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(CANCER RESEARCH 52, 1553-1560, March 15, 1992]
Tumor Types Derived from Epithelial and Myoepithelial
Rat Mammary Carcinoma1
Cell Lines of R3230AC
Anna Sapino, Mauro Papotti, Brunella Sanfìlippo,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
mary tumor. To test the hypothesis that these two cell types constitute
interactive but independent neoplastic populations, we obtained in vitro
cell lines with epithelial or myoepithelial patterns and transplanted them
in syngeneic animals. One stabilized line (EPI) and four cloned lines (A,
C, D, E) with epithelial characteristics, confirmed by positive reactions
for keratins in immunocytochemical and immunoblot tests, constantly
gave rise in vivo to carcinomas, which, however, lacked structural and
functional patterns typical of the original tumor. A fusiform shape and
immunocytochemical characteristics of myoepithelial cells were observed
in three clones (H, I, L), which in vivo gave rise to sarcomatous and
mixed carcinosarcomatous neoplasms. These data are consistent with the
above hypothesis and indicate that breast carcinomas derive from epithe
lial cells, while sarcomatous and carcinosarcomatous neoplasms can
originate from myoepithelial cell proliferation. This study provides data
suggesting myoepithelial cell involvement in the development of patho
logical entities occurring in the human breast and displaying mixed
epithelial and stromal neoplastic components, i.e.. cystosarcoma phylloides and sarcomatous metaplasia in carcinomas.
Tumor. R3230AC transplantable mammary tumor was obtained
from Biomeasure (Hopkinton, MA) and maintained by serial s.c. trans
plantations into the dorsal region of female Fischer 344 inbred rats
(100-120 g body weight; Nossan, Correzzana, Milan, Italy).
Tumor fragments were obtained from nonnecrotic areas of the donor
rat, mechanically dispersed in a Potter homogenizer and suspended in
RPMI. Approximately 1 mm3 of tumor (or 1-3 x IO6neoplastic cells)
INTRODUCTION
was used for monthly transplantations. The tumor consistently grew to
a 2-em mass within 20 days. Animals were killed by decapitation every
25-35 days; tumor tissue was in part inoculated in recipient rats as
described above, in part fixed in absolute ethanol or methacarn for
histology and immunohistochemistry (antibodies listed in Table 1), and
in part processed for cell culture lines (see below).
In order to evaluate the response to estrogen stimulation, tumorbearing animals were given for 1 month weekly s.c. injections of
estradici valerate (20 mg/kg body weight) according to the method of
Hilf (14).
Establishment of Primary Stabilized and Cloned Cultures. The tumor
tissue, removed aseptically, was minced into small pieces (~1 mm3)
with scalpels and digested with 125 units/mg collagenase (type II)
(Worthington Biochemical Corp., Freehold, NJ) in RPMI (GIBCOIsland Biological Co., Grand Island, NY), 10% PCS3 (GIBCO), 200
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
Fischer rats, was originally described by Hilf et al. (1) as a milkproducing adenocarcinoma. Under estrogen stimulation, it dis
plays marked increase of milk protein production and cellular
vacuolation. The biochemical properties and hormone depend
ency of this tumor have been investigated by several authors
(2-12).
We observed that the peculiar functional secretory similarity
of this carcinoma to the normal mammary gland is matched by
a parallel similarity in structure; both epithelial and basally
located myoepithelial cells are present, outlining neoplastic
pseudoglandular structures and showing the typical immuno
cytochemical and ultrastructural characteristics of these cells in
the normal breast (13).
The R3230AC mammary carcinoma therefore represents an
experimental model with unique morphological and functional
properties. We have established and cloned in vitro cell lines
from this tumor and studied their morphology, immunocytochemistry, and function. These cell lines were further investi
gated by transplanting them back into syngeneic rats, where
they gave rise to carcinomas, sarcomas, and mixed carcinosarcomas. This experimental model provides data that help to
understand the significance and genesis of peculiar and still
unexplained pathological entities occurring in the human
breast, i.e., cystosarcoma phylloides and sarcomatous meta
plasia in carcinomas.
Received 7/22/91; accepted 1/8/92.
The costs of publication of this article were defrayed in part by the payment
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.
1This work was supported by grants from the CNR (Rome), AIRC (Milan),
and the Ministry of the University.
2To whom requests for reprints should be addressed.
Single cell suspensions and several small tumor fragments were
washed twice in RPMI supplemented with 10% FCS without collagen
ase and plated in T25 flasks (Falcon Plastics, Los Angeles, CA) at a
concentration of about 3 x 10s cells/flask.
Incubation of the flasks for 24 h at 37'C in a 5% CO2 humidified
atmosphere allowed most of the fragments and free cells to attach. In
order to obtain an epithelial cell line (EPI) from the primary culture,
elongated cells were removed following trypsin-EDTA treatment (15)
with a minor modification proposed by Kuzumaki et al. (16). Briefly
the mixed cell sheet was rinsed once with Ca2+-and Mg2+-free PBS and
incubated for 3 min at 37*C in the presence of fresh trypsin-EDTA.
The less rapidly detaching cells, mostly islands of epithelium-like cells,
were rinsed and supplied with fresh medium. This procedure was
repeated every 3 days until epithelial like cells covered more than 90%
of the culture surface.
The stabilized EPI cells are presently at the 100th passage. Subcul
tures were routinely carried out in RPMI, 10% FCS, and antibiotic.
Cell Cloning. The mixed population obtained from primary cultures
(of R3230AC tumor) as well as the stabilized (EPI) epithelial cells were
cloned following the dilution plating technique proposed by Sato et al.
(17). A single cell suspension was appropriately diluted and 96-well
microtest plates (Falcon) were seeded; each well was inoculated with a
cell suspension yielding an average of 1 cell/well. Those wells contain
ing only one cell as ascertained by microscope inspection were consid
ered available for clone establishment; the others were discarded.
Conditioned media were prepared by adding to the routine medium
10% of the supernatant obtained from confluent cultures of epithelial
cells filtered through 0.22-/im Millipore filters and stored at 4*C before
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.
The cell lines, at early passages, were reinjected into syngeneic
animals and subcultured until passage 80.
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 P40; sm, smooth muscle.
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MAMMARY CARCINOMA CELL LINES
In Vitro Hormonal Stimulation. Primary cultures, stabilized EPI cells,
and clones D and E were cultured in Petri dishes and on glass coverslips
for 9 days in a basal medium containing 10 * M 17/3-estradiol (Merck,
Darmstadt, Germany) and 10% of charcoal-adsorbed PCS (18).
The medium was changed every 3 days.
a-Lactalbumin production was estimated immunocytochemically.
Light Microscopy. The morphological appearance of living cultures
in flasks was observed on a Leitz Labovert inverted microscope fitted
with phase contrast. Mixed cells from primary cultures, EPI cells, and
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,
fixed in methanol for 5 min at -20'C, permeabilized in acetone for 5 s
at —¿20*C,
and réhydratée!
with pig normal serum (diluted 1:5(1in PBS
for 20 min). Cells were incubated at 37'C for 60 min with the primary
antibodies listed in Table 1 and then with the appropriate fluoresceinlabeled secondary antisera (Sera-Lab, Ltd., Sussex, England) diluted
1:10 in PBS for 30 min at room temperature, following a standard
indirect immunofluorescence procedure.
Electron Microscopy. EPI cells and cells from clone I were fixed in
a solution of 1-2% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.3)
and 2% aqueous osmium tetroxide for l h at 4*C, dehydrated through
graded alcohols, cleared in propylene oxide, and embedded in EponAraldite. Ultrathin sections were counterstained with uranyl acetate
and lead citrate and examined with a Siemens Elmiskop 1 or a Philips
EM 401 electron microscope.
Cell Extracts. The established cell line EPI and the clones (C, D, H,
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
2-mercaptoethanol, and 2000 lU/ml Trasylol. Extracts were stored at
-80*C until used. Protein concentration was determined by the method
of Lowry as modified by Markwell et al. (19).
Immunoblotting. Total proteins (100 ng) were separated by sodium
dodecyl sulfate polyacrylamide gel electrophoresis on 8% acrylamide
gel slabs and transferred to nitrocellulose filters in a Ployblot apparatus
(ABN, Emeryville, CA) according to the manufacturer's instructions.
Nitrocellulose was then saturated in 3% bovine serum albumin in
NTEN for 3 h at 37'C, incubated in the same buffer containing the
anticytokeratin 19 monoclonal antibody 1165 (Amersham Interna
tional, England) diluted '/iooor an anti-a-sm-actin monoclonal antibody
diluted 1:800 (Sigma Chemical Co., St. Louis, MO) for 16-18 h at
4"C, washed twice for 10 min each in NTEN, incubated with the
secondary rabbit anti-mouse antibody (Dako) diluted 1:500 for 3 h at
4"C, washed twice for 10 min each in NTEN, incubated with '"!protein A (0.1 /¿Ci/ml)(Amersham), and then washed as before, air
dried, and exposed for 2 days at —¿80°C
to Hyperfilm MP autoradiographic films (Amersham).
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
early passages (3rd-8th) were injected s.c. into female Fischer rats. The
occurrence and time of growth of tumors were recorded. Tumors
growing in syngeneic rats from cultured lines were retransplanted into
other rats, following the procedure outlined above. Tissue blocks were
fixed in methacarn (20) and embedded in paraffin. Sections were
routinely stained with hematoxylin and eosin and processed for immunocytochemical characterization using the antibodies listed in Table
1. To detect immunological binding, the avidin-biotinylated peroxidase
complex procedure (21) was used, after treatment of the sections with
the appropriate biotinylated secondary antisemiti (Vector, Burlingame,
CA).
In Vivo Hormonal Stimulation. Animals bearing palpable tumors
obtained from EPI, D, E, H, and I cells were treated with estrogen
following the protocol used in R3230AC-bearing animals (see above).
RESULTS
Serially Transplanted R3230AC Tumors. The gross and mi
croscopic appearances of the R3230AC mammary tumor, de
scribed in detail elsewhere (13), were examined at each
transplantation.
The tumors were mostly well delimited, but focal invasion of
the adjacent muscular layers was observed in some cases. Dis
tant métastaseswere never recorded in the 52 rats transplanted
in this study. Uniform histological patterns were observed with
acinar and/or gland-like structures lined by polygonal epithelial
cells and basal, elongated myoepithelial cells. The original
tumor stained positively for «-sm-aetin in the elongated basal
cells, thus confirming their myoepithelial nature (Fig. la).
Epithelial secretory cells were stained with different antibod
ies to keratin. AE1, a monoclonal recognizing acidic cytokeratins, was positive only in single squamous cells while AE3, a
monoclonal to basic cytokeratins, recognized a few epithelial
cells and the majority of basal myoepithelial cells. Cytokeratin
8 (M, 52,000-55,000) recognized by 1166 monoclonal was
found in 70% of epithelial cells (Fig. \b) while cytokeratin 19
(M, 40,000), marked by 1165 monoclonal, was present in less
than 10% of epithelial cells. The basal membrane was positively
stained by anti-type IV collagen antibodies.
Primary and Stabilized Cell Cultures and Clones. Primary cell
cultures, large flat cells with epithelial appearance, were sur
rounded by thick bundles of fusiform cells, that tended to
overgrow the epithelial-like elements.
Cytokeratins were consistently detected in large epitheliallooking cells, while only 10% of elongated cells were positive
for the anti-sm-actin monoclonal. The majority (80%) of the
cells were vimentin positive. Collagen type IV was present as
small dots on the surface of the fusiform cells.
Stabilized EPI cells formed closely packed islands of large
cuboidal cells, sometimes surrounded by elongated cells (Fig.
la). Isolated single cells were fusiform with fibroblast-like
appearance (Table 2). All the cells were intensely positive for
the 1165 and 1166 monocle muÃ-sagainst cytokeratins. Bundles
of intermediate filaments at the periphery of the cytoplasm and
in a perinuclear arrangement were stained by the AE3 mono-
Table 1 Reagents used in immunohistochemistry and immunofluorescence
cellsEpithelialMyoepithelialEpithelialEpithelia
AntigenCytokeratin
40,000)Cytokeratin
19 (M,
EnglandAmersham
International,
undiluted1/100-1/101/40-1/11/800-1/1001/350-1/501/300-1/101/400-1/201/5000-1/50"
52,000-55,000)Cytokeratins
8 (M,
EnglandCambridge
International,
19-16-15-14-10Cytokeratins
(MAb)AE3
8-7-6-5-4-3-2-1..-Smooth
(MAb)1
actinCollagen
muscle
IVVimentina-LactalbuminRat
type
(MAb)AntiserumV9
A4
undilutedI/
'1(1
IO-
UnitedStates
Research Laboratory,
AmericaCambridge
of
UnitedStates
Research Laboratory,
AmericaSigmaHeyl,
of
GermanyDakopatts,
Berlin,
(MAb)AntiserumAntiserumSourceAmersham
DenmarkDr. 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
membraneMesenchymalSecretorySecretorymono
antibody.
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MAMMARY CARCINOMA CELL LINES
were moderately detectable with the 1165 monoclonal antibody
directed against keratin 19; actin micro lì
laments were clearly
detectable in some elements (Fig. 2e). Production of type IV
collagen was shown in Clone H, I, and L cells. Specific immunolocalization was locally revealed inside the cytoplasm and
patchy' deposits were detectable in the intercellular spaces (Fig.
2f>.
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.
6). Keratins were found in a variable proportion of carcinoma
!f»
tous cells, while vimentin stained the stromal component con
<•
sistently. Few cells reacted with rat milk fat globule membrane
antiserum. Type IV collagen was present in limited areas of
these tumors; cell membranes of single cells were outlined and
some neoplastic nests were marked at the basal membrane level.
No actin reactivity was observed.
Fig. 1. Original R3230AC rat mammary carcinoma. In a, nests of neoplastic
cells are lined by a peripheral layer of flattened actin-rich myoepithelial cells.
Tumors obtained from Clones H, I, and L had a sarcomatous
Immunoperoxidase staining with a-smooth muscle actin monoclonal, x 250. In
appearance with spindle-shaped neoplastic cells mixed with
b, immunostaining for cytokeratin 8 (monoclonal 1166) is positive in the majority
liposarcoma-like areas (Fig. 7). A second generation tumor,
of neoplastic epithelial cells arranged in nests or pseudoglandular structures, x
ISO.
obtained by previous injection of clone L cells, showed keratinpositive adenocarcinomatous foci while in a tumor originated
from Clone H, areas of chondromatous metaplasia were pres
clonal in 80% of the cells. The AE1 monoclonal was positive
ent. Clone I had a peculiar morphology, strongly resembling
in only 40% of the cells.
Clones A, C, D, and E (obtained from EPI cells) were cystosarcoma phylloides (Fig. 8, a and b). The epithelial com
ponent was strongly positive for cytokeratins and was supported
characterized by rounded clusters of cuboidal cells and isolated
cells with large cytoplasm (Fig. 2, b and r). Immunohistochemby 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
confirmed by positive actin staining (Fig. 8e).
(Table 2).
Type IV collagen was positive in myoepithelial cells but not
Cells of Clones I, L, and H (all derived from primary cultures
in the basal membrane of the phylloides-like tumor from clone
of R3230AC tumor) were elongated or star shaped (Fig. 2d).
I. Liposarcoma-like tumors were positive for this marker.
The cells formed an irregular network on the Petri dish. Gen
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
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MAMMARY CARCINOMA CELL LINES
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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 <•,
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 «-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++nt
IV
Vim.
1165-6++
MFGMnt++++ntntnt+
spindle-shapedRoundishRoundishRoundishRoundishStar-likeSpindle-shapedStar-likeRoundish
+
+++nt
-+
nt
++++++
j-+nt—-—++CytokeratinsAE3
—¿â€”
++
++++++
—¿nt
++
+++nt
-+++
nt
++—
—¿â€”¿+++
+++
-—
-+++
+++
+++
+++++
+++
+ spindle-shapedAE1"+nt+
++Coll.
+++
++Rat
cultureMorphologyRoundish
°AE1, AE3, 1165, 1166, cytokeratins (see references); Coll. IV, type IV collagen; Vim., \ ¡mentiti;«-sm-actin,«-smooth muscle actin monoclonal; rat MFGM,
EPIClone
AClone
CClone
DClone
EClone
HClone
IClone
LPrimary
serum against rat milk fat globule membrane antigens; EPI, stabilized cell line; nt, not tested.
changes and a-lactalbumin production, demonstrated by immunocytochemistry and immunoblotting. In contrast, estrogen
stimulation did not affect the morphology of any of the cultured
cell lines, and production of a-lactalbumin was never detectable
by immunocytochemical procedures.
In vivo estrogen stimulation, started as soon as a nodule was
palpable, did not induce milk protein production or change the
histológica! pattern.
DISCUSSION
We recently observed that the architectural organization of
the R3230AC tumor parallels that of the lactating gland since,
in addition to the luminal epithelial (secretory) cells, myoepi-
thelial cells are present (13). The neoplastic myoepithelium
shows: (a) typical peripheral arrangement against the basement
membrane; (b) typical ultrastructural cytoplasmic organization;
and (c) the presence of immunocytochemically detectable asmooth muscle actin. This isoform of actin is present, within
the epithelial domain, only in myoepithelial cells of the breast
and salivary glands (22, 23).
Similar biphasic patterns involving both myoepithelial and
epithelial neoplastic components have been described in dimethylbenz[a]anthracene-induced
mammary tumors in mice
(24). Two alternative hypotheses can be advanced to explain
the observed phenomena: myoepithelial and epithelial cell com
ponents represent differentiated terminal stages stemming from
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MAMMARY CARCINOMA CELL LINES
o
o
I - -I
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
nomas lacking both the myoepithelial component and the hor
mone responsiveness of the parent tumor; «-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 (3032). 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).
Tumors generated from cell lines with myoepithelial features
showed mainly sarcomatous patterns. Vimentin and collagen
type IV were demonstrated in these tumors; the latter finding
seems at variance with the behavior of human liposarcomas
which do not produce collagen type IV (37). Focal keratin
positivity and chondroid metaplasia were found in single tu
mors; of major interest was, however, the peculiar association
A
•¿1
B
Fig. 4. Electron microscopic study of Clone I. In 1. the cells show elongated
shape and lack desmosomes; the nuclei are irregular in outline. At higher mag
nification (B) a selected area (arrows. A) shows subplasmalemmal bundles of
microfilaments (arrowheads), with interposed dense bodies. A, x 2,000; B, x
16,000.
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MAMMARY CARCINOMA CELL LINES
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
1165-6EPIClone
'
lili.
-Carcinomatous
—¿â€¢Carcinomatous
+
++
++
++
+++
++
nt
—¿
-Carcinomatous (adenocarcinoma)
—¿Sarcomatous(anaplastic)
-metaplasia)Sarcomatous
(lipoblasts, with chondromatous
+
—¿
-
++
++
-
+++
+
-
+
—¿
++
—¿
+++
++Sarcomatous(area phylloides-like)
(lipoblasts, with Carcinomatous
LCarcinomatous —¿areas)+±++ntntnt
—¿
—¿
—¿
—¿
++
++
AClone
CClone
DClone
EClone
HClone
IClone
Õ T
U-3Ill-a\.lIII
IVtll
ITI!
VI
AE3
++
+++
+++
+++
Original R3230AC carcinoma
" Coll. IV, type IV collagen; AEl, AE3, 1165, 1166, cytokeratins (see references); Vim., vimentin; a-sm-actin, «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
neoplastic myoepithelial-like cells.
The literature provides examples of fibrosarcomas, rhabdomyosarcomas, and other types of sarcoma originating in ani
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
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
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
malignant mammary tumors of female canines, in which ohmi
ciro id 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.
Previously, other authors (44, 45) tried to establish cultures
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
form cells which have been regarded as the equivalent of the
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vi,-:'
^
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.
. JK
* -*»-.* ..-
!'^v
*
t
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
component in cases of human cystosarcoma phylloides has been
reported in the literature (39). Our study confirms these data
Fig. 7. Tumor with liposarcomatous patterns derived from Clone L infÃ-ltrales
muscle layer of the dorsal region of a Fischer rat. Spindle-shaped cells with
hyperchromatic nuclei are intermingled with polygonal vacuolated cells resem
bling lipoblasts. x 150.
1558
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.v."''Ã-.
;
!;î
•¿â€¢â€¢
•¿â€¢
m**
MAMMARY CARCINOMA CELL LINES
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
cells has also been described in cell lines derived from BALB/
CDH mouse mammary tumors (48) and from nitrosomethylurea-induced mammary tumors in WF rats (49). According to
the observations of Dulbecco et al. (49) and Warburton et al.
(32) these fÃ-broblast-like cells did not exactly correspond to
myoepithelial cells but showed immunocytochemical features
common to both stromal cells (presence of vimentin and of
Thy-1-1 antigen) and myoepithelial cells (production of type
IV collagen). The biphasic behavior of myoepithelial cells in
skin and salivary gland tumors, where these cells seem to be
able to form a stromal matrix, has been described and discussed
by several authors (50, 51). The nature of the myoepithelial cell
itself is indefinite: while its content of basic-type keratins sug
gests an epithelial nature, the presence of vimentin (52) and its
contractile ability, testified to by the presence of bundles of «smooth muscle actin (a unique feature among epithelial cells),
indicate its affinity to mesenchyme-derived cells. Even the
assumption that myoepithelial cells are terminally differen
tiated cells (53, 54) seems untenable, since we observed (55)
that in the developing mouse mammary gland myoepithelial
cells can proliferate.
All these data, in line with the reported experimental obser
vation in R3230AC rat tumor and related cell lines, support
the hypothesis that mixed epithelial-stromal tumors of the
mammary gland can originate from the myoepithelial cells.
ACKNOWLEDGMENTS
We thank Professor P. M. Cullino (University of Turin, Turin, Italy)
and Professor L. Ozzello (Columbia University, New York, NY) for
criticisms.
REFERENCES
1. Hilf, R., Michel, I., Bell, C., Freeman, J. J.. and Borman, A. Biochemical
and morphologic properties of a new lactating mammary tumor line in the
rat. Cancer Res., 25: 286-299, 1965.
Hilf, R., Michel, I., Bell, C., and Carrington, M. J. Influence of endocrine
organ ablation on the growth and biochemical responses of the R3230AC
mammary tumor to hormonal treatment. Cancer Res., 26:136S-1370, 1966.
Gardner, D. G., and Wittliff, J. L. Demonstration of a glucocorticoid hor
mone-receptor complex in the cytoplasm of a hormone-responsive tumor.
Br. J. Cancer, 27:441-444, 1973.
Costlow, M. E., Buschow, R. A., and McGuire, W. L. Prolactin receptors in
an estrogen receptor-deficient mammary carcinoma. Science (Washington
DC), 184: 85-86, 1974.
ShaFie, S. M., Gibson, S. L., and Hilf, R. Effect of insulin and estrogen on
hormone binding in the R3230AC mammary adenocarcinoma. Cancer Res.,
37; 4641-4649, 1977.
Smith, R. D., Hilf, R., and Senior, A. E. Prolactin binding to R3230AC
mammary carcinoma and liver in hormone-treated and diabetic rats. Cancer
Res., 3 7: 595-598, 1977.
Aylsworth, C. F., Hodson, C. A., Berg, G., Kledzik, G., and Meites, J. Role
of adrenals and estrogen in regression of mammary tumors during postpartum lactation in the rat. Cancer Res., 39: 2436-2439, 1979.
Hissin, P. J., and Hilf, R. Effects of estrogen to alter amino acid transport
in R3230AC mammary carcinomas and its relationship to insulin action.
Cancer Res., 39: 3381-3387, 1979.
Orloski, J. M., Fritz, P. J., and Liu, D. K. Pregnancy stimulates DNA
synthesis in R3230AC mammary adenocarcinoma. Eur. J. Cancer Clin.
Oncol., /*: 99-106, 1982.
Stein, T. P. Tumor induced changes in the host's protein metabolism. In: M.
10.
S. Arnott, J. Van Eyes, and Y. M. Want (eds.). Interrelations of Nutrition
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
status on binding of activated estrogen receptor to nuclei from R3230AC
and 7,12-Dimethylbenz[a]anthracene-induced
mammary tumors. Cancer
Res.,«: 1165-1170, 1989.
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
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
carcinoma. Virchows Arch. Abt. B Zeli Pathol., 55: 39-45, 1988.
Hilf,
R. Milk-like fluid in a mammary adenocarcinoma biochemical charac
14.
terization. Science (Washington DC), 155:826-827, 1967.
IS, Owens, R. B., and Hackett, A. J. Tissue culture studies of mouse mammary
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
mammary tumor cell lines from different mouse strains. Eur. J. Cancer, 16:
1181-1192, 1980.
17, Sato, H., Azoma, M., Hayashi, Y., Yoshida, H., Yanaga, W. A. T., and Yura,
Y. 5-Azacytidine induction of stable myoepithelial and acinar cells from a
1559
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research.
MAMMARY CARCINOMA CELL LINES
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
human salivary intercalated duct cell clone. Cancer Res., 47: 4453-4459,
1987.
Sapino, A., Pietribiasi, F., Bussolati, G., and Marchisio, P. C. Estrogen- and
tamox¡fun-induced rearrangement of cytoskeletal structures in breast cancer
MCF-7 cells. Cancer Res., 46: 2526-2531,1986.
Markwell, M. A. K., Haas, S. M., Bieber, L. C, and Tolbert, N. E. A
modification of Lowry procedure to simplify protein determination in mem
brane and lipoprotein samples. Anal. Biochem., 87: 206-210, 1978.
Mitchell, D., Ibrahim, S., and Gusterson, B. A. Improved immunohistochemical localization of tissue antigens using modified methacarn fixation. J.
Histochem. Cytochem., 33:491-495, 1985.
Hsu, S. M., Raine, L., and Fanger, H. Use of avidin-biotin-peroxidase
complex (ABC) technique. A comparison between ABC and unlabeled anti
body (PAP) procedures. J. Histochem. Cytochem., 29: 577-580, 1981.
Skalli, O., Ropraz, P., Trzeciak, A., Benzonana, G., Gillessen, D., and
Gabbiani, G. A monoclonal antibody against »-smoothmuscle actin. A new
probe for smooth muscle differentiation. J. Cell Biol., 103:2787-2796,1986.
Gugliotta, P., Sapino, A., Mucri, L., Skalli, O., Gabbiani, G., and Bussolati,
G. Specific demonstration of myoepithelial cells by anti-a smooth muscle
actin antibody. J. Histochem. Cytochem., 36:659-663, 1988.
Rehm, S. Chemically induced mammary gland adenomyoepitheliomas and
myoepithelial carcinomas of mice. Immunohistochemical and ultrastructural
features. Am. J. Pathol., 136: 575-584, 1990.
Kratochw ill, K. Organ specificity in mesenchymal induction demonstrated in
embryonic development of the mammary gland in the mouse. Dev. Biol., 20:
46-71, 1969.
Emmerman, J. T., Enami, J., Pitelka, D. R., and Nandi, S. Hormonal effects
on intracellular and secreted casein in cultures of mouse mammary epithelial
cells in floating collagen membranes. Proc. Nati. Acad. Sci. USA, 74:44664470, 1977.
Ormerod, J. E., and Rudland, P. S. Mammary gland morphogenesis in vitro:
formation of branched tubules in collagen gels by a cloned rat mammary cell
line. Dev. Biol., 91: 360-375, 1982.
Lee, E. Y-H., Parry, G., and Bissel, M. J. Modulation of secreted proteins
of mouse mammary epithelial cells by the collagenous substrata. J. Cell.
Biol., 98:146-155, 1984.
Stockdale, F. E., Wiens, D. J., and Levine, J. F. Cell-cell interactions in the
mediation of hormone dependent differentiation of mammary epithelium.
In: Progress in Developmental Biology, Part B, pp. 417-424. New York:
Alan R. Liss, Inc., 1986.
Warburton, M. J., Ferns, S. A., and Rudland, P. S. Enhanced synthesis of
basement membrane proteins during the differentiation of rat mammary
tumor epithelial cells into myoepithelial-like cells in vitro. Exp. Cell Res.,
137: 373-380, 1982.
Warburton, M. J., Mitchell, D., Ormerod, E. J., and Rudland, P. S. Distri
bution of myoepithelial and basement membrane proteins in the resting,
pregnant, lactating and involuting rat mammary gland. J. Histochem. Cyto
chem., 30: 667-676, 1982.
Warburton, M. J., Ferns, S. A., Hughes, C. M., Sear, C. H. J., and Rudland,
P. S. Generation of cell types with myoepithelial and mesenchymal phenotypes during the conversion of rat mammary tumor epithelial stem cells into
elongated cells. J. Nati. Cancer Inst., 78: 1191-1201, 1987.
Ozzello, L. Ultrastructure of the human mammary gland. Pathol. Annu., 6:
1-59, 1951.
Rennov-Jessen, L., van Deurs, B., Cells, J. E., and Petersen, O. W. Smooth
muscle differentiation in cultured human breast gland stromal cells. Lab.
Invest., 63: 532-543, 1990.
Lazarides, E. Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins. Annu. Rev. Biochem., 51: 219-250,
1982.
McGuire, L. J., Ng, J. P. V., and Lee, J. C. K. Coexpression of cytokeratin
and vimentin. Appi. Pathol., 7: 73-84, 1989.
37. Ogawa, K., Oguchi, M., Yamabe, H., Nakashima, \.. and Hamashima, Y.
Distribution of collagen type IV in soft tissue tumors. An immunohistochemical study. Cancer (Phila.), 5«:269-277, 1986.
38. Qizilbash, A. H. Cystosarcoma phyllodes with liposarcomatous stroma. Am.
J. Clin. Pathol., 65: 321-327, 1976.
39. Mechtersheiner, G., Krüger,K. H., Born, I. A., and Möller,P. Antigenic
profile of mammary fibroadenoma and cystosarcoma phillodes. A study using
antibodies to estrogen and progesterone receptors and to a panel of cell
surface molecules. Pathol. Res. Pract., 186:427-438, 1990.
40. Sanford, K. K., Dunn, T. B., Westfall, B. B., Covalesky, A. B., Dupree, L.
T., and luirle. W. R. Sarco matous change and maintenance of differentiation
in long term cultures of mouse mammary carcinoma. J. Nati. Cancer Inst.,
26:1139-1159, 1961.
41. Fasske, E., Fetting, R., Morgenroth, K., Jr., and Themann, H. The sarcomatous transformation of mammary carcinoma of mice in isologous transplan
tâtes,in cell cultures, and réimplantâtes.
Oncology (Basel), 21: 189-213,
1967.
42. Kahn, L. B., Uys, C. J., Dale, J., and Rutherfoord, F. Carcinoma of the
breast with metaplasia to chondrosarcoma: a light and electron microscopic
study. Histopathology, Z-93-106, 1978.
43. von Bombard, D., and von Sandersleben, J. Ãœberdie Feinstruktur con
Mammamischtumoren der Hündin.II. Das Vorkommen von Myoepithelzellen in chondroiden Arealen. Virchows Arch. Abt. A Pathol. Anat. Histol.,
362:157-167, 1974.
44. Warburton, J., Ormerod, E. J., Monaghan, P., Ferns, S., and Rudland, P. S.
Characterization of a myoepithelial cell line derived from a neonatal rat
mammary gland. J. Cell. Biol., 91: 827-836, 1981.
45. Dunnington, D. J., Hughes, C. M., Monaghan, P., and Rudland, P. S.
Phenotypic instability of rat mammary tumor epithelial cells. J. Nati. Cancer
Inst., 71:1227-1240,1983.
46. Bennett, D. C., Peachey, L. A., Durbin, H., and Rudland, P. S. A possible
mammary stem cell line. Cell, 15: 283-298, 1978.
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
intermediate between cultured mammary epithelial and myoepithelial-like
cells. Dev. Biol., 113: 388-405, 1986.
48. Hager, J. C., Fligiel, S., Stanley, W., Richardson, A. M., and Heppner, G.
H. Characterization of a variant-producing tumor cell line from a heteroge
neous strain BALB/cfC3H mouse mammary tumor. Cancer Res., 41:12931300, 1981.
49. Dulbecco, R., Henahan, M., Bowman, M., Okada, S., Battifora, H., and
Unger, M. Generation of fibroblast-like cells in vitro: a possible new cell
type. Proc. Nati. Acad. Sci. USA, 78: 2345-2349, 1981.
50. Azzopardi, J. G., and Smith, O. D. Salivary gland tumours and their mucins.
J. Pathol. Bacterio!., 77:131-140, 1959.
51. Varela-Duran, J., Diaz-Flores, L., and Varela-Nunez, R. Ultrastructure of
chondroid syringoma. Role of the myoepithelial cell in the development of
the mixed tumor of the skin, and soft tissues. Cancer (Phila.), 44: 148-156,
1979.
52. Warburton, M. J., Hughes, C. M., Ferns, S. A., and Rudland, P. S. Localiza
tion of vimentin in myoepithelial cells of the rat mammary gland. Histochem.
J.,2/: 679-685, 1989.
53. Ferguson, D. J. P. Ultrastructural characterization of the proliferative (stem?)
cells within the parenchyma of the normal "resting" breast. Virchows Arch.
Abt. A Pathol. Anat., 407: 379-385, 1985.
54. Joshi, K., Smith, J. A., Perusinghe, N., and Monoghan, P. Cell proliferation
if the human mammary epithelium. Differential contribution by epithelial
and myoepithelial cells. Am. J. Pathol., 124: 199-206, 1986.
55. Sapino, A., Macri, L., Gugliotta, P., and Bussolati, G. Immunohistochemical
identification of proliferating cell types in mouse mammary gland. J. Histo
chem. Cytochem., 38: 1541-1547, 1990.
1560
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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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