Production of Skeletal Muscle Elements by Cell Lines Derived from Neoplastic Rat Mammary Epithelial Stem Cells

[CANCER RESEARCH 44, 2089-2102, May 1984]

Philip S. Rudland,1 Damien J. Dunnington, Barry Gusterson, Paul Monaghan, and Christine M. Hughes

Ludwig Institute for Cancer Research (London Branch), Royal Marsden Hospital, Downs Road, Button, Surrey SM2 5PX, United Kingdom

ABSTRACT chymal elements seen in certain tumors of the mammary gland arise from preexisting, but more primitive, mesenchymal cells or Single-cell-cloned cell lines intermediate in morphology be from epithelial cells of the mammary parenchyma (3). tween the cuboidal epithelial and fully elongated myoepithelial- The carcinogen-induced tumors of the rat mammary gland like cells have been isolated from the single-cell-cloned epithelial usually contain both epithelial and myoepithelial-like cells (43), stem cell lines Rama 25 and Rama 37 originally obtained from and single-cell-cloned epithelial stem cell lines have been isolated dimethylbenz(a)anthracene-induced mammary tumors from from such tumors which were growing either in an outbred Sprague-Dawley and Wistar-Furth rats, respectively. These are Sprague-Dawley rat (6) or in an inbred Wistar-Furth rat (15). The designated Rama 25-11, Rama 25-I2, Rama 25-I4 (Sprague- former cell line is called Rama2 25, and the latter line is named Dawley) and Rama 50-55, Rama 59, and Rama 60 (Wistar- Rama 37. Both these single-cell-cloned epithelial cell lines can Furth), respectively. When growing as tumors in nude mice or give rise to elongated, myoepithelial-like cells in vitro. When syngeneic Wistar-Furth rats, respectively, many of the newly injected into nude mice (36) or syngeneic rats, respectively (15), cloned cell lines give rise to spindle and giant, multinucleated they form tumors which also contain both these cell types. That cells which stain immunocytochemically with antisera to myoglo- the parental cell line Rama 25 and the single-cell-cloned sublines bin and myosin and contain longitudinal fibrils, some of which developed from Rama 37, particularly Rama 37 CL-A3, are contain phosphotungstic acid-hematoxylin-staining cross-stria- epithelial in origin is confirmed by their characteristic ultrastruc tions. Ultrastructural analysis demonstrates the presence of A-, ture (6, 15, 29), the presence of intermediate filamental proteins I-, and H-bands and Z-discs and the hexagonal arrangement of of the prekeratin type (15, 25, 41), and the expression of MFGM thick and thin filaments characteristic of skeletal muscle. Similar antigens in tumors formed by injecting the cells into the appro results are obtained with selected cloned cell lines growing on priate rodents (10, 15, 36). Furthermore, Rama 25 cells can floating collagen gels in vitro. Thus, a developmental^ committed make small amounts of casein, the major protein product of the mammary epithelial cell can give rise, under suitable conditions, alveolar cell of the mammary gland, both in tissue culture with to a well-differentiated mesenchymal lineage, that of skeletal the appropriate hormones (40) and when growing as a tumor in muscle. It is suggested that such cells may be responsible for lactating nude mice (37), confirming its mammary epithelial iden the generation of the well-differentiated mesenchymal elements tity. In the course of this work, both single-cell-cloned cell lines seen in the mixed (epithelial and myoepithelial) tumors of glan also gave rise to a spectrum of cells with morphological forms dular origin. varying from cuboidal epithelial to fully elongated cells. We now report the isolation of some of these cells as single-cell-cloned INTRODUCTION lines from both epithelial precursors and show that they can give rise to tumors in rodents or structures on rafts of floating collagen In certain glandular systems, the myoepithelial cells which gels (17) that contain cells of another well-differentiated mesen surround the ductal epithelial cells have properties similar in chymal lineage, that of skeletal muscle. many respects to those of smooth muscle cells (21). The myo epithelial cell, however, is considered to arise from ductal epithe MATERIALS AND METHODS lial cells at least in the mammary (28, 33) and possibly in the salivary glands (13, 22). The mixed tumors which arise in the Tissue Culture. Rama 25 and Rama 29 cells isolated from a DMBA- salivary glands, the pleomorphic adenomas in particular, contain induced tumor in a Sprague-Dawley rat were grown in RM in an atmos both epithelial and myoepithelial cells together with varying phere of 10% CO2-90% air at 37Â°(6). Rama 25-25 cells were grown to amounts of well-differentiated mesenchymal products which in confluence, and fresh RM was added to the cultures 12 hr before cloning. clude banded collagen fibers, cartilage, and bone (21). In the Cells were detached by incubation with EDTA-trypsin solutions, and 96 mammary gland, tumors may arise in the epithelium that appar single cells were picked and grown up in wells containing cloning medium ently produce mucoid, chondroid, osteoid, and even bony inter (38). The overall cloning efficiency was 59%, and the percentages of colonies with cells of cuboidal, elongated, and intermediate morphologies cellular substances, and although extremely rare in humans they were 86, 2, and 12%. Three cell clones of intermediate morphology were are more frequent in dogs (18,19, 39). Usually, in humans these grown up in RM and frozen at passage 31. These were designated heterologous elements are thought to originate from neoplastic connective tissue elements (12), although there is evidence for 2 The abbreviations used are: Rama, rat mammary, followed by the single-cell transitional forms between carcinomatous and chondrosarcom- clone number and then a passage number; Rama 25-11, first cell line of intermediate morphology single cell cloned from Rama 25; Rama 37 CL, cell line of large cuboidal atous components suggesting a common ancestry (24). Thus, cells ring cloned from Rama 37; Rama 37 CL-A3, cell line single cell cloned from there is still doubt about whether the well-differentiated mesen Rama 37 CL (15); Rama 37 E, cell line of relatively elongated cells ring cloned from Rama 37; DMBA, 7,12-dimethylbenz(a)anthracene; MFGM, milk fat globule mem brane; PTAH, phosphotungstic acid-hematoxylin; RM, routine medium (Dulbecco's 1To whom requests for reprints should be addressed. modified Eagle's medium, 10% fetal calf serum, hydrocortisone (50 ng/ml), and Received October 25, 1983; accepted January 27, 1984. insulin (50 ng/ml).

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Rama37 grown for 3 to 4 days to a density of about 80 to 90% of that at (cuboidalepithelialcell) confluency. Two x 106 cells in 0.2 ml Ca2+- and Mg2+-free phosphate- buffered saline were then injected into the right inguinal fat pad, either through the skin of 6-week-old female nude mice or by making an incision and exteriorizing the relevant region in 62-day-old female rats (15, 38). Tumors were palpated and sized each week as before, and when about 1 cm in diameter they were removed and fixed for histology. At autopsy, ringcloning animals were examined for visible metastatic tumor deposits in the lungs, liver, spleen, kidneys, lymph nodes, and gonads. Any suspicious areas were processed for histology. To obtain lactating nude mice bearing tumors, female nude mice carrying tumors up to 1 cm in diameter were mated with male BALB/c mice and sacrificed 3 days after parturition. Both the tumor and the left inguinal mammary glands were removed and Rama37E Rama37CL processed for histology (37). (moreelongatedcells) (cuboidalepithelialcell) Histology of Tumors and Collagen Gels. Tumors were cut into slices less than 0.5 cm thick, and both tumor slices and collagen gels were fixed in 60% methanol-30% lnhibisol-10% acetic acid (v/v) for 24 hr at 4Â°and processed as before (41). Sections were stained by hematoxylin and eosin, Hoechst 33258, reticulin, Picro-Mallory's acid, Martius scarlet blue, and PTAH methods as described by Drury and Wallington (14), except that the Hoechst-stained sections were irradiated with UV and singlecellcloning restained with Giemsa (6, 27), and the reticulin method was that of Chadwin (11). The fluorochrome Hoechst 33258 was purchased from Riedel de Haen AG, Seelze-Hannover, West Germany. Photographs were taken on llford Pan F film using a Reichert Polyvar microscope. Immunocytochemical Staining. Immunocytochemical staining of sec tions of tumors and collagen gels was carried out by an indirect procedure Rama50to55 Rama37C A3 using an alkaline phosphatase-conjugated second antibody (2, 41). Pri Rama59and60 (cuboidalepithelialcell) mary antibodies raised in rabbits against rat MFGM; purified human keratins (molecular weights, 69,000, 57,000, 47,000, 45,000, and (elongatedcells) 43,000), pig uterine smooth muscle myosin (30), and actin3 (purchased

Chart 1. Summary of the isolation of different cell lines of the Rama 37 system. from Miles Chemical Co.) have been characterized and described previ Cell strains from the single-cell-cloned cell line Rama 37 were picked initially with a ously (41, 42). Rabbit antiserum to human skeletal muscle myoglobin ring clone; subsequently, cell lines were generated by picking single cells. Rama was purchased from Miles Yeda (Code 65-075, Lot R 542). In immuno- 37 E were more elongated than the large cuboidal (CL) epithelial cells, Rama 37 cytochemical tests, it reacted only with normal skeletal muscle and not CL, but still were not as fully elongated as Rama 29 or the equivalent elongated cells developed from Rama 37 CL-A3 (15). with smooth muscle or circulating RBC, and the staining of skeletal muscle could be abolished by prior incubation of this antibody with equine skeletal muscle myoglobin (Sigma Code M 0630). Rabbit antisera Rama 25-11, Rama 25-I2, and Rama 25-I4. to a mixture of the 3 separately purified caseins (molecular weights, The Rama 37 cell line was obtained from a DMBA-induced tumor in a 42,000, 29,000, and 24,000) from rat milk were raised and characterized Ludwig OLA strain of Wistar-Furth rats as reported previously (15). This as described previously (40). Purified IgGs (41) were diluted between cell line which had been single-cell cloned 3 times yielded elongated (E), 1:20 and 1:2000 with 0.5% bovine serum albumin in Ca2+- and Mg2+- small cuboidal (CS), and large cuboidal (CL) cells. A ring clone of 2 free phosphate-buffered saline before use. Sheep anti-rabbit IgG conju morphological types was picked and designated as Rama 37 E and gated to alkaline phosphatase with glutaraldehyde was purchased from Rama 37 CL. Rama 37 E and Rama 37 CL cultures were subsequently Sigma Chemical Co. Absorbed antisera were prepared by incubating single-cell cloned in cloning medium. Rama 37 E yielded only elongated antisera with 1 mg of antigen per ml at 37Â° for 3 hr. Sections were cell clones (70% efficiency), and some of these (Rama 50 to Rama 55, counterstained with Mayer's hemalum and photographed as before with Rama 59, and Rama 60) were grown up to bulk cultures in RM and a blue-green filter (Kodak Wratten No. 44) on Pan F film for the black frozen at passage 25 (Chart 1). Rama 37 CL-A3, a single-cell clone of and white photographs or with a blue daylight filter (Hoya, Japan No. cuboidal epithelial cells from Rama 37 CL, was frozen at passage 18 80A) on Kodacolor II film. (15). The specificity of the staining for each antiserum was checked by the Cellular morphology was determined by plating cells at 2000 to 5000 following controls: (a) sections from normal mammary glands from 70- per 9-cm Retri dish in RM and growing the cultures for 5 days. Colonies day-old female rats or from rat skeletal muscle and from glands of 3-day of each cell line were then scored for their predominant morphological lactating rats for antisera to myoglobin and casein, respectively, were type using an Olympus CK inverted microscope (x40), the results were stained in parallel with both histochemical and Â¡mmunocytochemical the average of 6 dishes, and 6 fields/dish of 20 to 40 colonies were reagents and compared with previous results for consistency of staining counted. Cell lines were seeded at approximately 105 cells/5-cm dish on (23, 37, 41); (b) complete abolition of staining was achieved by prior preformed 0.3% collagen gels, floated, and grown for 8 days as described incubation of each antiserum with the requisite antigen; (c) tumor cells previously (29). Cell monolayers growing on plastic Petri dishes were which failed to stain were recorded only as negative, providing that an photographed with a Zeiss ICM 405 automatic inverted microscope appropriate normal host (rat or mouse) cellular structure could be iden using phase-contrast optics on llford Pan F film using a green filter. All tified as staining on the same section; (d) finally, 2 sections from 2 tumors hormones were purchased from Sigma. or from 4 collagen gels of each cell line for each antibody were examined. Induction of Tumors in Rodents. Institute of Cancer Research C57BL Electron Microscopy. Tumors were cut into 1- to 2-mm cubes, fixed nude mice or the Ludwig Wistar OLA strain Wistar-Furth rats from Olac for 2 hr at room temperature in 2% glutaraldehyde, and postfixed for 8 (Banbury, England) were used as recipients for the Rama 25- and Rama hr at 4Â° in 1% osmium tetroxide. Both fixatives were buffered with 37-derived cell lines, respectively. Cultured cells, 2 passages after thaw ing, were seeded at 2 x 106 in 600-ml Falcon flasks in 50 ml RM and 3C. M. Hughes and M. J. Warburton, unpublished results.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1984 American Association for Cancer Research. Production of Skeletal Muscle Elements by Mammary Stem Cells phosphate to pH 7.3, and the osmotic pressure was adjusted to 350 All 3 morphologically intermediate cloned cell lines generated mOsmol by addition of sucrose. Tissues were dehydrated in ethanol and both elongated cells similar to Rama 29 and cuboidal cells similar embedded via propylene oxide in Epon-Araldite resin (26). Cultures of to Rama 25 at relatively low frequencies (Table 1). The number collagen gels were cut into 2- to 3-mm squares, fixed, and embedded of elongated cell colonies detected by single-cell clonal analysis as for the tumor samples except that both fixation and embedding times increased by successive factors of about 2-fold in the series were increased by at least 2-fold (29). Thick sections (1.5 to 2 Mm) were Rama 25-I2, Rama 25-11, Rama 25-I4; these results were inde stained with toluidine blue for light microscopy, and areas were selected for electron microscopy. Thin sections (about 70 nm) were cut with a pendent of the passage number of the cell lines up to passage diamond knife on a Reichert OMU4 microtome, stained with uranyl 40. It was difficult to estimate the number of cuboidal cell colonies acetate and lead citrate (34), and examined in a Phillips EM400 electron generated, since the cellular morphology of Rama 25-I2 and microscope. Rama 25 cuboidal epithelial cells was very similar. However, the number of cuboidal-like (I.e., both Rama 25 and Rama 25-I2) cell colonies was under 10% of the total for Rama 25-11 and Rama RESULTS 25-I4 for passage numbers under 40. When these morphologi Generation and Properties of Intermediate and Elongated cally intermediate cell lines were transferred beyond passage 42, Cell Lines. The single-cell-cloned cell line Rama 25 when grown they generated dramatically increased numbers of cuboidal-like on plastic Retri dishes resembles low, cuboidal epithelium (Fig. cell colonies (31) and hence were not used above passage 38. 1/4). Cultures were grown to confluency, and single-cell clones The single-cell-cloned cuboidal epithelioid cells, Rama 37 (5), were picked. These yielded cuboidal cells and fully elongated were virtually identical in appearance and behavior to Rama 25 cells which were morphologically similar to the mammary epithe cells in that they gave rise both to cuboidal epithelioid and to lial cell line Rama 25 and the myoepithelial-like cell line Rama 29, elongated cells at a low frequency in culture. Ring clones of both respectively (6). In addition, approximately 7% of the original types were picked and termed Rama 37 CL (15) and Rama 37 Rama 25 cells yielded colonies of cells with morphologies inter E (Fig. 1D), respectively, and then single-cell clones of each ring mediate between these 2 forms. Three representative single-cell clone were picked and grown up. One of the former epithelial clones were grown up and designated Rama 25-11, Rama 25-I2, cell lines was termed Rama 37 CL-A3 (15), while the latter cloned and Rama 25-I4. These represented single-cell-cloned cell lines cell lines which had a rather variable elongated morphology but with an increasing tendency towards the elongated morphology relatively distinctive for each cell line were termed Rama 50 to of Rama 29 cells, in the order Rama 25-I2, Rama 25-11 (Fig. 1B), Rama 55, Rama 59, and Rama 60 (Chart 1) (e.g., Rama 59; Fig. and Rama 25-I4. Thus, the peripheries of the cells became more 1E). Unlike the Rama 25-I cloned cell lines, the Rama 37 E- ragged and less distinct, increased numbers of gaps between derived cloned cell lines retained their morphology on extended the cells were observed, and longitudinal fibers gradually ap passage; they failed to revert to cuboidal-like cells in culture peared in the cell cytoplasm, although the overall morphology of when tested up to 10 passages from their frozen stocks. Some the cells was still cuboidal with a near circular nucleus. These of these cloned cell lines gave rise to relatively flat, giant, often morphologically intermediate cloned cell lines, particularly Rama multinucleated cells similar to those in Fig. 1C. In addition, very 25-11 and Rama 25-I4, appeared flatter than did the cuboidal large, highly refractile, strap-like cells were seen which appeared epithelial Rama 25 or the fully elongated Rama 29 types and as to be formed by the fusing of neighboring cells (Fig. 1F). None such became very large, often multinucleated, with much of the of the Rama 25-I or Rama 37 E cloned cell lines yielded droplet cytoplasm filled with longitudinal fibers (Fig. 1C). The morphology cells and domes under conditions where such structures were of Rama 25-I2, on the other hand, was much closer to that of seen with their parent cell lines, but all cloned cell lines formed Rama 25, and only the occasional cell was seen to break ridges composed of fully elongated cells as described previously contacts with its neighbors and to contain a few poorly organized for Rama 25 (6) and Rama 37 (15) cells. fibers. Formation of Tumors in Rodents. Since the Rama 25-I cloned cell lines originated from Rama 25 which came from a tumor in a randomly bred Sprague-Dawley rat while the Rama 37 E- derived cloned cell lines came from Rama 37 which in turn Table1 originated from a tumor in an inbred Wistar-Furth rat, we have Generation of different morphological cell types in Rama 25 series of intermediate tested these lines for their tumor-forming ability in immunodefi- cells % of nonintermediate cell colonies8 cient nude mice or in syngeneic Wistar-Furth rats, respectively. The Rama 25-derived and Rama 37 E-derived cloned cell lines Elongated cell colo- Cuboidal cell colo- Cell line Passage nies nies all caused tumors in nude mice, except Rama 29 which failed to Â±0.5*3.6 produce any tumors (not shown). However, the new cloned cell Rama25Rama Â±48.8 25-11Rama Â±1.31.8 -Â«-1.481 lines caused tumors to appear at different rates (Table 2). 25-I2Rama Â±0.66.4 Â±2Â°6.1 All tumors were macroscopically coarsely tabulated and ad 25-I4Rama Â±0.5100 Â±0.60 herent to the skin. The tumors were encapsulated in condensed 292532-4032-4032-40331.9 Â±095 Â±0 3 Isolated colonies of cells were scored for their cellular morphology as cuboidal, connective tissue. This pseudocapsule was breached with local elongated, or intermediate between these 2 extremes. The Rama 25 intermediate infiltration of the fat in tumors of Rama 25-I4, Rama 50, Rama cells were scored at 3 or 4 passage numbers between 32 and 40, and the average 52, Rama 59, and Rama 60. Only rodents inoculated with Rama results were taken. Rama 25 cuboidal epithelial cells and Rama 29 elongated myoepithelial-like cells were scored at only one passage number ("Materials and 54 or Rama 59 cells contained macrometastases at autopsy. Methods"). Nodular deposits were present in the lungs of one-third of the " Mean Â±S.E. 0 The difference between the morphology of Rama 25-I2 and Rama 25 cuboidal rats bearing Rama 59 tumors and in the lungs of both rats cells can be discerned only with higher-power phase-contrast microscopy; hence, bearing Rama 54 tumors. MÃ©tastases were not found in any with the counting system described here, they were classified as cuboidal. other organs or tissues examined.

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Histology of Tumors. The Rama 25, Rama 25-12, and Rama tion (Table 3). In some areas of the tumors, notably Rama 25-11, 37 CL-A3 (not shown) tumors were predominantly composed of Rama 37 E, Rama 50, Rama 52, Rama 55, Rama 59, and to a cuboidal-like cells with pleomorphic nuclei, a high mitotic rate, lesser extent in Rama 25-I4 and Rama 60 tumors, there were and a variable growth pattern. The degree of glandular morphol well-differentiated elements with a very low mitotic rate (below ogy varied both within different regions of the same tumor and one mitosis per 5 microscopic fields) which contained plump, between tumors. In some areas, there were well-formed duct- mononuclear and multinucleated giant, strap-like cells that mor like structures lined with one or more layers of cuboidal or low- phologically resembled rhabdomyoblasts (Fig. 2, ÃŸto D). These columnar epithelium (Fig. 3D) and solid sheets or clusters of areas underwent transitions into less-well-differentiated elements epithelial cells, sometimes with a papillary growth pattern (Fig. composed of ovoid or spindle-shaped cells with a higher mitotic 2A). In other areas, there were sheets of spindle cells. The duct- rate (greater than 2 per microscopic field) (Fig. 2C). Many of the like growth patterns were seen to a lesser extent in Rama 25-14 tumors, in particular those of Rama 25-11, Rama 52, Rama 55, tumors and were absent in all other tumors (Table 3). All tumors and Rama 60, also contained small, round cells and tadpole-like except Rama 25, Rama 25-12, Rama 37 CL-A3, and Rama 53 cells morphologically similar to those seen in rhabdomyosarco- showed a second pattern of varying degrees of differentiation, mas (Fig. 2D). The blood-borne metastatic deposits in the lungs that of skeletal muscle-like or rhabdomyoblastic-like differentia- of rats given injections of Rama 59 also showed the same rhabdomyoblastic-like pattern with additional spindle cell forms. Myxoid areas with perivascular cuffing were a notable feature in Table 2 tumors formed by Rama 60 (Table 3). Rama 53 tumors contained Kinetics of the formation of tumors in rodents only ovoid or spindle-shaped cells without any recognizable Time to form tumors in rodents skeletal muscle-like elements. Transitions between the glandular Incidence (%) (days) and rhabdomyoblastic differentiation patterns occurred in Rama Cell line3 in total tu total tu 25-I4 (Fig. 3D). jectedRama mors18-2437-4347-5359-6426-3241-4933-4128-3447-5360-6820-30NDC100%mors54-6064-7075-81154-16042-4862-6866-7244-5071-7773-7944-5020-30Tumors9510080904050902020509080MÃ©tastases00000000200300 25oRama In the nude mouse tumors, the glandular and rhabdomyoblas 25-I20Rama tic regions were composed of rat cells as demonstrated by the 1*Rama25-1 Hoechst 33258-Giemsa stain which showed a characteristic 25-I46Rama intense spotty pattern of heterochromatin in mouse fibroblasts 37ERama 50Rama (27) but not in the rat rhabdomyoblastic cell nuclei (Fig. 2E). 52Rama Immunocytochemical visualization of monoclonal antibodies to 53Rama the requisite histocompatibility leukocyte group antigen was not 54Rama 55Rama used because this elicited a very patchy stain in normal tissues 59Rama (32). 6050% Histological stains supported the above pathological descrip " Cell lines were injected into temale nude mice or female Wistar-Furth rats and followed for the appearance of tumors as described in "Materials and Methods." tion of the tumors. The reticulin staining accentuated the epithe The experiment was terminated after 160 to 180 days, and the animals were lial areas of Rama 25, Rama 25-I2, and Rama 25-I4 in the duct- examined for metastatic tumor deposits at autopsy. The number of animals given like regions with a pericellular distribution in the less-well-differ injections was 20 for Rama 25 and 10 each for the remaining cell lines. 6 Female nude mice. entiated and sarcomatous components. Trichrome stains dem c ND, not determined. onstrated very little collagen in the glandular and cuboidal-like

Table 3 Classification of tumors Cytology of tumors prÃ©sent0Glandular types

Cell line in epi- Round or class8Epithelial jectedRama thelialGiant++ ovoid Spindle 25Rama tumor with ovoid and spin ++++ ++++ dle cellcomponentsEpithelial 25-12Rama tumor with ovoid and spin â€”â€” ++++ ++++ dle cellcomponentsRhabdomyosarcomaCarcinosarcoma 25-11Rama ++++ +++ ++++ 25-I4Rama rhabdomy-osarcomatouswith +- ++ ++++ elementRhabdomyosarcomaRhabdomyosarcomaRhabdomyosarcomaUndifferentiated 37ERama +++â€” ++ ++++ +++â€” ++ ++++ 50Rama +++â€” +++ ++++ 52Rama â€”+++++ ++++ 53Rama tumorand spindle cell cellsRhabdomyosarcomaRhabdomyosarcomaRhabdomyosarcomaCellsome ovoid 55Rama ++â€” +++ ++++++++ ++++ 59Rama 60Pathology ++++ + 8 Tumors from Table 2 were classified by standard criteria using hematoxylin and eosin- and PTAH-stained histological sections. 6 Cell types present were recorded from an analysis of the average of 2 sections of each of 2 tumors from each cell line using x400 magnification and counting 6 microscopic fields per section. Scale (percentage of total number of cells in the microscopic field): -, nothing; +, 1 to 5; ++, 5 to 10; +++, 10 to 40; ++++, >40.

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cell areas, but it was more abundant in the spindle cell and anti-myosin serum, although some elongated cells stained in all muscle-like regions of the tumors. Some of the giant, strap-like tumors as for the Rama 25 and Rama 37 CL-A3 tumors (15, cells were stained red consistent with a muscle origin. PTAH 36). Most of the giant and strap-like cells were stained, whereas stains the actomyosin filaments in skeletal muscle cells (14). Only the majority of the round and spindle cells in the rhabdomyoblas cells in the rhabdomyoblastic areas of the tumors exhibited tic regions failed to stain (Fig. 3, F and G). The fraction of giant intensely staining longitudinal fibrils; most giant cells, strap-like cells staining and the intensity of stain in tumors of Rama 60 cells, and smaller elongated cells possessed these fibrils in cells were markedly reduced over those above (Table 4). varying amounts (Fig. 2F). Definite cross-striations were dis Antisera to rat MFGM (10) and to human keratins (25) were cerned in a minority (1 to 10%) of these cells in the well- used to help identify the epithelial elements within the tumors. In differentiated areas of Rama 25-11, Rama 25-I4, Rama 37 E, the normal rat mammary gland, antiserum to MFGM stains the Rama 50, Rama 52, Rama 55, and Rama 59 tumors (Fig. 2G). epithelial cells which line the ducts and alveoli; the myoepithelial They were very occasionally seen in tumors of Rama 52 but cells, fibroblasts, and smooth muscle cells fail to stain. Antiserum were not detected in tumors of Rama 25, Rama 25-I2, Rama 37 to human keratins stains the myoepithelial cells strongly and the CL-A3, Rama 53, Rama 60, or the lung mÃ©tastasesof Rama 59. epithelial cells weakly; all stromal cells fail to stain (41). Antiserum Cross-striations were present in both the tadpole-shaped cells to MFGM stained most of the duct-like regions and some cuboi (Fig. 2F) and the giant and strap-like cells (Fig. 2G). dal-like, ovoid cells in Rama 25-I2, Rama 25-11, and Rama 25-I4 Immunocytochemical Staining of Tumor Cells. Antisera to tumors and the very occasional focus of elongated cells in most both myoglobin and myosin were used to identify the skeletal of the other tumors as for the Rama 25 and Rama 37 CL-A3 muscle-like elements in the tumors. Antibodies to myoglobin tumors (15, 36). In addition, a variable fraction (5 to 70%) of the which are specific for muscle fibers (9) stain heterogeneously, giant and strap-like cells in the rhabdomyoblastic regions were giving rise to a checkerboard appearance (23). In rat skeletal stained, although normal rat skeletal muscle completely failed to muscle, approximately one-third of the fibers stained intensely stain (Table 4). Antiserum to human keratins stained the glan (type I fibers), approximately one-third did not stain (type II fibers), dular epithelial regions of Rama 25-I2, Rama 25-11, and Rama and the remainder stained weakly (Fig. 3A). Only the rhabdo 25-I4 tumors and focal areas of both cuboidal-like and elongated myoblastic elements of the tumors stained with antiserum to cell sheets as for the Rama 25 and Rama 37 CL-A3 tumors (15, myoglobin; glandular areas, cuboidal epithelioid or ovoid cells, 36). This antiserum also stained the occasional spindle cell or and the blocks of elongated cells failed to stain. Giant and strap- group of spindle cells and a small proportion (1 to 5%) of the like cells (Fig. 3, B and C) and cells morphologically intermediate giant and strap-like cells in the rhabdomyoblastic regions of between epithelial cells in duct-like structures and rhabdomyo Rama 50, Rama 52, Rama 55, and Rama 59 tumors (Table 4). blastic cells of the Rama 25-I4 tumors stained intensely (Fig. 3D). To confirm that the original single-cell-cloned cell line Rama 25 The proportion of giant cells in the tumors that stained varied which gave rise to Rama 25-11, Rama 25-I2, and Rama 25-I4 from cell line to cell line (Table 4). Sections of normal rat mam cells was still developmentally determined as a mammary epithe mary glands and DMBA-induced rat mammary tumors failed to lial cell, nude mice bearing Rama 25 tumors were mated, and stain (not shown). histolÃ³gica! sections of the tumors from 3-day lactating animals Antibodies to myosin stain smooth muscle cells, myoepithelial were stained with anti-casein serum (37). A small proportion of cells, and skeletal muscle, but they fail to stain epithelial cells or circular duct-like structures in these tumors specifically stained fibroblasts in the mammary glands in rodents (41). In the tumors, with this antiserum (Fig. 3Â£).Anti-casein serum failed to stain the glandular regions and cuboidal-like or ovoid cells of Rama Rama 25 tumors or Rama 25-I tumors growing in nonpregnant 25-I2, Rama 25-11, and Rama 25-I4 tumors failed to stain with nude mice or Rama 37 tumors or Rama 37 E-derived tumors growing in nonpregnant rats (not shown). Histology and Immunocytochemistry of Cells on Collagen Table4 Gels. To obviate the possibility that some of the cells seen in Immunocytochemical staining pattern of rhabdomyoblastic elements in tumors the tumors growing in rats were derived from host tissues rather Antiserum3 than from the injected cell lines, the Rama 50 series of cloned Cell line injected Myoglobin Myosin MFGM" Keratins" cell lines derived from Rama 37 were grown on floating collagen Rama 25-11 gels in culture. Cells formed multilayered structures on the Rama 25-I4 NDC surface of the gel with a few single cells penetrating the gel, but Rama 37 E no organized gland-like structures as described previously for Rama 50 Rama 52 Rama 25 and Rama 37 CL-A3 cells (15, 29) were observed. Rama 55 Histological sections of Rama 59 cells growing on the gels Rama 59 Rama 59 lung me showed the presence of small elongated cells, larger mononu- tastasis cleated plump cells, and giant multinucleated cells (Fig. 4A). Rama 60 Normal skeletal Similar sections that were stained with PTAH revealed longitu muscle tissue dinal filaments in many of these plump and giant cells, and in " Histological sections from tumors in rodents produced by the injection of the some instances cross-striations were observed (Fig. 48). cell lines shown in Table 2 were immunocytochemically stained with rabbit antiserum to the above antigens ("Materials and Methods ). Only the staining of the Immunocytochemical staining of the collagen gel cultures of Rama 59 with anti-myoglobin serum strongly stained 10 to 20% rhabdomyoblastic elements was scored (percentage of the skeletal muscle-like cells in the microscopic field): -, no staining; +, 1 to 5; ++, 5 to 10; +++, 10 to of the plump and giant cells (p and g, respectively, in Fig. 4C), 40; ++++, >40. These are the average results of scoring 6 microscopic fields the remainder were stained only weakly, while the smaller elon from each of 2 sections from each of 2 tumors of the same cell line. Cells were moderately rather than intensely stained. gated cells failed to stain (Fig. 4C). Staining with antiserum to c NO, not determined. actin which stained the normal gland in a manner similar to that

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1984 American Association for Cancer Research. P. S. Rudland et al. of myosin3 strongly stained the majority of the plump and giant myoepithelial-like) tumors. The epithelial nature of both single- cells, although only the occasional smaller elongated cell was cell-cloned precursor stem cell lines has been firmly established stained thus (Fig. 40, arrows). Staining of the Rama 59 cultures by electron microscopy for Rama 25 (6, 29) and confirmed for with antiserum to MFGM caused an intense cytoplasmic immu- Rama 37 in this paper. Further confirmation has been obtained noreactivity in some of the plump and giant cells (p and g, by the ability of Rama 25 and a subclone of Rama 37, Rama 37 respectively, in Fig. 4E), while others stained more weakly. The CL-A3, when grown as tumors in rodents, to express determi majority of the smaller elongated cells failed to stain (Fig. 4E). In nants characteristic of cells of an epithelial origin, in particular contrast, intense staining with anti-keratin serum was more prekeratin intermediate filaments and MFGM antigens (15, 36). frequently observed in the smaller, elongated cells, whereas the This paper also confirms our findings in vitro (40) that the single- plump and giant cells showed only moderate to weak staining cell-cloned cell line Rama 25 when growing as a tumor in nude (e, p, and g, respectively, in Fig. 4P). However, this moderate mice can produce a protein, casein, which is found only in and weak staining was completely abolished by absorption of differentiated epithelial cells of the mammary gland (44). the antisera with pure human keratins before use, thus ruling In this paper, we show that 2 new series of single-cell-cloned out the possibility of it being a simple artifact. cell lines derived from the epithelial stem cell lines Rama 25 and Ultrastructure of Cells on Collagen Gels and in Tumors. Rama 37 give rise to a third differentiation pattern. On the basis When grown on floating collagen gels and then examined by of the identification of spindle and giant, strap-like cells which transmission electron microscopy, the Rama 37 parental single- exhibit PTAH and myosin-staining longitudinal fibrils and PTAH- cell-cloned cell line at passage 15 displayed a cuboidal morphol staining cross-striations and which contain myoglobin, this dif ogy with somewhat convoluted nuclei, microvilli on the cell apical ferentiation pattern has been ascribed to that of striated muscle. surfaces, and intercellular junctional complexes typical of secre Ultrastructural analysis of the cross-striations demonstrates A-, tory epithelia (Fig. 5A). The Rama 59 cells examined under similar I-, and H-bands and Z-discs and shows the hexagonal arrange conditions showed a wide variation in cell size. The smaller cells ment of thick and thin filaments characteristic of striated muscle were elongated with indented nuclei and peripheral heterochro- (8). Since no intercalated discs are seen and the strap-like cells matin, and their cytoplasm contained abundant rough endo- do not branch, then this is more likely to represent skeletal plasmic reticulum, but usually no thick or thin filaments were muscle than cardiac muscle (1). These features clearly distinguish seen. In contrast, the larger plump cells frequently became giant tumors produced by the new single-cell-cloned cell lines from in size with multiple nuclei, and their cytoplasm was then filled those produced by the parent single-cell-cloned stem cell lines with arrays of longitudinal filaments. These filaments were oc or by the variant, fully elongated cell lines described previously casionally organized into bundles with cross-striations resem (6,15,36). bling the Z-discs of skeletal muscle (Fig. 5ÃŸ).This organization The possibilities that the well-differentiated skeletal muscle- was more clearly evident when the cells grew as tumors in like elements in the tumors may have been produced from host animals, especially those of Rama 50 (Fig. 6A), where cross- components and/or by contaminating skeletal muscle cells in bandings similar to A-, I-, and H-bands in addition to the Z-disc- advertently introduced into our single-cell-cloned cell lines must like structures were observed. The periodicity of the Z-disc-like be considered. The first possibility can be eliminated for 3 structures in these cells was 2.20 Â±0.01 (S.E.) /Â¿m;other cells reasons: (a) the rhabdomyoblasts have a neoplastic phenotype contained similar structures but were apparently devoid of the unlike that of normal regenerating muscle; (b) Rama 25-11 and thicker filaments of the A-band region (Fig. 6ÃŸ),and in this case Rama 25-I4 cells growing as rat tumors in nude mice produce the periodicity was 0.15 Â±0.01 MITI.Similar cells were also seen skeletal muscle-like elements which fail to stain with a relatively in collagen gel cultures of Rama 59 (not shown). When examined specific agent for mouse heterochromatin; (c) when grown on in cross-section, the filaments of Rama 59 cells both in tumors collagen gels in vitro, Rama 37 E-derived cloned cell lines pro and in collagen gel cultures were grouped in a pattern resembling duce giant and strap-like multinucleated cells similar to those that of skeletal muscle, with a central thick filament 15 to 20 nm seen in vivo. The second possibility is extremely unlikely for the in diameter surrounded by 6 thinner filaments, 7 to 11 nm in following 4 reasons: (a) the cuboidal epithelial stem cell lines diameter, and spaced in hexagonal arrangement (Fig. 6A, inset). have been cloned by picking a single cell on 3 successive occasions and characterized as epithelial after the third cloning DISCUSSION step; (b) 2 completely independent cellular systems have been subsequently picked by single-cell cloning from the cloned pre Previous results with the 2 cuboidal single-cell-cloned stem cursor epithelial cells that were derived 2 years apart from cell lines derived from DMBA tumors of the rat mammary gland, separate strains of rat. Both systems gave the same results; (c) Rama 25 (6, 16) and a subclone of Rama 37, Rama 37 CL-A3 multiple single-cell-cloned cell lines from both epithelial stem cell (15), have shown that they produce a spectrum of elongated cell systems yield muscle-like elements in their tumors; (d) when the types in culture. Some of these elongated cells have certain single-cell-cloned cell line Rama 25-11 was single-cell cloned properties characteristic of smooth muscle-like myoepithelial again and then injected into nude mice, it still yielded tumors cells (35). The parent cloned cell lines Rama 25 and Rama 37 which contained some skeletal muscle-like elements.4 CL-A3 give rise to mixed tumors in nude mice (36) or in syngeneic The histogenesis of true rhabdomyosarcomas in animals and rats, respectively (15), composed of both epithelial and myoepi- humans is not entirely clear, since they can occur in epithelial thelial-like elements, while certain single-cell-cloned elongated tissues such as the bladder, prostate, uterus, vagina, and mam cell lines produce tumors containing only spindle cells, some of mary glands as well as arising in skeletal muscle. There are 2 which exhibit myoepithelial characteristics. This has suggested main, although not mutually exclusive, classes: embryonic var- that the fully elongated cell in culture can contribute a proportion of the myoepithelial-like elements to the mixed (epithelial and ' A. Twiston Davies and P. S. Rudland, unpublished results.

2094 CANCER RESEARCH VOL. 44

Â¡antsinchildren and pleomorphic tumors in adults (1). The tumors the other. However, our single-cell-cloned epithelial stem cell produced in rodents by all the cell lines reported here, except lines seem to be capable normally of forming only 2 further cell those by the parental lines, Rama 25-I2, Rama 25-I4, Rama 53, types, myoepithelial-like and alveolar-like cells, both of which are and Rama 60, contain a mixture of cell types that is more part of the mammary parenchyma, and the new cell lines derived reminiscent of the those seen in pleomorphic rhabdomyosarco- from them form only one additional type, skeletal muscle-like mas. However, variable numbers of small, round undifferentiated cells; no epidermal cells in particular are seen. Thus, it seems cells have also been seen, similar to those seen in abundance in most likely that the cloned stem cells are committed as mammary the embryonic variant. Rama 60 tumors contain more of the epithelial cells and that the new clonal cell lines arise directly round cells and also myxomatous areas, thereby giving these from them. Hence, the generation of cells which can give rise to tumors a resemblance to the embryonic variants of this disease skeletal muscle is probably a consequence of an alteration (1). Normally, skeletal muscle is generated from myoblasts. imposed on the genome by the neoplastic process and/or by the These cells appear initially in development as small, round, or process of tissue culture and does not reflect a normal differen oval cells that rapidly become elongated, tadpole, and then tiation pathway. spindle shaped and subsequently develop longitudinal fibrils, Only the cell lines of intermediate morphology and conversion cross-striations, and multiple nuclei (4,8). The round or oval cells rate between that of the cuboidal epithelial and fully elongated described in our tumors may also give rise to the giant and strap- form of the Rama 25 system seem to generate the rhabdomyo like cells [e.g., Rama 50 (Fig. 2C)]. blastic elements. This fact may suggest that such cells are Naturally occurring tumors of the human mammary gland responsible for the epithelial, myoepithelial, and mesenchymal which show rhabdomyosarcomatous differentiation are ex elements that are seen in many of the mixed tumors of glandular tremely rare; they can be pure sarcomas without any epithelial origin. This idea is similar, in certain respects, to the concept of elements (7), cystosarcoma phyllodes with a benign epithelial a pluripotent myoepithelial cell which was first suggested by and a malignant mesenchymal element, or carcinosarcomas Hamper! (21) and was based solely on pathological examination containing both malignant epithelial and mesenchymal elements of these mixed tumors. Our model, however, suggests that it (20). Often, the skeletal muscle elements are accompanied by would be possible, under certain circumstances, to generate other mesenchymal differentiation patterns such as that of car differentiated mesenchymal elements without necessarily pro tilage and osteoid or bone (3). These differentiated mesenchymal ducing fully differentiated myoepithelial cells. Glandular epithelial elements are, however, seen more frequently in the mixed (epi cells which cannot differentiate even to a limited extent along thelial and myoepithelial) tumors that arise in canine mammary the myoepithelial pathway (31, 35) are, however, unlikely to glands and in the salivary glands of animals and humans (21). At produce differentiated mesenchymal elements in their tumors if the moment, these other differentiation patterns have not been this model were to be correct. Finally, the concept of classifying observed in our rodent systems. Strictly, only the tumors of tumors of glandular origin on the assumption that a strict division Rama 25-I4 can be classified as a carcinosarcoma in which the exists between the differentiation potential of epithelial and mes duct-like structures gradually merge morphologically with the enchymal tissues may have to be reexamined (13, 21, 22) in the rhabdomyoblastic elements. A similar transitional pattern has light of the finding that a metaplastic process can fundamentally also been identified recently in one human mammary tumor, but alter the differentiation program of a developmental^ committed this appears to be an isolated and rare case.5 The presence of epithelial cell. some keratin-containing cells in the Rama 50, 52, 55, 59, and 60 tumors does, however, raise the possibility that these tumors ACKNOWLEDGMENTS still retain an element of epithelial differentiation not detectable without this marker, and a case could possibly be made for We thank Anna Twiston Davies, Derek Winslow, and John Ellis for expert technical assistance; Linda Lovell and Simon Birch for looking after the animals; classifying them as carcinosarcomas on this basis. Or. M J. Warburton for gifts of MFGM and keratin antisera; Professor A. M. Neville Since our multiply single-cell-cloned stem cell lines (Rama 25 (all from Ludwig Institute for Cancer Research, London): and Dr. J. P. Sloane (Royal and Rama 37) have been unequivocally identified as epithelial Marsden Hospital, Button. United Kingdom) for advice and discussion. cells and one of them (Rama 25) is certainly committed to make casein, a mammary specific product, we have shown that a REFERENCES single neoplastic rat mammary epithelial cell can give rise, under 1. Ashley, D. J. B. Tumours of voluntary (skeletal) muscle. In: Evans' Histological suitable conditions, to a well-differentiated mesenchymal pattern, Appearance of Tumours, pp. 44-50. Edinburgh: Churchill Livingstone, 1978. that of skeletal muscle. It could be argued that, since no specific 2. Avrameas, S. Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies, immunocy tochemistry, mammary properties have been demonstrated in the cuboidal 6: 43-52, 1969. epithelial stem cells themselves and casein is produced only 3. Azzopardi, J. G. Problems in breast pathology. Major Probi. Pathol., 11: 346- 378, 1979. upon hormonal stimulation of these stem cells in vivo and in 4. Balinsky, B. I. Determination of the primary organ rudiments. In: An Introduction vitro, they could correspond to a cell type that normally occurs to Embryology, Ed. 4, pp. 197-202, 375-377. Philadelphia: W. B. Saunders earlier in development and is competent to form mammary Co., 1975. 5. Bennett, D. C. Rat mammary cell lines, their isolation and characterisation, pp. epithelium as well as skeletal muscle cells. This is highly unlikely. 69-104. Ph.D. Dissertation, University of London, London, England, 1979. Mammary epithelium develops directly from the embryonic epi 6. Bennett, D. C., Peachey, L. A., Durbin, H., and Rudland, P. S. A possible dermis, while skeletal muscle develops normally from the mes- mammary stem cell line. Cell, 75: 283-298,1978. 7. Bird, C. C. A breast sarcoma containing rhabdomyosarcomatous and other enchyme in the embryonic somites (4). Thus, any less committed metaplastic elements. J. Pathd., 101: 286-288,1970. epithelial cell might be expected to form at least epidermal 8. Bloom, W., and Fawcett, D. W. In: A Textbook of Histology, Ed. 10, pp. 328- 330. Philadelphia: W. B. Saunders Co., 1975. (keratinizing) cells on the one hand and cartilage and bone on 9. Brooks, J. J. Immunohistochemistry of soft tissue tumors: myogtobin as a tumor marker of rhabdomyosarcoma. Cancer (Phila.), 50: 1757-1763, 1982. 5 B. Gusterson, unpublished results. 10. Ceriam, R. L., and Paterson, J. A. Characterisation of different antigens of the

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mouse mammary epithelial cell (MME antigen) carried on the mouse milk fat England, 1983. globule. Cell Differentiation, 7: 355-366, 1978. 29. Ormerod, E. J., and Rudland, P. S. Mammary gland morphogenesis in vitro: 11. Chadwin, C. G. A method for the demonstration of reticulin fibres using a formation of branched tubules in collagen gels by a cloned rat mammary cell modified Glees silver solution. J. Sci. Technol., 75; 56-57,1969. line. Dev. Biol., 97: 360-375,1982. 12. Curran, R. C., and Dodge, O. G. Sarcoma of the breast with particular reference 30. Owen, M. J., Auger, J., Barber, B. H., Edwards, A. J., Walsh, f. S., and to its origin from fibroadenoma. J. Clin. Pathol., 15: 1-16, 1962. Crumpton, M. J. Actin may be present on the lymphocyte cell surface. Proc. 13. Dardick, I., van Nostrand, A. W. P., and Phillips, M. J. Histogenesis of salivary Nati. Acad. Sci. USA, 75: 4484-4488, 1978. gland pleomorphic adenoma (mixed tumor) with an evaluation of the role of 31. Paterson, F. C. Cellular and polypeptide changes associated with the differ the myoepithelial cell. Human Pathol., 13: 62-75, 1982. entiation of a mammary stem cell line, pp. 95-116. Ph.D. Dissertation, Univer 14. Drury, R. A. B., and Wallington, E. A. Carieton's Histological Technique, Ed. sity of London, London, England, 1983. 4. Cambridge, England: Oxford University Press, 1967. 32. Ponder, B. A. J., Wilkinson, M. M., Wood, M., and Westwood, J. Immunohis- 15. Dunnington, D. J., Hughes, C. M., Monaghan, P., and Rudland, P. S. Pheno- tochemical demonstration of H2 antigens in mouse tissue sections. J. Histo- typic instability of rat mammary tumor epithelial cells. J. Nati. Cancer Inst., 77: chem. Cytochem., 37: 911-919, 1983. 1227-1240, 1983. 33. Radnor, C. J. P. Myoepithelial cell differentiation in rat mammary glands. J. 16. Dulbecco, R., Henahan, M., Bowman, M., Okada, S., Battifora, H., and Unger, Anat., 777:381-398, 1972. M. Generation of fibroblast-like cells from cloned mammary epithelial cells in 34. Reynold, E. S. The use of lead citrate at high pH as an electron-opaque stain vitro: a possible new cell type. Proc. Nati. Acad. Sci. USA, 78: 2345-2349, in electron microscopy. J. Cell Biol., 77: 208-212, 1963. 1981. 35. Rudland, P. S., Bennett, D. C., Ritter, M. A., Newman, R. A., and Warburton, 17. Emerman, J. J., Enami, J., Pitelka, D. R., and Nandi, S. Hormonal effects on M. J. Differentiation of a rat mammary stem cell line in culture. In: L. Jimenez intracellular and secreted casein in cultures of mouse mammary epithelial cells de Asua, R. Levi Montalcini, R. Shields, and S. lacobelli (eds.), Control on floating collagen membranes. Proc. Nati. Acad. Sci. USA, 74: 4466-4470, Mechanisms in Animal Cells, pp. 341-365. New York: Raven Press, 1980. 1977. 36. Rudland, P. S., Gusterson, B. A., Hughes, C. M., Ormerod, E. J., and 18. Foote, F. R. W., and Stewart, F. W. A histological classification of carcinoma Warburton, M. J. Two forms of tumors in nude mice generated by a neoplastic of the breast. Am. J. Cancer, 40: 74-99, 1940. rat mammary stem cell line. Cancer Res., 42: 5196-5208,1982. 19. FrÃ¼hling,L.,Batzenschlager, A., and Blum, E. Epitheliosarcomes vrais (tumeurs 37. Rudland, P. S., Hughes, C. M., Twiston Davies, A. C., and Warburton, M. J. mixtes maligne) et cancers doubles didermiques de la vessie. Ann. Anat. Immunocytochemical demonstration of hormonally regulable casein in tumors Pathol. NS, 4:5-42, 1959. produced by a rat mammary stem cell line. Cancer Res., 43: 3305-3310, 20. Govan, A. D. T. Two cases of mixed malignant tumor of the breast. J. Pathol., 1983. 56:397-404,1945. 38. Rudland, P. S., Twiston Davies, A. C., and Warburton, M. J. Prostaglandin- 21. Hamper!, H. The myothelia (myoepithelial cells) "normal state" regressive induced differentiation or dimethyl sulfoxide-induced differentiation: reduction changes, hyperplasia, tumors. Curr. Top. Pathol., 53: 161-221, 1970. of the neoplastic potential of a rat mammary tumor stem-cell line. J. Nati. 22. Huber, G., Klein, H. J., Kleinsasser, O., and Schiefer, H. C. Role of myoepithelial Cancer Inst., 69: 1083-1093,1982. cells in the development of salivary gland tumors. Cancer (Phila.), 27: 1255- 39. Stewart, F. W. Tumors of the breast. In: Atlas of Tumor Pathology, Sect. IX, 1264, 1971. Fase. 34. Washington, DC: Armed Forces Institute of Pathology, 1950. 23. James, N. T. Histochemical demonstration of myoglobin in skeletal muscle and 40. Warburton, M. J., Head, L. P., Ferns, S. A., and Rudland, P. S. Induction of muscle spindles. Nature (Lond.), 279: 1174-1175,1968. differentiation in a rat mammary epithelial stem cell line by dimethyl sulphoxide 24. Kahn, L. B., Uys, C. J., Dale, J., and Rutherfoord, S. Carcinoma of the breast and mammotrophic hormones. Eur. J. Biochem., 733: 707-715,1983. with metaplasia to chondrosarcoma: a light and electron microscopic study. 41. Warburton, M. J., Mitchell, D., Ormerod, E. J., and Rudland, P. S. Distribution Histopathology, 2; 93-106,1978. of myoepithelial cells and basement membrane proteins in the resting, preg 25. Moll, R., Franke, W. W., Schiller, D. L, Geiger, B., and Krepier, R. The nant, lactating, and involuting rat mammary gland. J. Histochem. Cytochem., catalogue of human cytokeratins: patterns of expression in normal epithelial 30: 667-676, 1982. tumors and cultured cells. Cell, 31: 11-24, 1982. 42. Warburton, M. J., Ormerod, E. J., Monaghan, P., Ferns, S., and Rudland, P. 26. Mollenhauer, H. H. Plastic embedding mixture for use in electron microscopy. S. Characterisation of a myoepithelial cell line derived from a neonatal rat Stain Technol., 39: 111-114, 1964. mammary gland. J. 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Fig. 1. Morphology of cells in culture. Living cells growing on plastic Petri dishes are shown by phase-contrast optics. A, B, C, cells derived from the stem cell line Rama 25 showing cuboidal epithelial cells, Rama 25 (A), the intermediate cell line Rama 25-11 showing the cells tending to break off from their neighbors and the appearance of longitudinal fibers in their cytoplasm (B), and the intermediate cell line Rama 25-I4 showing large multinucleated cells with their cytoplasm full of fibers (C). A, B, x 190. Bar, 100 urn. C, x 490. Bar, 50 urn. D, E, F, cells derived from the stem cell line Rama 37 showing those from the ring clone strain Rama 37 E with a slightly more elongated morphology than that of Rama 25-11, but less than that of the fully elongated cells of Rama 37 which correspond to Rama 29 (6, 15) (D); a cell line, Rama 59 cloned from Rama 37 E, with similar morphology (E); and giant retractile cells formed in a culture of Rama 52 (F). D, E, f, x 120. Bar, 100 Â¡on. Fig. 2. Histology of tumors. Cell lines derived from Rama 25 and Rama 37 gave rise to tumors in rodents with different morphological forms. A, area of Rama 25-I2 tumor showing well-differentiated glandular form with papillary pattern. B, area of Rama 37 E tumor showing giant multinucleated cells in muscle-like formation. C, area of Rama 50 tumor showing small round (/â€¢)orovoid cells at the top and giant, multinucleated, strap-like cells (g) at the bottom with cells of intermediate morphology (0 in between; D, area of Rama 52 tumor showing giant, multinucleated cells admixed with small round or ovoid cells (r) and tadpole cells (/). Sections A to D stained with H & E. E, area of Rama 25-11 tumor stained with Hoechst-Giemsa showing the occasional mouse stromal cell with stained heterochromatin (arrows); the giant (g) and other cell types have pale-staining nuclei and are seen in phase contrast. A to E, x 330. Bar, 50 urn. F, Rama 50 tumor stained with PTAH showing a large multinucleated cell with only longitudinal filaments and a small cell with thick, dark cross-striations. x 1040. Bar, 20 ion. G, Rama 25-11 tumor stained with PTAH showing a giant, multinucleated cell with longitudinal filaments and thin cross-striations. x 840. Bar, 20 /jtn. Fig. 3. Immunocytochemical staining of tissues and tumors. Histological sections were incubated with the specific rabbit antibody; then, the antibody was located with a second sheep anti-rabbit serum conjugated to alkaline phosphatase and the subsequent development of a red coloration with naphthol dyes. Nuclei were stained blue with hemalum ("Materials and Methods"). In A, B, and D, sections of rat skeletal muscle (A) or Rama 25-I4 nude mouse tumors (8, D) were stained with anti- myoglobin serum, while in C, section of a Rama 25-I4 nude mouse tumor was stained with anti-myoglobin serum that had been previously absorbed with pure myoglobin. Note that in A some of the normal muscle fibers were stained and others were unstained, although smooth muscle cells surrounding blood vessels were always unstained (arrows); staining with absorbed antiserum was completely negative (not shown). Similarly, in B some giant cells were stained and others were not. In D, the duct-like (d) areas of the Rama 25-I4 nude mouse tumor containing cuboidal epithelioid cells (c) gradually merged with areas which contained more elongated and giant, multinucleated (g) cells, some of which were stained with anti-myoglobin serum. In E, sections from Rama 25 tumors growing in 3-day-lactating nude mice were stained with anti-casein serum. Only cells in the duct-like regions stained (arrows), and histological sections incubated with serum preabsorbed with pure rat /3-casein failed to stain (not shown). In F and G, sections of Rama 59 tumors growing in rats were stained with either anti-myosin serum (F) or anti-myosin serum that had been absorbed previously with pure myosin (G). A, B, C, E, F, and G, x 360. 8ars, 50 ^m. D, x 220. 8ar, 50 ion.

2096 CANCER RESEARCH VOL. 44

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Fig. 4. Staining of histological sections of Rama 59 grown on collagen gels. Rama 59 cells were grown on the surface of collagen gels (G) floating in medium (M) and then processed for histology ("Materials and Methods"). Black and white photographs of the colored sections were taken. In A and 6, sections were stained with either H & E (A) or PTAH (B). Note in A that the morphology of the cells varied from small elongated cells (e) through larger, plump, mononucleated cells of varying sizes (p) to giant multinucleated cells (g). Note in B the large cell with cross-striations (cs) plus smaller cells with only longitudinal fibrils (/). In C to F, sections were immunocytochemically stained with anti-myoglobin serum (C), anti-actin serum (D), anti-MFGM serum (E), or antiserum to human keratins (F). Note in C that a few large, plump cells were stained strongly (p) and some giant multinucleated cells (g) were moderately stained; in D, the larger plump and giant cells both in cross- and longitudinal sections were positively stained (arrows); in E, a few plump cells (p) stained strongly, and the giant multinucleated cells (g) stained moderately; in F, some smaller elongated (e) and larger plump cells (p) stained strongly, and the giant multinucleated cells (g) stained moderately and patchily. In C to F. absorptions of the antisera with the requisite antigens yielded a complete absence of staining (not shown). A, C, D, E, and F, x 420. Bars, 50 ^m. B, x 1040. Bars, 20 //m. 2100

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Fig. 5. Electron micrographs of cells on collagen gels. A, Rama 37 cells at passage 15 showing cuboidal morphology, apical microvilli (m), and junctional complexes between adjacent cells (arrows). 8, Rama 59 cells at passage 22 showing cytoplasmic filaments with A- and l-bands and Z-disc-like structures (arrows) A, x 6,900. Bar, 2 um. B, x 19,000. Bar, 1 (/m.

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Fig. 6. Electron micrographs of Rama 50 tumors growing in rats. A. cells with cytoplasmic filaments showing banding into regions similar to the A-, I-, H-, and Z- (arrows) bands of skeletal muscle. Inset, filaments in cross-section, illustrating hexagonal arrangement of thin filaments around a central thick filament. B, cell with Z- disc-like structures (arrows) but apparently devoid of associated thick filaments. A, x 9,000. Bar, 2 jjm. B, x 46,000. Bar, 0.5 ^m. Inset, x 130,000. Bar, 0.1 firn.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1984 American Association for Cancer Research. Production of Skeletal Muscle Elements by Cell Lines Derived from Neoplastic Rat Mammary Epithelial Stem Cells

Philip S. Rudland, Damien J. Dunnington, Barry Gusterson, et al.

Cancer Res 1984;44:2089-2102.

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