Loss of Production of Myoepithelial Cells and Basement Membrane

[CANCER RESEARCH 45, 3864-3877. August 1985]

Loss of Production of Myoepithelial Cells and Basement Membrane Proteins but Retention of Response to Certain Growth Factors and Hormones by a New Malignant Human Breast Cancer Cell Strain1

Philip S. Rudland,2 Richard C. Hallowes, Simon A. Cox, E. Jane Ormerod, and Michael J. Warburton

Ludwig Institute lor Cancer Research (London Branch), Royal Marsden Hospital, Downs Road, Sutton, Surrey SM2 5PX [P. S. R., E. J. 0., M. J. W.], and Imperial Cancer Research Fund, P. O. Box 123, Lincoln's Inn Fields, London, England WC2A 3PX [Ft. C. H., S. A. C.J

ABSTRACT strain, Ca2-83,3 obtained from a primary adenocarcinoma. We also examine the ability of this cell strain to generate myoepithe Digestion of primary breast cancers and their mÃ©tastaseswith lial cells and basement membrane proteins, the loss of which are collagenase yields cell clusters which can be selectively isolated often associated with the transition from benign to malignant from stromal cells and from the less malignant-looking epithelium breast disease (2, 5, 20, 21, 26). The capability of Ca2-83 cells of the primary tumors by their failure to attach as rapidly to to respond to systemic (53) and local (47) trophic agents that collagen gel. Continued passage in culture of one preparation of can stimulate the growth of normal mammary epithelial cells in cell clusters has yielded a continuously growing cell strain, culture (45, 46, 51, 57, 64) is also described. termed Ca2-83. This strain continues to grow mainly as cell clusters with doubling times of 10 to 14 days, although some clusters eventually adhere to plastic substrata. Two morpholog MATERIALS AND METHODS ical extremes of cell were observed, smaller polygonal or cuboidal cells and larger, often-multinucleated cells which contain fat Patient History. Cancer patients were predominantly postmenopausal droplets. Cell clusters grew in a gland-like pattern similar to those (23 of 30) women of varied ethnic origin with breast carcinomas covering the range of clinical staging. Histology of the majority showed infiltrating of the original carcinoma and formed small nodules in 50% of ductal carcinomas. Uninvolved peritumoral tissue from half the patients recipient nu/nu mice. Both morphological forms of Ca2-83 in and malignant pleural effusions from 2 patients were also removed. The culture or in tumor nodules stained immunocytochemically with remaining noncancerous tissues were from reduction mammoplasties of epithelial cell-specific antisera to epithelial membrane antigens otherwise healthy women and from lumpectomies of patients with benign and to human keratins but not to laminin or actin. Cultures of fibroadenomas. One particular 50-year-old patient presented with an Ca2-83 failed to synthesize laminin under conditions where its apparently benign lump in the left breast in June 1982. In December synthesis was observed in a rat myoepithelial cell line. Ultrastruc 1982, she represented with a second lump which was removed by lumpectomy. This lump, termed Ca2-83, was 2 cu cm and composed of tural analysis of the cell clusters has identified microvilli coated Grade II infiltrating ductal carcinoma (10) of rather undifferentiated ap with epithelial membrane antigens and junctional complexes pearance (Fig. 1,4). At subsequent mastectomy, 3 of 27 axillary lymph typical of secretory epithelia in both morphological forms, but no nodes also contained metastatic carcinoma. She presented again with a characteristics of myoepithelial cells or basement membranes histologically similar tumor nodule in the scar region in April and again in were observed. The DNA content of the cultures increased in September 1983 and with a right supraclavicular node largely replaced response to serum, a bovine pituitary fraction, and insulin. Num by a similar tumor in November 1983. She was treated by radiotherapy bers of cell clusters were also increased in the presence of but died of the disease in March 1984. culture medium exposed to preadipocytes, myoepithelial- or Tissue Preparation and Digestion. About a sixth of the surgically mesothelial-like cells/stromal cells, or to prostaglandin E2. removed carcinomas and lymph node mÃ©tastases were transported to the laboratory within 1 h in RPMI 1640 (Grand Island Biological Co., Paisley, Scotland), BSA Fraction V, 2.5 mg/ml (Sigma Chemical Co.). INTRODUCTION Each tumor was processed by a modification (25) of the procedure described for rat mammary tumors (24,45) and for human mammoplasty specimens (54). The specimen was sliced manually into 1- x 3- x 1-mm Although normal epithelial cells from human milk (7, 28, 48, pieces and then suspended in 20 ml of DM in a 50-ml flask fitted with a 56) and from enzymatic digestion of reduction mammoplasties suspended stirrer. The flask was gassed with a mixture of 5% CO2:95% (19, 23, 54) readily yield adherent cell cultures which can be air, sealed with an inverted beaker, and stirred at 20 rpm for about 5 h subcultured up to 4 times before they senesce, human malignant at 37Â°C. About 10% of the tumor fragments were digested, yielding mammary tumors are generally much more difficult to culture (9, numerous small clusters of cells. These short-digest clusters were sep 23,30). A few epithelial cell systems have been established from arated from undigested tissue by filtration through Nitex 1000-Mm cloth primary solid carcinomas (22, 32, 34, 39), but methods are still (H. Simon, Stockport, Cheshire, United Kingdom). Cell clusters were required for the isolation and characterization of malignant cells 3The abbreviations used are: Ca2-83. cell strain derived from lumpectomy of in the primary tumors before the properties of such cells can be main breast cancer patient; BSA, bovine serum albumin; DABA-2HCI, diaminoben- analyzed in any detail. We describe in detail the continuous zoic acid dihydrochloride; DM, digestion medium [RPM11640:fetal calf serum, 2.5% (Grand Island Biological Co.-Biocult):bovine insulin, 1 ng/ml:hydrocortisone, 10 ng/ propagation and characterization of an initially nonadherent cell ml:human transferrin, 5 nQ/m\ (all from Sigma):epidermal growth factor, 5 ng/ml (Collaborative Research Co., Waltham, MA):collagenase type VII, 120 units/ml ' Supported by internal funding from the Ludwig Institute for Cancer Research (Sigma)]; EGF, epidermal growth factor; EMA. human epithelial membrane antigens; and the Imperial Cancer Research Fund. PCS, fetal calf serum; GM, growth medium; PBS, phosphate-buffered saline, Ca2* 2 To whom requests for reprints should be addressed. and Mg2* free; PGE2, prostaglandin E2; Rama, rat mammary, followed by the single Received 8/30/84; revised 2/27/85; accepted 4/15/85. cell clone number.

CANCER RESEARCH VOL. 45 AUGUST 1985 3864

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1985 American Association for Cancer Research. PROPERTIES OF A NEW BREAST CANCER CELL STRAIN washed twice in warm DM:5% PCS without collagenase and left over ICM 405 inverted microscope fitted with phase-contrast optics and a night at room temperature (21 Â°C)in similar medium on a Labquake green filter, and photographs were recorded on llford Pan F film. shaker (Labindustries, Berkeley, CA) inverting 8 times/min. The remaining Production of Biological Material for Histology. The contents of 2 tissue was redigested overnight in fresh DM at 37Â°C as before. This T75 flasks of Ca2-83 (about 106 cells) were collected by centrifugation procedure resulted in more than 50% of the tissue being dispersed, but at 1 x g as before, washed twice in 20 ml PBS and fixed in Methacarn the yield of cellular clusters was less than after the short digest. The [60% methanol:30% inhibisoM 0% acetic acid (v/v)] for 24 h at 4Â°C.Cells clusters produced by overnight digestion were selected by filtration and were rinsed with 70% alcohol, embedded in agar before blocking in washed at 37Â°C for 4 h, as for the short digest. Cells from pleural paraffin wax, and then processed as before (60). Samples were also effusions were used directly without any digestion with collagenase, smeared onto subslides after the final collection step, air dried for 30 whereas peritumoral tissues, mammoplasties, and fibroadenomas were min, and left unfixed. digested as before (23, 54). To investigate their growth in nude mice, Ca2-83 cells at passage 11 Human Tissue Culture and Establishment of Ca2-83. Cell clusters were collected and washed twice in PBS as before. Approximately 0.5 were suspended in 4 ml of DM minus collagenase plus monothioglycerol, x 106 cells in 0.2 ml of PBS were injected into the right inguinal mammary 2 ng/ml, (Sigma) (61), and plated in 2 T25 flasks (Falcon Plastics, Los fat pad of 6-week-old female BALB/c nude (nu/nu) mice (Olac, Banbury, Angeles, CA) containing 2.5 ml of polyermized and medium-equilibrated England). After 12 weeks, the animals were sacrificed, and the injected 0.2% (w/v) collagen gel prepared from rat tails (38). The contents were areas plus potential metastatic sites were examined for visible tumor gassed with 5% CO2:95% air and incubated at 37Â°Cin the same water- deposits, sliced into pieces less than 0.5 cm thick, and fixed in Methacarn saturated atmosphere for 24 h. Unattached cells and clusters were as above. removed with the medium and replated in fresh T25 flasks containing Histology and Immunocytochemistry. Histological sections of the gel. After a further 24 h, this replating was repeated, but into one T25 paraffin-embedded material were stained by hematoxylin and eosin, and flask with and one T25 flask without collagen gel. This stage corre the unfixed smears were stained by oil red O, a nonspecific stain for sponded to day 0 of tissue culture. Two ml of fresh medium were added neutral lipids (35). to each T25 flask at weekly intervals. After 28 days, the floating cell Immunocytochemical staining of sections was carried out using an clusters were collected by centrifugation at 250 x g for 2 min, resus- alkaline phosphatase-conjugated second antibody (3, 60). Primary anti pended in 2 ml of medium, and plated into 2 T25 flasks without collagen bodies raised in rabbits against rat milk fat globule membrane, purified gel. Cell clusters that had attached to the plastic surface were released human keratins (Mrs: 69,000; 57,000; 47,000; 45,000; and 43,000), by pipetting, collected by centrifugation, and replated in 2 ml of medium bovine smooth muscle actin (Miles Chemical Co.), and mouse laminin into one T25 flask. Two ml of fresh medium were added at weekly have been described previously (21, 59, 60). Rabbit antisera to human intervals. Three weeks later, each of the 3 T25 flasks were subcultured EMA (52) was obtained from Dr. M. G. Ormerod (Institute for Cancer 1:2. Since then the stock flasks have been subcultured 1:2 at 4- to 5- Research, Sutton, Surrey, United Kingdom) (52). Purified IgGs (60) were weekly intervals by gently pipetting to release attached cells, collecting diluted between 1:20 and 1:2,000 with 0.5% BSA in PBS before use. cell clusters at 1 x g for 20 to 30 min, and replating the cultures in fresh Sheep anti-rabbit IgG conjugated to alkaline phosphatase with glutaral- medium. The entire population of cells growing in suspension from the dehyde was purchased from Sigma. Absorbed antisera were prepared original specimen was cultured for 5 passages, and a fraction of the by incubating antisera with 1 mg of antigen per ml at 37Â°C for 3 h. overall pool was passaged further; the remainder was stored as frozen Sections were counterstained with Mayers hemalum and photographed cell stocks as described previously (54). on a Reichert Polyvar microscope with a Kodak Wratten No. 44 filter on One cell strain from the Ca2-83 lumpectomy has been subcultured 23 llford Pan F film. times in the above way by January 1985, except that after 4 subcultures The specificity of staining for each antiserum was checked by the the monothioglycerol was omitted from the medium, and after the sixth following controls: (a) sections of normal mammary glands from a 19- subculture, phosphoethanolamine, 1.4 ng/m\ (Sigma) and Batch 5, 70 year-old woman or from 70-day-old female rats were stained in parallel /ig/ml, of a bovine pituitary extract from Dr. R. Ham (University of and compared with previous results for consistency of staining (21, 52, Colorado, Boulder, CO) (37) were included. The latter extract was the 59); (b) complete abolition of staining was achieved by prior incubation aqueous soluble fraction of a 0.15 M (NH4)2SO4 precipitation step at pH of each antiserum with the requisite antigen(s); (c) tumor cells in nude 7 and contained pituitary mammary and fibroblast growth factor (53). mice which failed to stain were only recorded as negative, providing that After 9 subcultures, some stocks of Ca2-83 (approximately, 2 x 105 an appropriate normal mouse cellular structure could be identified as cells) were suspended in 20 ml of GM (RPMI 1640, 2.5% FCS, 1 /Â¿g/ml staining on the same section; and finally, (d) 2 sections from 2 nude insulin, 10 ng/ml hydrocortisone, 5 ng/m\ human transferrin, 5 ng/ml mouse tumor nodules or from 4 separate isolates of cultured Ca2-83 EGF, 1 /ig/ml phosphoethanolamine, and an extra 2.4 mg/ml NaHCO3) cells were examined with each antibody. and grown in T75 Falcon flasks in an atmosphere of 10% CO2:90% air Electron Microscopy. Clusters of Ca2-83 cells were fixed and proc for 2 weeks, and then 20 ml fresh GM were added for a further 2 weeks essed for electron microscopy as described previously (40). Immunolo- before transfer as above. Ca2-83 was found to be free of mycoplasma calization of EMA at the ultrastructural level (41) was accomplished by by fluorescent staining with an indicator cell layer and did not contain incubating the cell clusters with 1:500 anti-EMA serum in PBS and 0.5% virus particles visible in the electron microscope. BSA for 1.5 h at room temperature. Cell clusters were washed in PBS, Routine Tissue Culture. The established cell lines from the rat mam then with PBS plus 0.5% BSA three times, before being incubated with mary gland, Rama 29 (6), Rama 401 (59), Rama 351 (47), and the SMT- 1:10 sheep anti-rabbit serum conjugated to horseradish peroxidase for 2A cell feeders (12, 14) were cultured as described previously. For 1.5 h. Cell clusters were rewashed as above and incubated with 0.05% preparation of feeder cells, 80 to 90% confluent cultures were incubated diaminobenzidine and 0.06% H2O2in PBS for 5 min at room temperature. with mitomycin C, 0.5 M9/ml. for 16 to 18 h, and then 4 x 105 cells were Cells were fixed and processed as described previously (40), except that transferred into T25 Falcon flasks in 5 ml of GM (6). For preparation of the staining with uranyl acetate and lead citrate was omitted, and viewed conditioned media, 80 to 90% confluent cultures were washed twice directly in the electron microscope. with PBS and incubated with RPMI 1640: NaHC03, 2.4 mg/ml, for 24 h Determination of Laminin Synthesis. Approximately 10E Ca2-83 at 37Â°C, filtered through 0.22-/Â¿mfilters and stored at 4Â°Cbefore use. passage 14 cells were plated in 10 ml of 90% Dulbecco's modified Eagle's medium without proline:5% Dulbecco's modified Eagle's me- Growth factors and hormones were then added directly to reconstitute serum-free conditioned GM. FCS was stripped of free steroids by treat dium:5% FCS dialyzed extensively against PBS: hydrocortisone, 50 ng/ ment with dextran-coated charcoal as described previously (50). ml: insulin, 50 ng/ml: i_-[3H]proline 10 ^Ci/ml (Amersham Int., United Morphological appearance of living cultures was observed in a Zeiss Kingdom) and grown for 7 days. Cell clusters were collected by centrif-

CANCER RESEARCH VOL. 45 AUGUST 1985 3865

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1985 American Association for Cancer Research. PROPERTIES OF A NEW BREAST CANCER CELL STRAIN ugation, lysed, and the dialyzed lysate and medium were immunoprecip- Table 1 itated with rabbit anti-mouse laminin serum or normal rabbit serum as Adherence of cultured digests of different mammary tissues to collagen gel described previously (59). Quantitation of laminin released into GM was No. of patients with different types of carried out by enzyme-linked immunoassay as described by Rennard ef epithelium in culture al. (44). Determination of Growth Rates of Ca2-83 Cells. For determination Total no. of adherent adherent of total DNA in cultures, cell clusters equivalent to about 2 ng of DNA patientsNormalMammary tissue8 colonies04010122500Slow-colonies00021292Nonadherentclusters"0002792 were suspended in 3 ml of the appropriate medium in each of 10 T25 mam-moplasty)Benign(reduction flasks, and 2 similar samples were retained for 0-time estimation. Further additions of appropriate medium were made as follows: 2 ml immediately; tumor (fibroade 2 ml on Day 7; 3 ml on Day 14; and 4 ml on Day 21. Duplicate cultures noma)Peritumoral (uninvolved)Primary were removed at each time point and harvested separately by first jetting carcinomaSecondary off any adherent colonies and then displacing any remaining cells with a inaxillary carcinoma silicone-rubber wedge in 5 ml PBS. Cells were collected by centrifugation nodeSecondarylymph aspleural carcinoma at 500 x g for 3 min, washed in 10 ml of PBS by centrifugation, and stored at -20Â°C. DNA was estimated by the binding of DABA-2HCI effusion401015a30*10*2Fast- 8 Mammary tissue was classified from histological sections, digested with col- (Aldrich Chemical Co., Gillingham, United Kingdom) to its purine deoxy- lagenase (except the pleural effusions), and the digest was cultured on collagen ribose portion as reported by Hinegardener (27), except that the DABA â€¢ gels as described in "Materials and Methods." 2HCI was purified in aqueous suspesion with activated charcoal and b Fast-adherent colonies of epithelial cells stuck to the collagen gel within 48 h dried slowly at 37Â°C instead of at 60Â°C, and 0.22-Mm filtered glass- after plating, and usually within 24 h. They always contained elongated, presumptive myoepithelial-like cells at their peripheries. distilled water was used throughout. DNA standards in the range 0.4 to c Slow-adherent colonies of epithelial cells took longer than 72 h to stick to the 2.0 ng and thawed-cell pellets were evaporated to dryness at 37Â°Cand collagen gel and did not contain elongated cells at their peripheries. incubated with 0.25 ml of freshly purified DABA-2HCI, 0.4 g/ml, at 60Â°C Nonadherent cell clusters of epithelial cells took longer than 21 days, if at all, to stick to the gel. for 60 min in sealed tubes. They were cooled, 5 ml 1 M HCI was added 8 A few samples (1 peritumoral, 3 primary carcinomas, and 1 lymph node to each, and the fluorescence was recorded in a modified Aminco metastasis) contained no visible epithelium after digestion. Bowman spectrofluorometer at 495 urn using excitation at 405 /Â¿m. For determination of the number of cell structures, duplicate cultures were seeded at 500 to 1000/T25 Falcon flask in either regular or Primaria 54). In these cases, both the polygonal epithelial-like cells and flasks in 5 ml of the specified medium. The average number of adherent the elongated presumptive myoepithelial-like cells were cytolog- and total cell structures was determined by counting 20 randomly ically normal in that there was no marked nuclear pleomorphism, selected microscopic fields in an Olympus CK inverted microscope (x40, few if any bi- or multinucleated cells, and no aberrant mitotic 14 sq mm microscopic field). Adherent colonies were assessed by their figures. In addition, variable numbers of cell clusters were also ability to remain attached to the substratum after 2 shakes of the culture seen that rarely attached to the collagen gel within 72 h (Fig. flasks. For determination of the total protein content, the adherent and suspended cells were collected by centrifugation at 200 x g for 5 min 16), and those that eventually did were easily released by gentle and assayed by the method of Lowry ef al. (36). pipetting (Table 1). Any cellular colonies that formed from these For autoradiography, 2-day-old cultures in GM were incubated with 3 clusters contained only polygonal epithelial-like cells (Fig. 1C); iiCi/ml [3H]thymidine, at 1 ^M, and duplicate flasks were removed after no elongated cells were observed. Cells in these slow-adherent 48 and 72 h. Cell clusters were collected by centrifugation and processed colonies and nonadherent clusters were cytologically malignant as described previously (45) before being smeared onto subslides. The in that they showed a marked nuclear pleomorphism, usually slides were air dried for 30 min, coated with Nuclear Immulsion, exposed, high nuclear to cytoplasmic ratios, and were often bi- or multi- developed, and fixed as described by Williams (62), and finally counter- nucleated with some aberrant mitotic figures. Only a few of these stained by hematoxylin and eosin. They were viewed and photographed cells proliferated and then at a rate slower than those in the fast- in a Reichert Polyvar microscope as above. Results for the percentage adherent colonies. In contrast, cultured lymph node mÃ©tastases of cells with radioactively labeled nuclei were the average of duplicate and malignant pleural effusions yielded only the nonadherent cell smears, 10 fields of not less than 100 cells being counted. clusters which occasionally formed the slow-adherent colonies; no fast-adherent colonies containing elongated cells were ob RESULTS served (Table 1). In all cases, the stromal cells attached readily to the gel so that in 3 daily transfers their total number was Culture of Human Breast Cancer Cells and Establishment greatly reduced. The same pattern of finding only fast-adherent of Ca2-83. Collagenase digests of primary and secondary tu colonies of epithelial and elongated presumptive myoepithelial mors from patients with infiltrating ductal carcinomas behaved cells from normal mammary glands, benign breast lesions and differently on collagen gels in our general GM (see "Materials peritumoral but pathologically "normal tissues," and of finding and Methods"). Digestion of the primary tumors usually yielded only nonadherent or loosely adherent epithelial cell clusters de organoids which adhered to the collagen gel after 24 to 48 h. void of elongated myoepithelial cells from secondary breast These fast-adherent structures grew rapidly for the next 7 to 21 cancers has now been observed for many more tissue samples.4 days to produce cellular islands or colonies containing polygonal, Although a few of the nonadherent cell clusters appeared to epithelial-like cells and at their peripheries, elongated, presump multiply initially and at least to double their cellular volume, only tive myoepithelial-like cells (Table 1). Three-dimensional tubes 5 of the 30 primaries gave rise to short-term cell strains that were also seen to protrude below some of the colonies into the could be subcultured up to 2 times during the following 6 months, gel. These cellular colonies bore a striking morphological resem but even these were eventually lost. Most of the primary cultures blance to those obtained from digests of reduction mammoplas- were eventually lost to fungal infections. However, nonadherent ties of otherwise healthy women, benign fibroadenomas, and uninvolved peritumoral tissues of the breast (Table 1) (23, 25, 4R. C. Hallowes,unpublishedresults.

CANCER RESEARCH VOL. 45 AUGUST 1985 3866

Å“il clusters from the primary tumor of Ca2-83 (Fig. 14) continued Table 2 to proliferate in culture with a doubling time of 10 to 14 days Immunocytochemical staining pattern of cultured cells and tumor nodules in nude mice without any such infections, and this cell strain has now been passaged routinely 23 times (see "Materials and Methods"). Percentage of cells staining8 Many of these cells continued to grow in suspension as cell hu nude mouse nodules"70-80 clusters, some of which possessed bulbous projections (Fig. 1S). usedHumanAntiserum mangland40-60C cells70-80 gland0 Some clusters eventually adhered to the plastic substratum, and EMA 40-70Â°50-70" polygonal epithelioid cells spread out as a monolayer forming Rat milk fat globule mem 025-35c 045-55 045-55 small compact islands (Fig. 1C). However, transfer of slow- brane Keratins 20-30" 25-35" adherent colonies alone to fresh medium yielded the same pro Actin 0 0 25-35"Cultured 25-40"Tumor portion of slow-adherent and suspended cell clusters, and wee LamininNormal 0Normal 0 versa. Ca2-83 cells did not attach to 0.15 to 0.2% collagen gels " Histological sections were immunocytochemically stained with rabbit antiserum to the requisite antigens (see "Materials and Methods"), but only the staining of the and failed to produce tubular protrusions when embedded in mammary parenchymal cells were scored in the normal mammary glands. All similar gels, although expanding cell clusters were observed in results are expressed as a range of percentages from determinations made upon 6 microscopic fields from each of 2 sections of 2 separate specimens. 0.05 to 0.1% gels. Two morphological extremes of cell were 6 Tumor nodules were caused by the injection of Ca2-83 cells into the mammary observed in the slow-adherent colonial cells at all passage num glands of female nude mice (see "Materials and Methods"). bers, small (10 to 20 ^m), dark, isometric cells with a large pale c Only the epithelial and not the myoepithelial cells stained. Only the myoepithelial and not the epithelial cells stained. nucleus, often with more than one prominent nucleoli (Fig. 1Â£), 8 Mouse myoepithelial cells stained intensely, although some epithelial cells were and large (>40 /Â¿m),more irregularly shaped, often multinu- also stained rather more weakly. cleated cells with a very granulated or foamy cytoplasm (Fig. 1D). There were also morphological intermediates between these epithelial cells and fibroblasta failed to stain, whereas antiserum 2 extremes (Fig. 1C). No permanently elongated or flbroblastic to laminin stained the basement membrane adjacent to the cells were observed. Ca2-83 cells were aneuploid with a mean myoepithelial cells as reported previously (21). However, no chromosomal number of 63 Â±11 (SD). Stocks of Ca2-83 were cellular staining with either antibody could be detected in any slowly frozen to -70Â°C as described previously (54) and then cultured cell clusters (Fig. 3, A and B). To confirm that laminin stored in liquid nitrogen. These stocks were successfully thawed, was not synthesized, Ca2-83 cells were labeled with [3H]proline, grown up, and found to be indistinguishable from nonfrozen Ca2- and the culture medium was immunoprecipitated with antilaminin 83. serum. No radioactive peptide corresponding to laminin was Histology and Immunocytochemical Staining of Cultured detected in the immunoprecipitate, although laminin was de Cells. HistolÃ³gica!examination of the nonadherent cell clusters tected in the culture medium of the rat myoepithelial cell line revealed circular structures of tightly compact, cuboidal cells of Rama 401 (not shown). Similarly, although Rama 401 cells varying sizes with a tendency to multilayering. They thus formed secreted 1.4 to 5.1 Â¿igof laminin/24 h/mg cell protein as mea a gland-like pattern of a rather undifferentiated adenocarcinoma sured by an enzyme-linked Â¡mmunoassay,that secreted by Ca2- (Fig. 2A) similar to that of the original tumor (Fig. 14). The cultured 83 cells was not detectable, the lower limit of detection being cells were cytologically malignant with a marked nuclear pleo- 0.02 Mg/24 h/mg protein. morphism, usually high nuclear-to-cytoplasmic ratios, often bi- Induction of Tumor Nodules in Nude Mice. Injection of Ca2- or multinucleated with some aberrant mitotic figures. The larger 83 cells in PBS into the right inguinal fat pad of female nude cells often possessed a granulated cytoplasm which stained mice yielded no palpable tumors after 12 weeks. However, 50% intensely with oil red O (Fig. 2B), a stain for neutral lipids (35). (4 of 8) of the animals contained a small nodule of cells, less Even some of the smaller cuboidal cells possessed finely staining than 1 mm diameter, at the point of injection (Fig. 3, C to F). lipid droplets (Fig. 2B). Nude mice injected with PBS alone formed no nodules. No Antisera to human EMA and to human keratins were used to additional tumor deposits were detected on inspection or in identify the epithelial cells. Antibodies to EMA stained epithelial histological sections of the left inguinal mammary fat pad, lungs, cell surfaces lining the ducts and lobules of the human mammary liver, spleen, kidneys, heart, lymph nodes, or gonads of any of gland (52) (Fig. 2C). The myoepithelial and stromal cells failed to the 4 mice with tumor nodules. Tumor nodules were well circum stain. Antisera to human keratins stained the human glandular scribed by condensed connective tissue elements and were epithelial cells strongly but heterogeneously and the myoepithe composed of a relatively solid cord of cells occasionally surround lial cells more weakly; the stromal cells failed to stain (21) (Fig. ing small, lumen-like structures. The cells varied in size, were 2D). The majority of Ca2-83 cells in the circular structures could cytologically malignant, and were often bi- or multinucleated (Fig. be stained specifically by antisera to both EMA (70 to 80% of 3, C to F). cells) (Fig. 2, E and F) and human keratins (45 to 55% of cells) The majority of tumor cells in the nodules stained immunocy (Fig. 2, G and H), yielding appreciably higher percentages of cell tochemically with antiserum to human EMA, whereas no cells of staining than in the human mammary gland (Table 2). The the nude mouse host stained with this antibody (Fig. 3C). More strongest staining for EMA was observed at the cellular peri over, tumor cells failed to stain with antiserum to rat milk fat pheries, although weaker cytoplasmic staining was also ob globule membrane, whereas the majority of the luminal epithelial served; staining for keratins was confined to the cytoplasm. cells of the nude mouse mammary gland stained intensely (Fig. Antisera to actin and to the basement membrane protein 3D). Approximately one half of the tumor cells and most of the laminin were used to identify any myoepithelial cells. In the normal mouse mammary gland cells also stained with antihuman kera mammary gland, antiserum to actin stained the myoepithelial tins serum (Fig. 3E). No staining of the tumor cells for laminin cells and smooth muscle cells which lined blood vessels; the (Fig. 3F) or for actin (not shown) was observed under conditions

CANCER RESEARCH VOL. 45 AUGUST 1985 3867

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1985 American Association for Cancer Research. PROPERTIES OF A NEW BREAST CANCER CELL STRAIN where basement membranes and myoepithelial cells of the ad jacent host mouse mammary glands were stained. The percent ages of tumor cells staining with these 5 antisera were virtually identical when growing in culture or as nodules in nude mice (Table 2). Ultrastructure of Cultured Cells. When observed in the elec tron microscope, the cells in clusters of Ca2-83 were predomi nantly cuboidal, although they varied in size, and were closely apposed with interdigitating membranes between adjacent cells (Fig. 4/4). Where cells were exposed to the medium, some junctional complexes typical of epithelia were seen at apical- lateral borders, although their 3 characteristic elements were not always distinguishable. Desmosomes containing a recognizable intercellular contact layer were also present, and typical tonofilaments issued from both halves (Fig. 46). Where cells were exposed to the medium, microvilli were also seen, and these varied from being short and sparse to being long and in parallel arrays (Fig. 4, A and C). The nuclei varied from having a rounded and smooth morphology to an indented shape associated with prominent heterochromatin. Prominent, sometimes multiple, nu cleoli were seen. Mitochondria, rough endoplasmic reticulum, 10 20 30 polyribosomes, and Golgi apparati were present to varying de Time (days) grees in the cytoplasm. Some cells contained large numbers of Chart 1. Effects of pituitary extract, insulin, EGF, and hydrocortisone on DNA content of Ca2-83 cultures. Cultures were set up in RPMI 1640 as described in fat droplets, either concentrated in the perinuclear region or "Materials and Methods." The fold increase in DNA for a given time in culture = scattered throughout the cytoplasm. Some fat droplets were as DNA content of the cultures at that time â€¢*â€¢DNAcontent after 0 days for a given large as 2 to 3 um diameter and could occupy up to 70% of the set of additives. Results are the average of duplicate cultures which agreed to cytoplasm (Fig. 4/4). Individual filaments, 10 to 12 nm diameter within 10%. In A. the medium consisted of RPMI 1640, BSA alone, 2.5 mg/ml (â€¢}. or with pituitary extract, 70 pg/m\ and insulin, 1 /Â¿g/ml(Ã€);controls with RPM11640 and probably of the intermediate type (43), were abundantly and 2.5% FCS (â€¢)were also included. In B, the medium consisted of RPMI 1640 scattered throughout the cytoplasm of some cells (Fig. 46). and 2.5% FCS alone (â€¢)or with the following additives: V, EGF (5 ng/ml); O, However, features characteristic of myoepithelial cells such as hydrocortisone (10 ng/ml); A, insulin (1 tÂ¡g/m\);Y, pituitary extract (70 fig/ml); A, pituitary extract (70 Mg/ml) plus insulin (1 Â»ig/ml). myofilaments, pinocytotic vesicles, hemidesmosomal connec tions to basement membranes (43), and even basement mem Table 3 branes themselves were absent. Relative effects of estradici, progesterone, and pituitary extracts on DNA content Ultrastructural localization of the binding of anti-EMA serum to of Ca2-83 cultures Fold increase in DNA in cultures containing" the cultured cell clusters revealed that this antigen occurred Additionsmedium3None to FCSC5.3 mainly on the surfaces of cells that were exposed to the medium, FCS12.1 particularly in areas where there were microvilli (Fig. 4D). In these areas, the reaction product coated the whole of the outer sur Progesterone 7.5 15.2 faces of the microvilli (Fig. 4C). The large, fat-droplet-containing Estradici 10.1 16.5 Pituitary extractStripped 11.3Untreated 18.0 cells also posssessed extensive arrays of microvilli on their Cultures of Ca2-83 cells were set up as described in "Materials and Methods" surfaces that were coated with EMA (Fig. 5). in a medium consisting of RPMI 1640, 2.5% FCS, insulin (1 ng/ml), hydrocortisone Effect of Different Medium Components on Growth in Cul (10 ng/ml), EGF (5 ng/ml), plus one of 1 nM progesterone, 1 nM estradiol, or pituitary extract (70 i*g/m\) as indicated. ture. The total DNA content of cultures of Ca2-83 cells increased 6 Fold increase in DNA = DNA content in cultures after 21 days â€¢+â€¢DNAcontent throughout the 28 days tested, provided that additional volumes after 0 days for a given set of additives. Results are the average of duplicate cultures which agreed to within 10%. of media were added to feed the increasing numbers of cells. In c FCS was stripped by incubating with charcoal (see "Materials and Methods") the presence of RPMI 1640 and 2.5% FCS, the DNA content prior to its addition to the medium. per culture increased by 4 times after the initial 14 days and by 14 times after 28 days (Chart 1/4). Addition of insulin, 1 ^g/m\, without effect. When FCS was replaced by BSA, 2.5 mg/ml, the and bovine pituitary extract, 70 ^g/m\, increased these totals by DNA content failed to increase with time, although the pituitary about a further 3 times in both cases. Insulin alone at 1 pg/m\ extract and insulin still stimulated a 5-fold increase by 28 days had no effect, whereas the pituitary extract alone increased the (Chart 1/4). Growth of Ca2-83 cells with charcoal-stripped FCS DNA content by 25 to 55% (Chart 18). The optimal concentration resulted in a lower DNA content per culture than with nonstripped of insulin was 1 fig/m\, and higher concentrations were inhibitory, serum, and in both cases, 1 nM estradiol or 1 nM progesterone whereas the effect of the pituitary extract was discernible at 10 increased slightly the DNA content of the cultures (Table 3). i/g/ml and increased proportionally up to about 400 /Â¿g/ml(not Their combination, however, was inhibitory (not shown). shown). Addition of hydrocortisone, 10 ng/ml, EGF, 5 ng/ml The total number of Ca2-83 cells growing in culture was (Chart 16), or a combination of insulin, hydrocortisone, and EGF linearly related to the number of cell clusters, since the number (Table 3) all inhibited slightly the DNA-stimulatory effect of the of cells in a cluster was approximately constant at 100. Hence, FCS, whereas phosphoethanolamine and transferrin, all compo the progress of the cultures could be followed readily by counting nents of the original GM (see "Materials and Methods") were such structures. The total number of structures in GM without

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Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1985 American Association for Cancer Research. PROPERTIES OF A NEW BREAST CANCER CELL STRAIN serum declined slightly over a period of 32 days. With increasing Table 4 concentrations of serum up to 10%, the number of structures Effect of different feeder cells on the number of cell structures in culture was dramatically increased by up to 5-fold for the first 10 days Number of structures/field" of culture. Beyond 32 days, however, the optimum serum con cells8None Feeder centration for producing the maximum number of structures and Â±0.8 Â±256 for the highest cellular protein content was 5% (Chart 2B). The SMT-2A feeders (mesothelial-like/stromal 1.1Â±0.427 Â±448 mesothelial-like/stromal) structure doubling time was about 10 days in GM. That the cells Rama29 (myoepithelial-like) Â±2 Â±2 in GM were synthesizing DNA was confirmed by autoradiography Rama351 (preadipocyte)Adherent6.3 19Â±3Total22 45 Â±4 (Fig. 6). Exposure of cells for 48 or 72 h to pHJthymidine yielded * Near confluent (80 to 90%) cultures of rat feeder cells were inactivated by 43 Â±3% or 64 Â±1% radioactively labeled cell nuclei, respectively, treatment with mitomycin C, and 4 x 105feeder cells were plated per 26-sq cm normal tissue culture flask. corresponding to a cell division time of about 10 days. Increasing 6 Approximately 1000 cell structures/26-sq cm flask were added in 5 ml of GM, concentrations of PCS caused an even more dramatic increase and after 10 days an extra 5 ml of fresh GM was added. The average number of in the number of adherent structures, rising to almost 50% of cell structures Â±SE/14-sqmm microscopicfield from 2 flasks, 20 fields/flask, was recorded after a total of 28 days. Both the total number of structures and the the total number in 10 or 20% PCS after 10 days (Chart 2A). number of structures sticking to the surface of the flask (adherent structures) were Incubation of Ca2-83 cells with nondividing rat feeder cells, recorded. The total cellular protein content (Â±SE)ofthe flasks after 28 days was the mesothelial-like/stromal mesothelial-like/stromal feeder SMT- for no feeders, SMT-2A feeders, Rama 29 cells, and Rama 351 cells: 350 Â±10, 750 Â±40,700 Â±40,and 680 Â±30^g of total protein, respectively. 2A feeder cells (13, 14), the myoepithelial-like Rama 29 cells (6), and the mammary preadipocytic Rama 351 cells (47) also stim ulated the total number of structures and protein content by about 2-fold over that observed in GM alone. However, whereas 15 the SMT-2A feeders reduced the number of adherent structures of Ca2-83, Rama 29 and Rama 351 cells stimulated their ap pearance by 3- to 4-fold (Table 4). When serum-free medium was exposed to any one of the feeder cells for 24 h (see "Materials and Methods") and then incubated with Ca2-83 cul tures in GM without serum, the total number of cell structures was increased by about 2-fold after 10 days, and thereafter declined (Chart 3B). This increase in 10 days was approximately

10 20 30 Time (days) Chart 3. Effect of growth-promoting agents released by different cultured cells on the number of cell structures in culture. Cultures were seeded at 550 cell structures/26-sq cm Primaria tissue culture flask in GM without serum and with various additives. The average total number of structures (both floating in the medium and attached to the substratum) Â±SE(Ã¶ars)/14-sqmm microscopic field from 2 flasks, 20 fields/flask, were recorded. A. medium with different concentra 10 20 30 tions of serum: O, no PCS;T, 2.5% PCS;A, 5% PCS;and â€¢10%FCS. The total cellular protein content (Â±SE)ofthe flasks after 32 days were for 0, 2.5, 5, and Time (days) 10% PCS:22 Â±1, 231 Â±1,300 Â±15,and380 Â±20/jg of total protein,respectively. Chart 2. Effect of different concentrations of serum on the number of cell B, mediumwith no added serum but exposed to no cells (O),SMT-2A feeder cells structures in culture. Cultures were seeded at 550 cell structures/standard 26-sq (rat mesothelial-like/stromalcells) (â€¢),Rama29 cells (rat myoepithelial-likecells) cm tissue culture flask in GM without serum, and various concentrations of serum (A), and Rama 351 cells (rat mammary preadipocytes) (â€¢).Theusual hormones were added back. The average number of structures Â±SE (bars)/14-sq mm and growth factor fractions in GM were added to the medium exposed to the microscopic field from 2 flasks, 20 fields/flask were recorded at the times shown. different cells immediatelyprior to addingthe Ca2-83cells.The total protein content In A, the number of structures adhering to the plastic substratum, and in 8, the (Â±SE)ofthe flasks after 32 days were for no cells. SMT-2A feeder cells, Rama29, total number of structures (both floating in the medium and attached to the and Rama 351 cells: 30 Â±5, 60 Â±5, 101 Â±8, and 125 Â±13 /ig of total protein. substratum) were recorded for: O, no PCS; T, 2.5% PCS; A, 5% PCS; â€¢10% respectively. C, medium with no added serum but with the usual hormones and PCS;and â€¢.20%PCS. In 8, the total cellular protein contents [Â±SE(bars)] of the growth factor fractions and various concentrations of PGE2as follows: O, Ong/ml; flasks on termination of the experiment after 32 days were for 0, 2.5, 5, 10, and T, 0.5 ng/ml; A, 5 ng/ml; â€¢50ng/ml; and â€¢,500ng/ml. The total protein content 20% PCS:24 Â±3,320 Â±30,420 Â±50,360 Â±30.and 390 Â±6Mgof total protein, (Â±SE)ofthe flasks for PGE2after 32 days was for 0, 0.5. 5. 50, and 500 ng/ml: 29 respectively. Â±5,106 Â±4, 140 Â±15,122 Â±4,and 59 Â±5Â»goftotal protein, respectively.

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Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1985 American Association for Cancer Research. PROPERTIES OF A NEW BREAST CANCER CELL STRAIN the same as that achieved by 2.5 to 5% PCS, although the antisera to a similar rat determinant (60). No evidence for the effects of serum were maintained over longer periods of time other major cell type of the mammary parenchyma, the myoepi (Chart 3A). To obviate the possibility that trace amounts of serum thelial cell, can be found at the light or electron microscopic level were released from the feeder cells, so causing the stimulatory in cultures or in tumor nodules of Ca2-83 in nude mice (43). That effects, the conditioned media were added separately to cultures Ca2-83 cells are the malignant cells from the original lumpectomy of Ca2-83 cells in GM with the usual 2.5% FCS. However, all 3 specimen is suggested by the following findings: (a) the cells are conditioned media still stimulated the number of cell structures cytologically malignant; (b) the histology of the cell clusters in by about 2-fold (not shown). The trophic agent for rat mammary culture is similar in many respects to clusters in the original epithelial cells that is released by Rama 351 preadipocytes is specimen; (c) the 2 major cellular pleomorphic variants seen in PGE2 (47). PGE2 at 0.5 to 5 ng/ml also stimulated the number of culture can be identified in the original lumpectomy specimen cell structures by 2-fold or more after 10 days in GM without and in the recurrent mÃ©tastasesof the patient; and (d) there is serum; no further stimulation was achieved at 50 ng/ml, whereas little or no synthesis of laminin or deposition of basement mem 500 ng/ml was slightly inhibitory (Chart 3C). brane by the cultured cells, consistent with the loss of the basement membrane being a marker for human mammary car cinomas (5, 21). DISCUSSION The ability to form small tumor nodules of Ca2-83 in nude mice is not necessarily a property of most malignant breast Breast cancer cells from the primary tumors or mÃ©tastases carcinoma cells (29). However, Â¡mmunocytochemical analysis have been grown in culture only with difficulty (17, 23, 30, 33). has been undertaken to confirm the results found in vitro where Several reports, however, have indicated that unlike the epithe full expression of the differentiation potential of cells, particularly lium and fibroblasts derived from normal or near-normal human myoepithelial cells (6), is more limited. Despite the various cellular mammary glands, clusters of slow-adherent or nonadherent pleomorphic forms of Ca2-83, there is still a complete absence epithelial cells are often obtained both from collagenase digestion of any elongated, myoepithelial-like cells that are in any way of solid tumors (31, 32, 34) and from pleural effusions of malig equivalent to those generated by the normal and benign tumor nant disease (11,15,49,58,61 ), and these results are consistent stem cells of rat mammary epithelial origin. In this respect, Ca2- with our findings reported here. The slow or lack of adherence 83 parallels our malignant metastasizing rat mammary tumors of epithelial cell clusters obtained from secondary breast carci and cell lines (12-14,63). This absence of myoepithelial-like cells nomas does not necessarily prove that epithelial cells from has been noted previously in other cultures of malignant breast normal breast tissues adhere, whereas those from secondary cancer cells (22, 23, 30) and is also consistent with their virtual carcinomas do not, since the adherent properties of the epithelial absence in histolÃ³gica! sections of infiltrating ductal carcinoma cell clusters may depend more on the presence or absence of in humans (1, 4, 20, 21, 26). myoepithelial cells and/or their products (54). Normally, most Infiltrating ductal carcinomas are composed of cells that are breast cancer cells eventually die out on repeated transfer in highly heterogeneous, and their relationship to the malignant/ vitro (16, 39, 42). Those rare instances where epithelial cells metastastic progression is not known (4). Whether Ca2-83 is a emerge as permanently growing cell strains usually involve their good representative of the original tumor cells of the patient is passage through a period of crisis which is often characterized not entirely clear. However, all the cells growing in suspension by a switch from very slow-growing, nonadherent cell clusters from the original specimen of the patient have been retained up to more rapidly growing cultures of adherent cell sheets (15, 34, to passage 5, and further cultures have been made from such 49). In the case of Ca2-83, there has been little change in pooled stocks. Furthermore, the main pleomorphic variants of adherent, morphological, or growth rate properties throughout the epithelial cells in the primary tumor and secondary mÃ©tas its entire period of culture. Indeed, even the estrogen receptor tases of the patient, particularly the fat-containing epithelial cells, content of the primary tumor and of early and late-passage cells can also be distinguished in the cultures of Ca2-83, indicating was roughly constant at 22, 14, and 17 fmol/mg cell protein that Ca2-83 can reconstitute the major epithelial cell types ob respectively.5 The reason why this particular cell strain grew served in the primary tumor and recurrent mÃ©tastasesof the continuously in culture, whereas the remainder of the isolates patient. Since Ca2-83 has been developed from one of 30 tumors did not, may be due in part to the relatively aggressive behavior cultured, its relationship to tumor cells occurring in other primary of the original cancer. carcinomas and their mÃ©tastasesis also not entirely clear. How The small polygonal cells of cultures of Ca2-83 have been ever, the vast majority of epithelial cells from secondary carci characterized as cells of an epithelial origin by their staining with nomas grow only as loosely adherent or nonadherent aggre epithelial cell-specific antisera to keratin (18) and to EMA (52) and the occurrence of luminal-like cells in the clusters which gates, whereas those from normal mammary tissues or benign mammary tumors grow only as fast-adherent sheets of epithe display microvilli and junctional complexes typical of glandular lium. In this respect, Ca2-83 is similar to the epithelial cells epithelial cells (8, 43). Although large, fat-containing cells are obtained from the malignant metastatic lesions of infiltrating seen in these cultures, they also have an epithelial ultrastructure ductal carcinomas of the human breast. and possess microvilli coated with EMA. Further confirmation of the epithelial nature of Ca2-83 cells comes from their possession Often, the growth of malignant human mammary epithelial of estrogen receptors. That Ca2-83 cells are of human origin is cells is relatively unaffected by changes in the concentrations of serum or of growth factors (particularly EGF) and hormones in confirmed by their staining with antisera to EMA, which is specific the medium (30, 55, 57). In the case of Ca2-83, it is moderately for human epithelial cells (52), and their failure to stain with responsive to changes in the serum concentration but still shows â€¢R.J. B. King, unpublished results. little response to EGF. However, the major growth stimulator

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Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1985 American Association for Cancer Research. PROPERTIES OF A NEW BREAST CANCER CELL STRAIN tested is contained in pituitary extracts, together with insulin and Fogh (ed.), Human Tumor Cells in Vitro, pp. 79-114. New York: Plenum Publishing Corp., 1975. possibly estradiol and progesterone. This pituitary activity may 12. Dunnington, D. J. Ph.D. Dissertation, University of London, London, England. be related to the new pituitary mammary growth factor, which 1984. stimulates normal and benign rat mammary epithelial cells to 13. Dunnington, D. J., Kim, U., Hughes, C. M., Monaghan, P. Ormerod, E. J., and Rudland, P. S. Loss of myoepithelial cell characteristics in metastasizing rat grow in culture (53). However, the apparent lack of response to mammary tumors relative to their nonmetastasizing counterparts. J. Nati. other hormones and factors may merely reflect the design of the Cancer Inst., 72: 455-466,1984. 14. Dunnington, D. J., Kim, U., Hughes, C. M., Monaghan, P., and Rudland, P. S. assays or the combinations tested. For example, although the Lack of production of myoepithelial variants by cloned epithelial cell lines Ca2-83 cells maintained for 1 month in serum-free medium derived from the TMT-081 metastasizing rat mammary tumor. Cancer Res 44:5338-5346,1984. containing BSA can be stimulated to grow somewhat with the 15. Engel, L W., Young, N. A., Tralka, T. S., Lippman, M. E., O'Brien, S. J., and pituitary extract, if the cultures are left for 2 months or more in Joyce, M. J. Establishment and characterization of three new continuous cell BSA the cells will not respond.4 Ca2-83 cells can also be stimu ines derived from human breast carcinomas. Cancer Res., 38: 3352-3364 lated to grow by media from fibroblasts-adipocytes and from 1978. 16. Feller, W. F., Stewart, S. E., and Kanton, J. Primary tissue culture expiants of myoepithelial cells that surround the epithelium within the normal human breast cancer. J. Nati. Cancer. Inst., 48: 1117-1120, 1972. rat mammary gland and by medium from rat mesothelial-like/ 17. Foley, J. F., and Aftonomos, B. T. Growth of human breast neoplasms in cell culture. J. Nati. Cancer Inst., 34: 217-229,1965. stromal cells that are required for the growth in culture of the 18. Gabbiani, G., Kapanci, Y., Barazzone, P., and Franke, W. W. Immunocyto- metastasizing cell strain SMT-2A (12, 13, 14, 29). One of the chemical identification of intermediate-sized filaments in human neoplastic major mitogenic components synthesized by the fibroblasts- cells: a diagnostic aid for the surgical pathologist. Am. J. Pathol., 704: 206- 216,1981. adipocytes is PGE2 (47). That some of the components released 19. Gaffney, E. V., Polanowski, F. P., Blackburn, S. E., LambÃase,J. T., and Burk, by the SMT-2A cell line are different from those released by the R. E. Cultures of normal human mammary cells. Cell Differ., 5: 69-81, 1976. myoepithelial-like or preadipocytic cells can be seen from their 20. Gould, V. E., Jao, W., and Battifora, H. Ultrastructural analysis in the differential diagnosis of breast tumours. The significance of myoepithelial cells, basal differential effects in stimulating attachment of the cell clusters lamina, intracytoplasmic lumina and secretory granules. Pathol. Res. Pract., to plastic substrata (Table 4). This result, however, does not 767:45-70,1980. necessarily prove that the growth-stimulating agents themselves 21. Gusterson, B. A., Warburton, M. J., Mitchell, D., Ellison, M., Neville, A. M., and Rudland, P. S. Distribution of myoepithelial cells and basement membrane are different. proteins in the normal breast and in benign and malignant breast diseases. In conclusion, we have established in culture a cell strain, Ca2- Cancer Res., 42: 4763-4770, 1982. 83, which, although not yet single-cell cloned, still retains some 22. Hacket, A. J., Smith, H. S., Springer, E. L., Owens, R. B.. Nelson-Rees, W. A., Riggs, J. L., and Gardner, M. B. Two syngeneic cell lines from human of the properties of the original epithelial cells from a human breast tissue: the aneuploid mammary epithelial (Hs578T) and the diploid breast cancer. We also show that Ca2-83 cells fail to produce myoepithelial (Hs578Bst) cell lines. J. Nati. Cancer Inst., 58:1795-1806,1977. myoepithelial-like cells and basement membrane proteins and 23. Hallowes, R. C., Millis, R., Pigott, D.. Shearer, M., Stoker, M. G. P., and Taylor- Papadimitriou, J. Results on a pilot study of cultures of human lacteal secre that they are primarily responsive to novel growth factors for the tions and benign and malignant breast tumors. J. Clin. Oncol., 3: 81 -90,1977. mammary gland. 24. 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O., Capony, F., Garcia, M., Chanteland, J., Pujol, H., Veith, A. Z., and Enzyme-linked Â¡mmunoassay(ELISA) for connective tissue components. Anal. Rochefort, H. Release of estrogen-induced glycoprotein with a molecular Biochem., 704: 205-214, 1980. weight of 52,000 by breast cancer cells in primary culture. Cancer Res., 43: 45. Rudland, P. S., Hallowes, R. C.. Durbin, H., and Lewis, D. Mitogenic activity 1861-1868, 1983. of pituitary hormones on cell cultures of normal and carcinogen-induced tumor 59. Warburton, M. J., Ormerod, E. J., Monaghan, P., Ferns, S., and Rudland, P. epithelium from rat mammary glands. J. Cell Biol., 73: 561-577,1977. S. Characterization of a myoepithelial cell line derived from a neonatal rat 46. Rudland. P. S., Bennett, D. C., and Warburton, M. J. Hormonal control of mammary gland. J. Cell Biol., 97: 827-836,1981. growth and differentiation of cultured rat mammary epithelial cells. Cold Spring 60. Warburton, M. J., Mitchell, D., Ormerod, E. 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Fig. 1. Morphology of Ca2-83 cells. A, histological section of the lumpectomy specimen of the primary breast tumor from which Ca2-83 cells were cultured showing areas typical of a rather undifferentiated adenocarcinoma with cells arranged in cords and circular structures. Some of the cells were multinucleated with a granulated, foamy cytoplasm (g). H & E, x300. B to E, living Ca2-83 cells growing in plastic flasks were photographed with phase-contrast optics. B, cells growing in suspension in clusters, some of which possessed bulbous projections (b). x 190. C, cells spreading and growing from a slow-adherent colony (o) on the surface of the flask giving rise to a cohesive island of isometric cells, some of which were binucleated (b) and others which possessed a very granulated or foamy cytoplasm (g). x 190. D, higher magnification of large, granulated cells showing extensive arrays of vacuoles or granules in their cytoplasm, x 490. Â£,higher magnification of epithelioid cells growing on plastic showing variations in cellular size from small cuboidal cells to larger, more irregularly shaped cells with multiple nuclei, x 490. Fig. 2. Histological and immunocytochemical staining of cultured Ca2-83 cells. Cells growing in suspension were harvested by centrifugation and embedded in paraffin wax. A, histological section showing circular arrays of cells surrouding a central cavity; some of the cells are multinucleated (m), and some possess a lighter, granulated, foamy, or speckled region within their cytoplasm (g). H & E. B, similar sections stained with oil red O, a stain for neutral lipids, showing accumulation of lipid (/) and/or lipid droplets (d) in the cytoplasm of some of the cultured cells. In C to H, the histological sections were incubated with the specific rabbit antibody. The first 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. Hemalum (see "Materials and Methods"). In C and D. sections of normal human breast from a mastectomy specimen of a 19-year-old woman were stained with antisera to EMA and to human keratins, respectively. Anti-EMA serum stained mainly the luminal epithelial cells (arrows), whereas antikeratin serum stained strongly the epithelial cells lining the ducts (e) and only weakly stained the myoepithelial cells (m). Antisera absorbed with the requisite antigens yielded no immunocytochemical staining (not shown). In E and F, sections of Ca2-83 cells were stained with antiserum to EMA and with the same antiserum preabsorbed with EMA, respectively. Strongest specific staining was observed at the cellular peripheries (arrows), although weaker cytoplasmic staining was also observed. In G and H, sections of Ca2-83 cells were stained with antiserum to human keratins and with antiserum preabsorbed with human keratins. Only specific staining of the cytoplasm was observed, mainly in the perinuclear regions. All sections, x 300. Fig. 3. Immunocytochemical staining of Ca2-83 cells in culture and in tumor nodules in immunodeficient mice. In A and B, sections of Ca2-83 cells were incubated with antisera to actin and laminin, respectively, but no specific staining was observed, x 300. C to F are histological sections of inguinal mammary glands of 18-week- old female nude mice at the site of inoculation of the Ca2-83 cells. In C, sections were stained with antiserum to human EMA, and the majority of the tumor cells in the nodule were stained positively (arrows), whereas adjacent mouse mammary gland ducts were unstained (insert). In D, sections were stained with antiserum to rat milk fat globule membrane, and all the tumor cells in the nodule were unstained, although the luminal epithelial cells of an adjacent mouse mammary gland duct were stained intensely (arrows in insert). In E, sections were stained with antiserum to human keratins, and about half the tumor cells in the nodule were stained (arrows). All the myoepithelial cells of an adjacent mouse mammary gland duct were stained intensely (arrows in insert) while the luminal epithelial cells were stained more weakly, if at all (/nsert). In F, sections were stained with antiserum to laminin; no tumor cells were stained. Basement membrane round adjacent mouse mammary gland ducts were stained (not shown). In sections C to F and their inserts, antisera preabsorbed with the requisite antigens abolished completely any immunocytochemical staining (not shown). All in C to F and inserts, x 240. Fig 4. Electron micrograph of cultured Ca2-83 cells. A, portion of a cell cluster with microvilli (m) on the cell surface lining the medium-filled cavity (c). An occasional larger, distended cell with fat droplets (f) was also observed. Cells were closely apposed with interdigitating membranes and apical junctional complexes (/), and desmosomes (d) were present between adjacent cells, x 3,400. B, higher magnification showing a desmosome (d) with a thickening of the 2 cell membranes, an intercellular contact layer (/), and associated tonofilaments (f). Individual filaments, 10 to 12 nm diameter, were also scattered throughout the cytoplasm (f). x 53,600. C and D, immunoperoxidase staining with anti-EMA serum. In the higher-power micrograph (C), EMA was located on the surface of the cells, particularly on the outer surfaces of the microvilli (arrows), x 11.500. In the lower-power micrograph (D), EMA was found on the surface of the cells exposed to the medium and was particularly concentrated where there were microvilli (arrows), x 4,410. Fig. 5. Immunoperoxidase localization of EMA on fat-containing cells. Spherical cell containing large numbers of fat droplets was stained with antisera to EMA and observed in the electron microscope. The cell surface exposed to the medium was coated with reaction product (arrows), particularly in those areas with extensive numbers of microvilli. x 3,530. Fig. 6. Autoradiograph of [3H]thymidine-labeled Ca2-83 cells. Cultured cells were exposed to [3H]thymidine for 48 h in normal growth medium, then harvested by centrifugation. The cells were smeared onto subslides, exposed to photographic emulsion, developed, and also counterstained by H & E as described in "Materials and Methods." Those nuclei which were synthesizing DNA in the 48-h period are black (arrows), x 300.

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Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1985 American Association for Cancer Research. Loss of Production of Myoepithelial Cells and Basement Membrane Proteins but Retention of Response to Certain Growth Factors and Hormones by a New Malignant Human Breast Cancer Cell Strain

Philip S. Rudland, Richard C. Hallowes, Simon A. Cox, et al.

Cancer Res 1985;45:3864-3877.

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