ethanol or methacarn for in three clones (H, I, L), which in vivo gave rise to sarcomatous and histology and immunohistochemistry (antibodies listed in Table 1), and mixed carcinosarcomatous neoplasms. These data are consistent with the in part processed for cell culture lines (see below). above hypothesis and indicate that breast carcinomas derive from epithe In order to evaluate the response to estrogen stimulation, tumor- lial cells, while sarcomatous and carcinosarcomatous neoplasms can bearing animals were given for 1 month weekly s.c. injections of originate from myoepithelial cell proliferation. This study provides data estradici valerate (20 mg/kg body weight) according to the method of suggesting myoepithelial cell involvement in the development of patho Hilf (14). logical entities occurring in the human breast and displaying mixed Establishment of Primary Stabilized and Cloned Cultures. The tumor epithelial and stromal neoplastic components, i.e.. cystosarcoma phyl- tissue, removed aseptically, was minced into small pieces (~1 mm3) loides and sarcomatous metaplasia in carcinomas. with scalpels and digested with 125 units/mg collagenase (type II) (Worthington Biochemical Corp., Freehold, NJ) in RPMI (GIBCO- Island Biological Co., Grand Island, NY), 10% PCS3 (GIBCO), 200 INTRODUCTION units/ml penicillin, 200 Mg/ml streptomycin (EUROBIO), and 2.5 mg/ ml Fungizone (GIBCO) for 2 h at 37*C. The R3230AC mammary carcinoma, transplantable in Single cell suspensions and several small tumor fragments were Fischer rats, was originally described by Hilf et al. (1) as a milk- washed twice in RPMI supplemented with 10% FCS without collagen producing adenocarcinoma. Under estrogen stimulation, it dis ase and plated in T25 flasks (Falcon Plastics, Los Angeles, CA) at a plays marked increase of milk protein production and cellular concentration of about 3 x 10s cells/flask. Incubation of the flasks for 24 h at 37'C in a 5% CO2 humidified vacuolation. The biochemical properties and hormone depend ency of this tumor have been investigated by several authors atmosphere allowed most of the fragments and free cells to attach. In (2-12). order to obtain an epithelial cell line (EPI) from the primary culture, elongated cells were removed following trypsin-EDTA treatment (15) We observed that the peculiar functional secretory similarity with a minor modification proposed by Kuzumaki et al. (16). Briefly of this carcinoma to the normal mammary gland is matched by the mixed cell sheet was rinsed once with Ca2+-and Mg2+-free PBS and a parallel similarity in structure; both epithelial and basally incubated for 3 min at 37*C in the presence of fresh trypsin-EDTA. located myoepithelial cells are present, outlining neoplastic The less rapidly detaching cells, mostly islands of epithelium-like cells, pseudoglandular structures and showing the typical immuno were rinsed and supplied with fresh medium. This procedure was cytochemical and ultrastructural characteristics of these cells in repeated every 3 days until epithelial like cells covered more than 90% the normal breast (13). of the culture surface. The R3230AC mammary carcinoma therefore represents an The stabilized EPI cells are presently at the 100th passage. Subcul tures were routinely carried out in RPMI, 10% FCS, and antibiotic. experimental model with unique morphological and functional Cell Cloning. The mixed population obtained from primary cultures properties. We have established and cloned in vitro cell lines (of R3230AC tumor) as well as the stabilized (EPI) epithelial cells were from this tumor and studied their morphology, immunocyto- cloned following the dilution plating technique proposed by Sato et al. chemistry, and function. These cell lines were further investi (17). A single cell suspension was appropriately diluted and 96-well gated by transplanting them back into syngeneic rats, where microtest plates (Falcon) were seeded; each well was inoculated with a they gave rise to carcinomas, sarcomas, and mixed carcinosar- cell suspension yielding an average of 1 cell/well. Those wells contain comas. This experimental model provides data that help to ing only one cell as ascertained by microscope inspection were consid understand the significance and genesis of peculiar and still ered available for clone establishment; the others were discarded. unexplained pathological entities occurring in the human Conditioned media were prepared by adding to the routine medium 10% of the supernatant obtained from confluent cultures of epithelial breast, i.e., cystosarcoma phylloides and sarcomatous meta cells filtered through 0.22-/im Millipore filters and stored at 4*C before plasia in carcinomas. use. We obtained 3 clones (I, H, L) from the mixed population of primary culture and 4 clones (A, C, D, E) from the EPI cell line. Received 7/22/91; accepted 1/8/92. The costs of publication of this article were defrayed in part by the payment The cell lines, at early passages, were reinjected into syngeneic of page charges. This article must therefore be hereby marked advertisement in animals and subcultured until passage 80. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by grants from the CNR (Rome), AIRC (Milan), 3The abbreviations used are: FCS, fetal calf serum; PBS, phosphate-buffered saline; NTEN, 50 miviTris, pH 7.4-0.15 M NaCI-2 mM EDTA-0.1% Nonidet P- and the Ministry of the University. 2To whom requests for reprints should be addressed. 40; sm, smooth muscle. 1553

In Vitro Hormonal Stimulation. Primary cultures, stabilized EPI cells, routinely stained with hematoxylin and eosin and processed for im- and clones D and E were cultured in Petri dishes and on glass coverslips munocytochemical characterization using the antibodies listed in Table for 9 days in a basal medium containing 10 * M 17/3-estradiol (Merck, 1. To detect immunological binding, the avidin-biotinylated peroxidase Darmstadt, Germany) and 10% of charcoal-adsorbed PCS (18). complex procedure (21) was used, after treatment of the sections with The medium was changed every 3 days. the appropriate biotinylated secondary antisemiti (Vector, Burlingame, a-Lactalbumin production was estimated immunocytochemically. CA). Light Microscopy. The morphological appearance of living cultures In Vivo Hormonal Stimulation. Animals bearing palpable tumors in flasks was observed on a Leitz Labovert inverted microscope fitted obtained from EPI, D, E, H, and I cells were treated with estrogen with phase contrast. Mixed cells from primary cultures, EPI cells, and following the protocol used in R3230AC-bearing animals (see above). clones were also grown on glass coverslips; fixed in methanol; and stained by the Papanicolaou method for cytological examination. For immunohistochemical tests, cells were grown on glass coverslips, RESULTS fixed in methanol for 5 min at -20'C, permeabilized in acetone for 5 s at â€”¿20*C,andrÃ©hydratÃ©e!withpig normal serum (diluted 1:5(1in PBS Serially Transplanted R3230AC Tumors. The gross and mi for 20 min). Cells were incubated at 37'C for 60 min with the primary croscopic appearances of the R3230AC mammary tumor, de antibodies listed in Table 1 and then with the appropriate fluorescein- scribed in detail elsewhere (13), were examined at each labeled secondary antisera (Sera-Lab, Ltd., Sussex, England) diluted transplantation. 1:10 in PBS for 30 min at room temperature, following a standard The tumors were mostly well delimited, but focal invasion of indirect immunofluorescence procedure. the adjacent muscular layers was observed in some cases. Dis Electron Microscopy. EPI cells and cells from clone I were fixed in tant mÃ©tastaseswere never recorded in the 52 rats transplanted a solution of 1-2% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.3) in this study. Uniform histological patterns were observed with and 2% aqueous osmium tetroxide for l h at 4*C, dehydrated through acinar and/or gland-like structures lined by polygonal epithelial graded alcohols, cleared in propylene oxide, and embedded in Epon- cells and basal, elongated myoepithelial cells. The original Araldite. Ultrathin sections were counterstained with uranyl acetate tumor stained positively for Â«-sm-aetin in the elongated basal and lead citrate and examined with a Siemens Elmiskop 1 or a Philips cells, thus confirming their myoepithelial nature (Fig. la). EM 401 electron microscope. Epithelial secretory cells were stained with different antibod Cell Extracts. The established cell line EPI and the clones (C, D, H, ies to keratin. AE1, a monoclonal recognizing acidic cytokera- I, L) were homogenized in a buffer 20 mM Tris (pH 7.4), 0. l M NaCl, 5 mM MgCl2, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1 mM tins, was positive only in single squamous cells while AE3, a 2-mercaptoethanol, and 2000 lU/ml Trasylol. Extracts were stored at monoclonal to basic cytokeratins, recognized a few epithelial -80*C until used. Protein concentration was determined by the method cells and the majority of basal myoepithelial cells. Cytokeratin of Lowry as modified by Markwell et al. (19). 8 (M, 52,000-55,000) recognized by 1166 monoclonal was Immunoblotting. Total proteins (100 ng) were separated by sodium found in 70% of epithelial cells (Fig. \b) while cytokeratin 19 dodecyl sulfate polyacrylamide gel electrophoresis on 8% acrylamide (M, 40,000), marked by 1165 monoclonal, was present in less gel slabs and transferred to nitrocellulose filters in a Ployblot apparatus (ABN, Emeryville, CA) according to the manufacturer's instructions. than 10% of epithelial cells. The basal membrane was positively stained by anti-type IV collagen antibodies. Nitrocellulose was then saturated in 3% bovine serum albumin in NTEN for 3 h at 37'C, incubated in the same buffer containing the Primary and Stabilized Cell Cultures and Clones. Primary cell anticytokeratin 19 monoclonal antibody 1165 (Amersham Interna cultures, large flat cells with epithelial appearance, were sur tional, England) diluted '/iooor an anti-a-sm-actin monoclonal antibody rounded by thick bundles of fusiform cells, that tended to diluted 1:800 (Sigma Chemical Co., St. Louis, MO) for 16-18 h at overgrow the epithelial-like elements. 4"C, washed twice for 10 min each in NTEN, incubated with the Cytokeratins were consistently detected in large epithelial- secondary rabbit anti-mouse antibody (Dako) diluted 1:500 for 3 h at looking cells, while only 10% of elongated cells were positive 4"C, washed twice for 10 min each in NTEN, incubated with '"!- for the anti-sm-actin monoclonal. The majority (80%) of the protein A (0.1 /Â¿Ci/ml)(Amersham), and then washed as before, air dried, and exposed for 2 days at â€”¿80Â°CtoHyperfilm MP autoradi- cells were vimentin positive. Collagen type IV was present as small dots on the surface of the fusiform cells. ographic films (Amersham). Stabilized EPI cells formed closely packed islands of large Transplantation of Cell Lines into Animals. Cultured cells (approxi mately 0.8-4 x IO6, suspended in 0.5-0.8 ml RPMI), obtained from cuboidal cells, sometimes surrounded by elongated cells (Fig. early passages (3rd-8th) were injected s.c. into female Fischer rats. The la). Isolated single cells were fusiform with fibroblast-like occurrence and time of growth of tumors were recorded. Tumors appearance (Table 2). All the cells were intensely positive for growing in syngeneic rats from cultured lines were retransplanted into the 1165 and 1166 monocle muÃsagainst cytokeratins. Bundles other rats, following the procedure outlined above. Tissue blocks were of intermediate filaments at the periphery of the cytoplasm and fixed in methacarn (20) and embedded in paraffin. Sections were in a perinuclear arrangement were stained by the AE3 mono-

Table 1 Reagents used in immunohistochemistry and immunofluorescence AntigenCytokeratin cellsEpithelialMyoepithelialEpithelialEpithelialEpithelialMyoepithelialMyoepithelialBasal '1(1undilutedI/ 40,000)Cytokeratin19 (M, EnglandAmershamInternational,

52,000-55,000)Cytokeratinsundiluted1/100-1/101/40-1/11/800-1/1001/350-1/501/300-1/101/400-1/201/5000-1/50"8 (M, EnglandCambridgeInternational, IO-

19-16-15-14-10Cytokeratins (MAb)AE3 UnitedStatesResearch Laboratory, ofAmericaCambridge 8-7-6-5-4-3-2-1..-Smooth (MAb)1 UnitedStatesResearch Laboratory, ofAmericaSigmaHeyl, actinCollagenmuscle A4(MAb)AntiserumV9 IVVimentina-LactalbuminRattype GermanyDakopatts,Berlin, membraneMesenchymalSecretorySecretorymonoclonal (MAb)AntiserumAntiserumSourceAmershamDenmarkDr.Glostrup, DOurVonderhaar. Bethesda, M MFGMReagent1165(MAb)1166(MAb)AE1 laboratoryDilution"1. Data referred to dilutions in immunoperoxidase and immunofluorescence, respectively.Ali,1554Target MFGM, milk fat globule membrane; M antibody.

were moderately detectable with the 1165 monoclonal antibody directed against keratin 19; actin micro lÃ¬lamentswere clearly detectable in some elements (Fig. 2e). Production of type IV collagen was shown in Clone H, I, and L cells. Specific immu- nolocalization was locally revealed inside the cytoplasm and 2f>.patchy ' deposits were detectable in the intercellular spaces (Fig.

The immunocytochemical results were further checked in immunoblots. All clones, as well as the EPI line, when tested by Western blotting for the presence of cytokeratin 19 with the antibody 1165, showed a M, 43,000 band, characteristic of this intermediate filament; only Clone H was always negative for keratin (Fig. 3). Electron Microscopy. The EPI cells showed typical features of epithelial cells with desmosomes and numerous microvilli; bundles of intermediate filaments were present in the cytoplasm of some cells. On the contrary Clone I cells had an elongated shape with large mitochondria and numerous ribosomes; no desmosomes were observed but tight junctions were occasion ally present. Microfilaments were detected, often arranged in bundles close to the cell membrane. Dense bodies along such bundles were observed (Fig. 4). Growth Patterns of Transplanted Clones. EPI cells injected into Fischer rats generated approximately 2 cm tumors within 30 days. Histologically, polygonal or round cells were arranged in gland-like or trabecular structures, delimited by thin stromal bundles (Fig. 5). No actin-rich cells were observed. Necrosis and foci of squamous metaplasia were present. Cytokeratins and rat milk fat globule membrane were abundant in all tumor cells. Type IV collagen and vimentin were not detectable. All seven clones were injected into animals and generated tumors in a time range of 20 days to 7 months (Table 3). Animals were killed when tumors grew up to 2 cm or more (largest diameter) and the tumor material was reinjected into other syngeneic rats for one to three passages. Clone A, D, and E tumors had frank epithelial carcinomatous features with solid nests or gland-like structures haphazardly intermingled with a spindle-shaped cell component. Foci of squamous metaplasia were seen in Clone A and E tumors (Fig. !fÂ» 6). Keratins were found in a variable proportion of carcinoma "â€¢$4tous cells, while vimentin stained the stromal component con %Â«&3K& <â€¢>i.ms* sistently. Few cells reacted with rat milk fat globule membrane &vyK&?r Â«r.->iw?.,â€¢¿antiserum. Type IV collagen was present in limited areas of ":'-b>;,*,v.*. v^..^:. these tumors; cell membranes of single cells were outlined and some neoplastic nests were marked at the basal membrane level. Fig. 1. Original R3230AC rat mammary carcinoma. In a, nests of neoplastic No actin reactivity was observed. cells are lined by a peripheral layer of flattened actin-rich myoepithelial cells. Immunoperoxidase staining with a-smooth muscle actin monoclonal, x 250. In Tumors obtained from Clones H, I, and L had a sarcomatous b, immunostaining for cytokeratin 8 (monoclonal 1166) is positive in the majority appearance with spindle-shaped neoplastic cells mixed with of neoplastic epithelial cells arranged in nests or pseudoglandular structures, x liposarcoma-like areas (Fig. 7). A second generation tumor, ISO. obtained by previous injection of clone L cells, showed keratin- positive adenocarcinomatous foci while in a tumor originated clonal in 80% of the cells. The AE1 monoclonal was positive from Clone H, areas of chondromatous metaplasia were pres in only 40% of the cells. ent. Clone I had a peculiar morphology, strongly resembling Clones A, C, D, and E (obtained from EPI cells) were cystosarcoma phylloides (Fig. 8, a and b). The epithelial com characterized by rounded clusters of cuboidal cells and isolated ponent was strongly positive for cytokeratins and was supported cells with large cytoplasm (Fig. 2, b and r). Immunohistochem- by a highly cellular stromal proliferation reactive to vimentin ically, these clones showed a pattern similar to the EPI cells only. A basal layer of myoepithelial cells lined the ducts, as (Table 2). confirmed by positive actin staining (Fig. 8e). Cells of Clones I, L, and H (all derived from primary cultures Type IV collagen was positive in myoepithelial cells but not of R3230AC tumor) were elongated or star shaped (Fig. 2d). in the basal membrane of the phylloides-like tumor from clone The cells formed an irregular network on the Petri dish. Gen I. Liposarcoma-like tumors were positive for this marker. erally, the cells were proliferating intensely. In Vitro and in Vivo Hormonal Stimulation. Estrogen treat Clone L grew as islands of large star-shaped cells. Keratins ment of control R3230AC tumor induced typical lactational 1555

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Fig. 2. /n vitro cultures of the cell lines obtained from R3230AC rat mammary carcinoma. In a, a stabilized cell line (EPI), observed in phase contrast, forms closely packed islands of elongated and cuboidal cells. In b. Clone D cells, observed in phase contrast, grow as small roundish colonies of cuboidal cells. In <â€¢, immunofluorescence for cytokeratins (monoclonal 1165) in Clone D cells gives a strong positive reaction. In d, cells of Clone H, examined in phase contrast, present a star-like shape. In e, intense reaction for Â«-smoothmuscle actin displayed by cells of clone I.. In/, immunocytochemical detection of type IV collagen in clone H reveals mainly extra- and pericellular deposits, a, e, and d, x ISO; c and e, x 240: / x 480.

Table 2 Morphological and immunochemical features 0/R3230AC mammary carcinoma cell lines

a-sm-actin++ntIV Vim. MFGMnt++++ntntnt+ 1165-6++ EPIClone spindle-shapedRoundishRoundishRoundishRoundishStar-likeSpindle-shapedStar-likeRoundish+ +++nt AClone ++++++ -+ nt CClone j-+ntâ€”-â€”++CytokeratinsAE3++++++ â€”¿â€” ++ DClone +++nt â€”¿nt ++ EClone ++â€” -+++ nt HClone â€”¿- â€”¿+++ +++ IClone -â€” -+++ +++ LPrimary +++ +++++ +++ cultureMorphologyRoundish+ spindle-shapedAE1"+nt+ ++Coll. +++ ++Rat Â°AE1, AE3, 1165, 1166, cytokeratins (see references); Coll. IV, type IV collagen; Vim., \ Â¡mentiti;Â«-sm-actin,Â«-smoothmuscle actin monoclonal; rat MFGM, serum against rat milk fat globule membrane antigens; EPI, stabilized cell line; nt, not tested. changes and a-lactalbumin production, demonstrated by im- thelial cells are present (13). The neoplastic myoepithelium munocytochemistry and immunoblotting. In contrast, estrogen shows: (a) typical peripheral arrangement against the basement stimulation did not affect the morphology of any of the cultured membrane; (b) typical ultrastructural cytoplasmic organization; cell lines, and production of a-lactalbumin was never detectable and (c) the presence of immunocytochemically detectable a- by immunocytochemical procedures. smooth muscle actin. This isoform of actin is present, within In vivo estrogen stimulation, started as soon as a nodule was the epithelial domain, only in myoepithelial cells of the breast palpable, did not induce milk protein production or change the and salivary glands (22, 23). histolÃ³gica! pattern. Similar biphasic patterns involving both myoepithelial and epithelial neoplastic components have been described in di- DISCUSSION methylbenz[a]anthracene-induced mammary tumors in mice We recently observed that the architectural organization of (24). Two alternative hypotheses can be advanced to explain the R3230AC tumor parallels that of the lactating gland since, the observed phenomena: myoepithelial and epithelial cell com in addition to the luminal epithelial (secretory) cells, myoepi- ponents represent differentiated terminal stages stemming from 1556

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43 Kd -

Fig. 3. Sodium dodecyl sulfate-gel electrophoresis of protein extract of the clone cell lines obtained from R3230AC rat mammary adenocarcinoma. Total extracts, run on 12% polyacrylamide gel, were then transferred on nitrocellulose paper and reacted with anti-cytokeratin 1165 monoclonal antibody as described in "Materials and Methods." A strong positive reaction is observed at M, 43,000 (43 Kd) in Clones C and D and EPI, while Clones I and L give a weak positivity and Clone H is negative. a common uncommitted precursor; or they constitute interac tive but independent neoplastic populations. To test these hy potheses, we obtained separate clones of the epithelial and the myoepithelial cells. Primary and stabilized cell populations as well as seven clones were derived from the R3230AC carcinoma; they presented either epithelial or myoepithelial-like features. Transplantation s.c. gave rise to carcinomas, to sarcomas, or to mixed (carcinosarcomatous) neoplasms (Table 3). Epithelial cell lines (EPI, A, D, E) generated in vivo carci A nomas lacking both the myoepithelial component and the hor â€¢¿1 mone responsiveness of the parent tumor; Â«-lactalbumin pro duction was not stimulated by estrogen administration. A pos sible interpretation of these findings is that the complex structural organization of the primary tumor is necessary to complete functional differentiation. Moreover, a number of investigators have shown that the matrix on which the mam mary epithelial cells attach in vitro modulates their response to hormones (25-29). Myoepithelial-like cells (lines H, I, and L) were elongated; all cells were producing collagen type IV, which is selectively produced in the mammary gland by myoepithelial cells (30- 32). In addition, tight junctions and subplasmalemmal bundles of microfilaments with interposed dense bodies (characteristic features of myoepithelial cells) (33) were observed in Clone I and a-sm-actin was detected in Clone L. The presence of this isoform of actin in cultured cells is not per se proof of this myoepithelial nature, since it was recently demonstrated in mammary stromal cells evolving into myofibroblasts (34). How ever, in line with the myoepithelial nature of the cloned cells, we detected cytokeratin of basal type by both immunofluorescence and immunoblotting procedures in Clone L and by immunoblotting alone in Clone I cells. All cell lines contained vimentin, but in cultured cells this latter finding is not proof of nonepithelial nature (35) and coexpression of both keratin and vimentin intermediate filaments has been reported in numerous epithelial tumors (36). B Tumors generated from cell lines with myoepithelial features showed mainly sarcomatous patterns. Vimentin and collagen Fig. 4. Electron microscopic study of Clone I. In 1. the cells show elongated type IV were demonstrated in these tumors; the latter finding shape and lack desmosomes; the nuclei are irregular in outline. At higher mag nification (B) a selected area (arrows. A) shows subplasmalemmal bundles of seems at variance with the behavior of human liposarcomas microfilaments (arrowheads), with interposed dense bodies. A, x 2,000; B, x which do not produce collagen type IV (37). Focal keratin 16,000. positivity and chondroid metaplasia were found in single tu mors; of major interest was, however, the peculiar association 1557

Table 3 Morphological and immunochemical features of in vivo growth ofR3230AC cell lines Cytokeratins .t '.'"'111 VI MU ('l'Ut. Il 1 ITIAEl V^VHli Ã• T ' lili. U-3Ill-a\.lIII IVtll ITI! VI 1165-6EPIClone AE3 -Carcinomatous + ++ +++ - - AClone â€”¿â€¢Carcinomatous ++ ++ ++ nt â€”¿ CClone DClone -Carcinomatous (adenocarcinoma) + ++ +++ + - EClone â€”¿Sarcomatous(anaplastic) â€”¿ ++ + â€”¿ â€”¿ HClone -metaplasia)Sarcomatous(lipoblasts, with chondromatous - - - ++ +++

IClone ++Sarcomatous(area phylloides-like) â€”¿ â€”¿ ++ ++ +++ LCarcinomatous â€”¿areas)+Â±++ntntnt(lipoblasts, with Carcinomatous â€”¿ â€”¿ ++ +++ +++

Original R3230AC carcinoma " Coll. IV, type IV collagen; AEl, AE3, 1165, 1166, cytokeratins (see references); Vim., vimentin; a-sm-actin, Â«smooth muscle actin monoclonal; Rat MFGM, serum against rat milk fat globule membrane antigens; EPI, stabilized cell line; *. not grown after injection; nt, not tested.

and provides experimental evidence of derivation of sarcoma tous and mixed carcinosarcomatous tumors of the breast from >â€¢ neoplastic myoepithelial-like cells. The literature provides examples of fibrosarcomas, rhabdo- ..â€¢.. v myosarcomas, and other types of sarcoma originating in ani .. A---'- * mals transplanted with fragments of spontaneously developed mouse mammary tumors and with long term cultures of mouse mammary cancer cells (16, 40, 41). Peculiarly, mammary tu % mors seem to be the only ones for which this phenomenon (of â€ž¿-â€¢ a sarcoma originating by serial transplantation or by culture of an epithelial tumor) has been described. This peculiarity of experimental mammary tumors has some relationship with â€”¿. vi,-:' certain well known features described in human breast neo ^ plasms. Areas of sarcomatous (osteosarcoma, chondrosarcoma) metaplasia are known to occur in carcinomas of the human breast (42); a similar phenomenon is seen rather commonly in Fig. 5. Tumor derived from EPI cells. Pseudoglandular structures and laminae of polygonal epithelial cells are arranged in a cellular stroma. H&E, x 150. malignant mammary tumors of female canines, in which ohmi ciroid elements are regarded as being of myoepithelial derivation (43). Our studies support the hypothesis that these sarcomatous areas might be related to proliferation of neoplastic myoepithe lial cells. . JK Previously, other authors (44, 45) tried to establish cultures * -*Â»-.* .-. of myoepithelial cells from dimethylbenz[a]anthracene-induced rat mammary tumors and from the mammary gland of a neo natal rat. The line called RAMA 25 showed a peculiar sponta neous transformation in vitro from cuboidal to elongated fusi !'^v * t form cells which have been regarded as the equivalent of the

Fig. 6. Carcinoma derived from Clone E. Solid nests and pseudoglandular structures are separated by thin stromal bundles. Squamous metaplasia can be focally observed, x 150. in Clone I tumors of liposarcomatous areas with features of cystosarcoma phylloides, a lesion with mixed epithelial and stromal growth regarded as the malignant counterpart of the more common fibroadenoma of the breast. Liposarcomatous differentiation is rather common in human malignant phyl loides tumors (38). Ultrastructural and immunocytochemical evidence of myoepithelial derivation of the stromal sarcomatous Fig. 7. Tumor with liposarcomatous patterns derived from Clone L infÃltrales muscle layer of the dorsal region of a Fischer rat. Spindle-shaped cells with component in cases of human cystosarcoma phylloides has been hyperchromatic nuclei are intermingled with polygonal vacuolated cells resem reported in the literature (39). Our study confirms these data bling lipoblasts. x 150. 1558

Fig. 8. Tumor derived from Clone I. At low magnification (a), the typical architecture of cytosarcoma phylloides is evident. Leaf-like projections of sarcomatous stromu are lined by epithelium. At high power (b) gland-like structures are lined by a double layer of epithelial and myoepithelial cells. Spindle-shaped cells with hyperchromatic and irregular nuclei can be observed in the strenna. In c, immunostaining for a-smoolh muscle actin reveals a regular layer of actin-rich myoepithelial cells basally located in gland-like structures. No staining is detectable in the spindle-shaped sarcomatous cells, a, x 70; b and c,x 150. normal myoepithelial cells (32, 46, 47). Formation of fusiform rat. Cancer Res., 25: 286-299, 1965. Hilf, R., Michel, I., Bell, C., and Carrington, M. J. Influence of endocrine cells has also been described in cell lines derived from BALB/ organ ablation on the growth and biochemical responses of the R3230AC CDH mouse mammary tumors (48) and from nitrosomethylu- mammary tumor to hormonal treatment. Cancer Res., 26:136S-1370, 1966. rea-induced mammary tumors in WF rats (49). According to Gardner, D. G., and Wittliff, J. L. Demonstration of a glucocorticoid hor mone-receptor complex in the cytoplasm of a hormone-responsive tumor. the observations of Dulbecco et al. (49) and Warburton et al. Br. J. Cancer, 27:441-444, 1973. (32) these fÃbroblast-like cells did not exactly correspond to Costlow, M. E., Buschow, R. A., and McGuire, W. L. Prolactin receptors in an estrogen receptor-deficient mammary carcinoma. Science (Washington myoepithelial cells but showed immunocytochemical features DC), 184: 85-86, 1974. common to both stromal cells (presence of vimentin and of ShaFie, S. M., Gibson, S. L., and Hilf, R. Effect of insulin and estrogen on Thy-1-1 antigen) and myoepithelial cells (production of type hormone binding in the R3230AC mammary adenocarcinoma. Cancer Res., IV collagen). The biphasic behavior of myoepithelial cells in 37; 4641-4649, 1977. Smith, R. D., Hilf, R., and Senior, A. E. Prolactin binding to R3230AC skin and salivary gland tumors, where these cells seem to be mammary carcinoma and liver in hormone-treated and diabetic rats. Cancer able to form a stromal matrix, has been described and discussed Res., 3 7: 595-598, 1977. Aylsworth, C. F., Hodson, C. A., Berg, G., Kledzik, G., and Meites, J. Role by several authors (50, 51). The nature of the myoepithelial cell of adrenals and estrogen in regression of mammary tumors during postpar- itself is indefinite: while its content of basic-type keratins sug tum lactation in the rat. Cancer Res., 39: 2436-2439, 1979. gests an epithelial nature, the presence of vimentin (52) and its Hissin, P. J., and Hilf, R. Effects of estrogen to alter amino acid transport contractile ability, testified to by the presence of bundles of Â«- in R3230AC mammary carcinomas and its relationship to insulin action. Cancer Res., 39: 3381-3387, 1979. smooth muscle actin (a unique feature among epithelial cells), Orloski, J. M., Fritz, P. J., and Liu, D. K. Pregnancy stimulates DNA indicate its affinity to mesenchyme-derived cells. Even the synthesis in R3230AC mammary adenocarcinoma. Eur. J. Cancer Clin. Oncol., /*: 99-106, 1982. assumption that myoepithelial cells are terminally differen Stein, T. P. Tumor induced changes in the host's protein metabolism. In: M. 10. tiated cells (53, 54) seems untenable, since we observed (55) S. Arnott, J. Van Eyes, and Y. M. Want (eds.). Interrelations of Nutrition that in the developing mouse mammary gland myoepithelial and Cancer, pp. 137-150. New York: Raven Press, 1982. 11. Klinge, C. M., Bambara, R. A., Zain, S., and Hilf, R. Effects of host hormonal cells can proliferate. status on binding of activated estrogen receptor to nuclei from R3230AC All these data, in line with the reported experimental obser and 7,12-Dimethylbenz[a]anthracene-induced mammary tumors. Cancer vation in R3230AC rat tumor and related cell lines, support Res.,Â«: 1165-1170, 1989. the hypothesis that mixed epithelial-stromal tumors of the 12. Feldman, J. M., Cave, W. T., Jr., and Hilf, R. Relationship of dietary lipid to mammary tumor growth and insulin binding. Proc. Am. Assoc. Cancer mammary gland can originate from the myoepithelial cells. Res., 25:211, 1984. 13. Papotti, M., Coda, R., Ottinetti, A., and Bussolati, G. Dual secretory and myoepithelial differentiation in the transplantable R3230AC rat mammary ACKNOWLEDGMENTS carcinoma. Virchows Arch. Abt. B Zeli Pathol., 55: 39-45, 1988. 14. Hilf, R. Milk-like fluid in a mammary adenocarcinoma biochemical charac We thank Professor P. M. Cullino (University of Turin, Turin, Italy) terization. Science (Washington DC), 155:826-827, 1967. and Professor L. Ozzello (Columbia University, New York, NY) for IS, Owens, R. B., and Hackett, A. J. Tissue culture studies of mouse mammary criticisms. tumor cells and associated viruses. J. Nati. Cancer Inst., 48: 1321-1332, 1972. 16 Kuzumaki, N., More, 1. A. R., Cochran, J., and Klein, G. Thirteen new REFERENCES mammary tumor cell lines from different mouse strains. Eur. J. Cancer, 16: 1181-1192, 1980. 1. Hilf, R., Michel, I., Bell, C., Freeman, J. J.. and Borman, A. Biochemical 17, Sato, H., Azoma, M., Hayashi, Y., Yoshida, H., Yanaga, W. A. T., and Yura, and morphologic properties of a new lactating mammary tumor line in the Y. 5-Azacytidine induction of stable myoepithelial and acinar cells from a 1559

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1992 American Association for Cancer Research. MAMMARY CARCINOMA CELL LINES human salivary intercalated duct cell clone. Cancer Res., 47: 4453-4459, and vimentin. Appi. Pathol., 7: 73-84, 1989. 1987. 37. Ogawa, K., Oguchi, M., Yamabe, H., Nakashima, \.. and Hamashima, Y. 18. Sapino, A., Pietribiasi, F., Bussolati, G., and Marchisio, P. C. Estrogen- and Distribution of collagen type IV in soft tissue tumors. An immunohistochem- tamoxÂ¡fun-induced rearrangement of cytoskeletal structures in breast cancer ical study. Cancer (Phila.), 5Â«:269-277, 1986. MCF-7 cells. Cancer Res., 46: 2526-2531,1986. 38. Qizilbash, A. H. Cystosarcoma phyllodes with liposarcomatous stroma. Am. 19. Markwell, M. A. K., Haas, S. M., Bieber, L. C, and Tolbert, N. E. A J. Clin. Pathol., 65: 321-327, 1976. modification of Lowry procedure to simplify protein determination in mem 39. Mechtersheiner, G., KrÃ¼ger,K. H., Born, I. A., and MÃ¶ller,P. Antigenic brane and lipoprotein samples. Anal. Biochem., 87: 206-210, 1978. profile of mammary fibroadenoma and cystosarcoma phillodes. A study using 20. Mitchell, D., Ibrahim, S., and Gusterson, B. A. Improved immunohistochem- antibodies to estrogen and progesterone receptors and to a panel of cell ical localization of tissue antigens using modified methacarn fixation. J. surface molecules. Pathol. Res. Pract., 186:427-438, 1990. Histochem. Cytochem., 33:491-495, 1985. 40. Sanford, K. K., Dunn, T. B., Westfall, B. B., Covalesky, A. B., Dupree, L. 21. Hsu, S. M., Raine, L., and Fanger, H. Use of avidin-biotin-peroxidase T., and luirle. W. R. Sarco matous change and maintenance of differentiation complex (ABC) technique. A comparison between ABC and unlabeled anti in long term cultures of mouse mammary carcinoma. J. Nati. Cancer Inst., body (PAP) procedures. J. Histochem. Cytochem., 29: 577-580, 1981. 26:1139-1159, 1961. 22. Skalli, O., Ropraz, P., Trzeciak, A., Benzonana, G., Gillessen, D., and 41. Fasske, E., Fetting, R., Morgenroth, K., Jr., and Themann, H. The sarcom- Gabbiani, G. A monoclonal antibody against Â»-smoothmuscle actin. A new atous transformation of mammary carcinoma of mice in isologous transplan probe for smooth muscle differentiation. J. Cell Biol., 103:2787-2796,1986. tÃ¢tes,in cell cultures, and rÃ©implantÃ¢tes.Oncology(Basel), 21: 189-213, 23. Gugliotta, P., Sapino, A., Mucri, L., Skalli, O., Gabbiani, G., and Bussolati, 1967. G. Specific demonstration of myoepithelial cells by anti-a smooth muscle 42. Kahn, L. B., Uys, C. J., Dale, J., and Rutherfoord, F. Carcinoma of the actin antibody. J. Histochem. Cytochem., 36:659-663, 1988. breast with metaplasia to chondrosarcoma: a light and electron microscopic 24. Rehm, S. Chemically induced mammary gland adenomyoepitheliomas and study. Histopathology, Z-93-106, 1978. myoepithelial carcinomas of mice. Immunohistochemical and ultrastructural 43. von Bombard, D., and von Sandersleben, J. Ãœberdie Feinstruktur con features. Am. J. Pathol., 136: 575-584, 1990. Mammamischtumoren der HÃ¼ndin.II. Das Vorkommen von Myoepithelzel- 25. Kratochw ill, K. Organ specificity in mesenchymal induction demonstrated in len in chondroiden Arealen. Virchows Arch. Abt. A Pathol. Anat. Histol., embryonic development of the mammary gland in the mouse. Dev. Biol., 20: 362:157-167, 1974. 46-71, 1969. 44. Warburton, J., Ormerod, E. J., Monaghan, P., Ferns, S., and Rudland, P. S. 26. Emmerman, J. T., Enami, J., Pitelka, D. R., and Nandi, S. Hormonal effects Characterization of a myoepithelial cell line derived from a neonatal rat on intracellular and secreted casein in cultures of mouse mammary epithelial mammary gland. J. Cell. Biol., 91: 827-836, 1981. cells in floating collagen membranes. Proc. Nati. Acad. Sci. USA, 74:4466- 45. Dunnington, D. J., Hughes, C. M., Monaghan, P., and Rudland, P. S. 4470, 1977. Phenotypic instability of rat mammary tumor epithelial cells. J. Nati. Cancer 27. Ormerod, J. E., and Rudland, P. S. Mammary gland morphogenesis in vitro: Inst., 71:1227-1240,1983. formation of branched tubules in collagen gels by a cloned rat mammary cell 46. Bennett, D. C., Peachey, L. A., Durbin, H., and Rudland, P. S. A possible line. Dev. Biol., 91: 360-375, 1982. mammary stem cell line. Cell, 15: 283-298, 1978. 28. Lee, E. Y-H., Parry, G., and Bissel, M. J. Modulation of secreted proteins 47. Rudland, P. S., Paterson, F. C., Monaghan, P., Twiston-Davies, A. C., and Warburton, M. J. Isolation and properties of rat cell lines morphologically of mouse mammary epithelial cells by the collagenous substrata. J. Cell. intermediate between cultured mammary epithelial and myoepithelial-like Biol., 98:146-155, 1984. 29. Stockdale, F. E., Wiens, D. J., and Levine, J. F. Cell-cell interactions in the cells. Dev. Biol., 113: 388-405, 1986. 48. Hager, J. C., Fligiel, S., Stanley, W., Richardson, A. M., and Heppner, G. mediation of hormone dependent differentiation of mammary epithelium. H. Characterization of a variant-producing tumor cell line from a heteroge In: Progress in Developmental Biology, Part B, pp. 417-424. New York: neous strain BALB/cfC3H mouse mammary tumor. Cancer Res., 41:1293- Alan R. Liss, Inc., 1986. 1300, 1981. 30. Warburton, M. J., Ferns, S. A., and Rudland, P. S. Enhanced synthesis of 49. Dulbecco, R., Henahan, M., Bowman, M., Okada, S., Battifora, H., and basement membrane proteins during the differentiation of rat mammary Unger, M. Generation of fibroblast-like cells in vitro: a possible new cell tumor epithelial cells into myoepithelial-like cells in vitro. Exp. Cell Res., type. Proc. Nati. Acad. Sci. USA, 78: 2345-2349, 1981. 137: 373-380, 1982. 50. Azzopardi, J. G., and Smith, O. D. Salivary gland tumours and their mucins. 31. Warburton, M. J., Mitchell, D., Ormerod, E. J., and Rudland, P. S. Distri J. Pathol. Bacterio!., 77:131-140, 1959. bution of myoepithelial and basement membrane proteins in the resting, 51. Varela-Duran, J., Diaz-Flores, L., and Varela-Nunez, R. Ultrastructure of pregnant, lactating and involuting rat mammary gland. J. Histochem. Cyto chondroid syringoma. Role of the myoepithelial cell in the development of chem., 30: 667-676, 1982. the mixed tumor of the skin, and soft tissues. Cancer (Phila.), 44: 148-156, 32. Warburton, M. J., Ferns, S. A., Hughes, C. M., Sear, C. H. J., and Rudland, 1979. P. S. Generation of cell types with myoepithelial and mesenchymal pheno- 52. Warburton, M. J., Hughes, C. M., Ferns, S. A., and Rudland, P. S. Localiza types during the conversion of rat mammary tumor epithelial stem cells into tion of vimentin in myoepithelial cells of the rat mammary gland. Histochem. elongated cells. J. Nati. Cancer Inst., 78: 1191-1201, 1987. J.,2/: 679-685, 1989. 33. Ozzello, L. Ultrastructure of the human mammary gland. Pathol. Annu., 6: 53. Ferguson, D. J. P. Ultrastructural characterization of the proliferative (stem?) 1-59, 1951. cells within the parenchyma of the normal "resting" breast. Virchows Arch. 34. Rennov-Jessen, L., van Deurs, B., Cells, J. E., and Petersen, O. W. Smooth Abt. A Pathol. Anat., 407: 379-385, 1985. muscle differentiation in cultured human breast gland stromal cells. Lab. 54. Joshi, K., Smith, J. A., Perusinghe, N., and Monoghan, P. Cell proliferation Invest., 63: 532-543, 1990. if the human mammary epithelium. Differential contribution by epithelial 35. Lazarides, E. Intermediate filaments: a chemically heterogeneous, develop- and myoepithelial cells. Am. J. Pathol., 124: 199-206, 1986. mentally regulated class of proteins. Annu. Rev. Biochem., 51: 219-250, 55. Sapino, A., Macri, L., Gugliotta, P., and Bussolati, G. Immunohistochemical 1982. identification of proliferating cell types in mouse mammary gland. J. Histo 36. McGuire, L. J., Ng, J. P. V., and Lee, J. C. K. Coexpression of cytokeratin chem. Cytochem., 38: 1541-1547, 1990.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1992 American Association for Cancer Research. Tumor Types Derived from Epithelial and Myoepithelial Cell Lines of R3230AC Rat Mammary Carcinoma

Anna Sapino, Mauro Papotti, Brunella Sanfilippo, et al.

Cancer Res 1992;52:1553-1560.

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