Coexpression of Cytokeratins Characteristic for Myoepithelial And

(CANCER RESEARCH 51, 5943-5950. November 1. 1991] Coexpression of Cytokeratins Characteristic for Myoepithelial and Luminal Cell Lineages in Rat 13762NF Mammary Adenocarcinoma Tumors and Their Spontaneous MÃ©tastases R. B. Lichtner,1 J. A. Julian,2 S. M. North,3 S. R. Classer,4 and G. L. Nicolson5

Department of Tumor Biology, The University of Texas M. D. Anderson Cancer Center [R. B. L., S. M. N., G. L. N.J, and Department of Cell Biology, Baylor College of Medicine fJ. A. J., S. K. G.J. Houston, Texas 77030

ABSTRACT tumor originated, but substantiation of this assumption neces sitates precise definition of the normal and malignant We used innnunohistochemical procedures to study the cellular expres phenotypes. sion and distribution of cytokeratins (CKs) in rat 13762NF mammary As defined by morphological, biochemical, and functional adenocarcinoma cells growing at mammary fat pad sites and at sponta neous lymph node and lung sites. In order to establish CK distribution in criteria, the mammary gland contains at least four types of normal rat mammary epithelia, immature, resting, and lactating rat epithelial cells: luminal ductal, basal ductal, luminal alveolar mammary glands were probed with a panel of monospecific antibodies (secretory), and myoepithelial cells. The predominant pheno that recognize individual CKs. Basal/myoepthelial cells were distin types of these cell populations are distinct in the mature mam guished by expression of CKs 5 and 14 and coexpression of t inienti n mary gland. Luminal ductal and luminal alveolar epithelia are from luminal cells, which expressed CKs 8, 18, and 19. Antibody to CK recognized by antibodies specific for CKs found in simple 7 recognized luminal epithelium of immature and resting, but not lactat epithelia, whereas basal ductal and myoepithelial cells are rec ing, mammary glands. Myoepithelial cells of lactating mammary gland ognized by antibodies specific for CKs of stratified squamous were weakly recognized by antibodies to CKs 7 and 19. Tumors formed epithelia (8, 9) and coexpress vimentin (10, 11). There also by cell lines and clones derived from parental 13762NF tumor (MTPa, MTC, MTA, and MTF7) were not recognized by any of the anti-CK exists a limited population of cells in the basal epithelia that antibodies. Only vimentin was expressed in these tumors and their coexpresses simple and stratified CKs (5, 10, 12). It has been mÃ©tastases. In tumors and mÃ©tastases generated from cell lines and suggested that these constitute a population of pluripotent stem clones derived from lymph node (MTLY) and lung mÃ©tastases(M l'I,ii2 cells from which the four distinct epithelia are derived (12) and and M 11 .n.*) of the 13762NF tumor we observed heterogeneous CK generate genetically unstable cells during the carcinogenic proc phenotypes. Expression of CKs 5 and 18 was greatly reduced or lacking, ess (13). In humans the majority of carcinomas lack cells while CK 14 was coexpressed with CKs 7, 8, and 19 with or without expressing the basal/myoepithelial CK phenotype (5, 7, 14- vimentin. Tumors from the highly metastatic clone MTLn3 had a domi 18), and this also appears to characterize the acquisition of nant cellular phenotype, expressing CKs 7, 8, 14, and 19 and vimentin, a metastatic potential in rat mammary tumor cells (13, 19). pattern that did not match normal mammary epithelia, whether luminal, basal/myoepithelial, or the dual-phenotype stem cell, in which CKs 5, 8, We recently reported that cell extracts enriched for interme diate filaments of double cloned or uncloned cell lines isolated 14, and 18 were coexpressed. MTLn3 lymph node and lung mÃ©tastases expressed the same cellular phenotype as the s.c. growing MTLn3 tumor. from the rat 13762NF mammary adenocarcinoma, and its The results appear to contradict the belief that malignant mammary spontaneous lymph node and lung mÃ©tastasesvaried phenotyp- tumors may be distinguished from benign tumors or hyperplastic growths ically in the expression of CKs (20). These rat tumor cells failed by the lack of basal/myoepithelial markers. to express CKs or expressed CKs characteristic for both simple and stratified epithelia. This was an unexpected finding. To INTRODUCTION obtain positional information about the cells expressing CKs we used monospecific antibodies on frozen sections of tumors. Analysis of CK6 expression is frequently used in tumor di We have demonstrated that CK-positive rat mammary tumor agnosis, particularly of epithelial tumors (1-3). The definition cells exhibit pronounced phenotypic heterogeneity of CK of a tumor as a carcinoma is often based on the presence of expression at the cellular level. CKs, and detection of specific CKs has allowed the subclassifi cation of certain tumors, such as those arising in such complex MATERIALS AND METHODS epithelial tissues as the mammary gland (4-7). It is assumed Cell Lines and Culture Conditions. The tumor cells used in this study that, in general, the profile of CK expression in the tumor cells were derived from the 13762NF mammary adenocarcinoma syngeneic qualitatively reflects the phenotype of the cell from which the to F344 rats (21, 22). The 13762NF cell lines and clones vary repro- Received 10/5/89; accepted 8/14/91. ducibly in their metastatic properties during in vitro growth (23) and The costs of publication of this article were defrayed in part by the payment display heterogeneity in a number of other properties (24, 25). Tumor of page charges. This article must therefore be hereby marked advertisement in cells were used at passages T14-16 (MTLn3), Tl 1-13, (MTLY), T42- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Present address: Research Laboratories of Schering AG. 1000 Berlin 65, 44 (MTLn2), and T8-12 (MTPa, MTC, MTF7). Tumor cells were Federal Republic of Germany. grown as described previously (21,25) in Â»modified minimum essential 2Present Address: Department of Biochemistry and Molecular Biology, The medium supplemented with 5% fetal bovine serum (HyClone, Logan, University of Texas, M. D. Anderson Cancer Center, Houston. TX 77030. UT) without antibiotics. At 60-80% cell confluency, tumor cells were 5 Present Address: College of Veterinary Medicine, Texas A & M University. harvested by using 0.125% trypsin-2 mM EDTA in phosphate-buffered College Station, TX 77843. 4 Supported by United States Department of Health and Human Services saline, pH 7.4 (containing, in g/liter: NaCl, 8; KCI, 0.2; Na2HPO4, Grants R01-HD07495 and HD25189 from the National Institute of Child Health 1.14; KH2PO4, 0.2), and diluted in fresh medium without fetal bovine and Development. serum. Cells (1 x IO6) were injected into the mammary fat pads of *To whom requests for reprints should be addressed. Supported by United F344 rats. Within 2-3 weeks tumors developed, reaching a size of 1- States Department of Health and Human Services Grant R35-CA44352 from the to 2-cm diameter after 4 weeks. National Cancer Institute. 'The abbreviations used are: CK(s), cytokeratin(s); MAb, monoclonal anti Histochemical Procedures and Immunofluorescence. Tumors were body; Vim, vimentin. excised, snap frozen in liquid nitrogen, and stored at -20'C. Normal 5943

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1991 American Association for Cancer Research. CYTOKERATINS IN METASTATIC RAT MAMMARY TUMORS rat mammary tissue was obtained from 21-day-old female rats or mature Table 1 Expression of CKs and Vim in the mature lactating and immature lactating female rats. Tissue sections of 4-fim thickness were cut on a resting rat mammary gland cryostat (International Equipment Co., Needham, MA) and processed CK type (No.) or Vim as described previously (26). Tissue 14 8 18 19 Vim Antibody Source and Description. Anti-CK 5, anti-CK 6, and anti- Lactating gland CK 14 are monospecific polyclonal antibodies generated in rabbits Basal and myoepithelial cells â€¢¿" against peptide sequences located in the carboxyl termini that are Luminal cells - unique to CKs 5, 6, and 14, respectively (20, 27-29). They were the gift of Dr. Dennis Roop (Baylor College of Medicine, Houston, TX). Immature gland Basal and myoepithelial cells + Anti-CK 7, anti-CK 8, and anti-CK 19 are monospecific mouse MAbs Luminal cells - obtained commercially from Amersham Corp. (Arlington Heights, IL), Â°+,positive for antibody staining; -, negative for antibody staining; (+), weak which was also the source of the mouse MAb to Vim. Endo B, a staining for antibody or expression varying from cell to cell. polyclonal rabbit antibody monospecific for CK 18 (30), was provided by Dr. Robert G. Oshima (La Jolla Cancer Research Foundation, La Jolla, Ã‡A).X-260, a polyclonal rabbit antibody recognizing CK 18 and (data not shown). These cells may represent a subpopulation of CK 19, was the gift of Dr. Warren N. Schmidt (Vanderbilt University dual phenotype stem cells like those described by Allen et al. School of Medicine, Nashville, TN) and was characterized by us. In (31). All epithelia of both of both mature and immature mam probes of human or rat tissues it recognized simple epithelia; in rat mary glands were negative for CK 6 expression (data not mammary gland this antibody recognized luminal epithelia. Although shown); however, low levels of CK 6 were detected in hair X-260 has been shown biochemically to recognize CKs 18 and 19, in follicles of adjacent skin, indicating that the antibody could extracts of tumor cells in the present study it detected only CK 19 (20). bind to CK 6 in the same tissue section. To demonstrate coexpression of CKs we performed double labeling by CK Expression and Distribution in Locally Growing Mammary pairing a monospecific mouse MAb (antibody to CK 7, 8, 19, or Vim) with a monospecific rabbit polyclonal antiserum (to CK 5, 6, 14, or Tumors. Four cell lines and clones (MTPa, MTF7, MTC, and 18). Fluorescein isothiocyanate-donkey anti-rabbit IgG and Texas red- MTA) isolated from the 13762NF parental tumor were previ conjugated sheep anti-mouse IgG were species specific antibodies pur ously shown to be CK negative in studies using an antibody chased from Amersham Corp. All designations of CKs in this study that recognized a single CK (26) or in biochemical analyses correspond to the numbering system used for human CK studies (1) to (20). Tumors generated by injection of these cell lines and facilitate comparison between human and rat. Microscopic observations clones into syngeneic rats were probed with the entire panel of were performed by using an Orthoplan Leitz photomicroscope equipped antibodies to detect any low-level CK expression that went for transmitted and incident light fluorescence. undetected in the previous studies. Tumor cells were negative with all CK antibodies tested, and Vim was the only interme RESULTS diate filament protein expressed in these tumors (data not shown). The small clusters of CK-positive cells previously found Normal Rat Mammary Glands. Comprehensive biochemical in the mesenchyme at the tumor margin (26) were identified in or immunohistochemical studies on CK expression and cellular the present study as residual normal mammary gland (data not distribution are not available for the rat mammary gland. To shown). These structures possessed luminal cells positive for evaluate CK expression by rat mammary tumor cells, it was CKs 7, 8, 18, and 19, and they were surrounded by basal/ necessary first to establish the pattern of CK expression and myoepithelial cells positive for CKs 5 and 14 and Vim. Spon distribution in normal rat mammary gland by using the same taneous lymph node mÃ©tastasesarising occasionally in rats panel of antibodies subsequently applied to the tumors. given injections of the MTC clonal line were also negative for Frozen sections of mature and immature rat mammary glands CK expression, using the same panel of antibodies (data not were probed with combinations of the monospecific mouse shown). MAb and monospecific rabbit polyclonal antibodies. Antibodies CK Expression and Distribution in Locally Growing Mammary to CK 5, 6, 14, or 18 were paired with antibodies to CK 7, 8, Tumors Generated from Spontaneous MÃ©tastases.Three cell 19, or Vim, respectively; Table 1 summarizes our findings. lines and clones, isolated from a spontaneous lymph node Coexpression of CKs defining simple epithelial (CKs 8 plus 18) metastasis (cell line MTLY) and lung mÃ©tastases (clones or basal epithelial (CKs 5 plus 14 and Vim) cells was restricted MTLn2 and MTLn3) in the same animal as the parental tumor, to luminal or myoepithelial cells, respectively, in mature and were found to be CK positive (26), expressing CKs tentatively immature rat mammary glands (Fig. 1, A-E, G, I). However, identified as CKs 7, 8, 14, 15 or 17, and 19 (20). When probed tissue distribution patterns of the other CKs investigated were with an antibody recognizing CK 8, the three lines varied in the less clear cut or in some cases more variable when the two proportion of CK-positive cells in the total cell population in tissues were compared. For example, CK 19 in luminal epithelia vitro (26). In the present investigation, serial sections of tumors of the immature and resting adult mammary glands was uni generated by injection of MTLY, MTLn2, or MTLn3 cells formly distributed (Fig. IF), whereas in lactating mammary were probed with paired antibodies to specific CKs, which glands expression of CK 19 varied from cell to cell (data not allowed us to locate the CK-positive cells and estimate their shown). In both tissues basal and myoepithelial cells were fraction of the cell population. In general, tumors exhibited a weakly stained by antibody to CK 19. Similarly, luminal cells variety of CK expression phenotypes, while the CK-negative of both ducts and terminal end buds of immature mammary cells in these tumors expressed Vim. In CK-positive tumor glands were positive for CK 7 expression (Fig. I//), whereas in cells, the combination of individual CKs varied considerably, adult lactating mammary glands antibody to CK 7 reacted and detailed staining patterns for individual tumor cells are weakly with basal and myoepithelial cells and was unreactive presented only for MTLn3 tumors (Fig. 2) (summarized in with luminal cells (Fig. I/). Occasional cells in the terminal Table 2). Based on the estimated frequency of cells that coex end buds of immature glands and in ducts of resting adult pressed individual CKs, a dominant CK-positive phenotype mammary glands coexpressed CKs 5 and 8 normally restricted could be established in MTLn3 tumors but not in tumors to the basal/myoepithelial or luminal cell types, respectively formed from MTLn2 or the uncloned line MTLY. This domi- 5944

Fig. 1. Expression and distribution of CKs and Vim in immature (A-H) and lactating (/, J) rat mammary glands demonstrated by fluorescence microscopy. Sections of tissues were simultane ously probed with pairs of antibodies and detected by either fluorescein isothiocyanate-donkey anti- rabbit IgG or Texas red-sheep anti-mouse IgG in order to demonstrate exclusive or coexpression of CKs in single cells. The antibodies used are: anti- CK 14 (A, E); anti-Vim (B); Endo B, recognizing CK 18 (C); anti-CK 8 (D); anti-CK 19 (F): anti- CK 5 (G, I), and anti-CK 7 (H, J). x 400.

5945

Fig. 2. Expression and distribution of CKs and Vim in locally grown (mammary fat pad) rat MTLn3 mammary adenocarcinoma tumors demonstrated by fluorescence microscopy. Sections of MTLn3 tumors were simultaneously probed with paired antibodies as in Fig. I in order to demonstrate exclusive or coexpression of CKs in the same cell. The antibodies used are: anti-CK 14 (A) and anti-Vim (A); anti-CK 14 (C) and anti-CK 7 (O); anti-CK 14 (Â£")andanti-CK 8 (F); anti-CK 14 (C) and anti-CK 19 (Â«).x 400. 5946

Table 2 Coexpression ofCKs and Vim in local mammary fat pad tumors and undetectable quantities. When tumor sections were probed with spontaneous mÃ©tastasesofrat 13762NF tumors antibody to CK 5, a limited population of cells were positive. CK type (No.) or Vim For example, in fat pad MTLn3 tumors less than 2% of CK- BTumorLocal A" positive cells were positive for CK 5, whereas CK 5* cells did not exceed 20 cells per section of MTLn3 inguinal lymph node 14*8* 19* 14* 19*8* g-+' Vim* 7* VinT+ mÃ©tastases(Fig. 3A). However, cells from axillary lymph nodes or lung mÃ©tastaseswere negative for CK 5. Less than 5% of fat padtumorsMTLY CK-positive cells were positive for CK 5 in fat pad MTLn2 (>90%)cMTLn2 Â±+ Â± + ++ (<40%)MTLn3 0+ + + +Â± tumors, while positive cells could not be identified in local (70-90%)MÃ©tastasesInguinal0+ + + 0Â± MTLY tumors. When CK 5 was detected, it was coexpressed with CKs 7, 8, and 19, which appeared to be expressed at LN' 0+ + + 00 reduced but detectable levels. All of these tumors were negative AxillaryLN7Lung*18*14** + +oÂ± 00C18*14-Â±+ for CK 6. + + 014*19'Â±+00 0D14*Vim"++Â±0Â±0 " Classification of epithelial cell type: A, mixed: B. basal/myoepithelial-like: Various tumors were also probed with Endo B to test for the C, liiiiiin.il like; and D. undefined phenotype. presence of CK 18, a type I CK normally coexpressed with CK * +, positive for antibody staining; -, negative for antibody staining. 8 in the luminal cells of rat mammary glands. A low level of ' Percentage of tumor cells that were CK-positive. diffuse, positive staining for CK 18 was present in 10% of CK* * Antibody reactivity: 0, no cells of this phenotype; Â±,very low frequency (<10%); +, frequency above 10% in cell population. cells in local MTLn3 tumors, but the staining pattern of CK 18 ' LN, spontaneous inguinal lymph node mÃ©tastasesof M 11 n

Fig. 3. Expression and distribution of CK 5 and 18 in rat MTLn3 mammary adenocarcinoma tumors demonstrated by fluorescence microscopy. Sections of M 11 n.i inguinal lymph node mÃ©tastases(A, B) and locally grown (mammary fat pad) MTLn3 tumor (C, D) were simultaneously probed with paired antibodies as in Fig. 1 to demonstrate exclusive or coexpression of CKs in the same cell. The antibodies used are: anti-CK 5 (A); anti-CK 8 (B); Endo B recognizing CK 18 (C) and anti- Vim (D). x 400.

basal/myoepithelial cells (7, 11). The intensity of expression of the other basal/myoepithelial markers (12, 13). Since the varies, with ductal basal cells that strongly express CK 17 and dominant cellular phenotype in the metastatic cell line, MTLn3, occasionally express Vim, whereas myoepithelial cells of alveoli expressed a mixed CK profile intermediate between myoepithe exhibit the inverse relationship between CK 17 and vimentin lial and stem cell phenotype, no strict definition of the cell of expression. Although CK 15 has been biochemically detected origin of MTLn3 tumor cells can be made on the basis of CK in human mammary gland extracts (1), determination of its expression. It might be argued that derivation of MTLn3 tumor cellular distribution awaits development of a monospecific anti- cells from a lung metastasis and the subsequent cloning resulted CK 15 antibody. As established by the present study, CK in selection of a unique cellular phenotype not generally present expression in normal rat mammary gland, with the exception in the primary tumor or normal cells (38). The dominant of CKs 15 and 17, which were not included because of the MTLn3 cellular phenotype (expressing CKs, 7, 8, 14, and 19 unavailability of monospecific antibodies, appears to be similar but lacking CKs 5 and 18 and coexpressing Vim) could repre to that of normal human mammary gland. Indeed, the only sent a myoepithelial or basal cell that has partially dedifferen- notable difference in the normal mammary glands of the two tiated, or alternatively, a stem cell which has progressed to an species is the location of putative stem cells, that is, cells intermediate stage. MTLn2 and MTLY cells showed even coexpressing CKs characteristic of the luminal or basal/myo greater phenotypic heterogeneity than MTLn3 cells; therefore, epithelial lineages. In human mammary glands, these cells were the cell of origin of these two cell lines also cannot be assessed. found in the luminal cell population (discussed in Ref. 8), Examination of these tumors for other markers of the basal/ whereas they were located in the basal cell population of the myoepithelial cell lineage may resolve this issue. A preliminary rat mammary gland (12). survey has detected significant amounts of laminin around cells The mere presence of an individual determinant does not coexpressing Vim in MTLn3 and MTLn2 tumors, particularly define a particular cellular phenotype, because each determinant in regions containing CK+ cells. In MTLY tumors laminin may be shared by several cell types. Rather, it is the coordinated surrounded tumor cell foci.7 expression of several determinants combined with particular Several investigators have suggested that characteristics of morphological or ultrastructural characteristics that define a the basal/myoepithelial lineage might serve as the criterion for distinct phenotype. The basal/myoepithelial cellular phenotype, a differential diagnosis of human mammary tumors when his in addition to expressing CKs 5, 14, 17, and possibly 15 (plus tolÃ³gica! analyses are not sufficient (39-41). This suggestion low levels of CKs 7 and 19), coexpress Vim (7, 10, 13), smooth was supported by the observation that dysplastic proliferations muscle a-actin (36), smooth muscle myosin, laminin, and col and benign tumors of the mammary gland contained a variety lagen IV (10, 12, 37). Stem cells coexpress CKs 5 and 14, of normal and intermediate CK expression phenotypes, whereas characteristic of the basal/myoepithelial lineage, with CKs 8 and 18, characteristic for luminal cells, but do not express most ' Unpublished observations. 5948

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1991 American Association for Cancer Research. CYTOKERATINS IN METASTATIC RAT MAMMARY TUMORS tumors, dysplasias and breast cancer. Int. J. Cancer, 42: 147-153. 1988. the majority of carcinomas lack cells expressing the basal/ Taylor-Papadimitriou, J., and Lane, E. B. Keratin expression in the mam myoepithelial CK phenotype (5, 7, 14-18). The loss of cells mary gland. In: M. C. Neville and C. Daniels (eds.), The Mammary Gland. expressing the basal/myoepithelial CK phenotype also ap Development, Regulation and Function, pp. 181-215. New York: Plenum peared to characterize the acquisition of metastatic potential in Publishing Co.. 1987. 9. Purkis, P. E., Steel, J. B., Mackenzie, I. C., Nathralh, W. B. J.. Leigh. I. M., rat mammary tumor systems (13, 19, 39). However, in a mouse and Lane, E. B. Antibody markers of basal cells in complex epithelia. J. Cell mammary tumor model using an antibody that recognized CK Sci., 97:39-50, 1990. 10. Dulbecco, R., Linger, M.. Armstrong, B., Bowman, M., and Syka, P. Epithe 14 the basal/myoepithelial phenotype was detected in the fat lial cell types and their evolution in the rat mammary gland determined by pad tumors and cells in the hematogenously disseminated mÃ© immunological markers. Proc. Nati. Acad. Sci. USA, 80: 1033-1037, 1983. tastases (42). Furthermore, 10% of invasive human mammary 11. Mark, C., van Deurs, B., and Petersen, O. W. Regulation of vimentin expression in cultured human mammary epithelial cells. Differentiation, 43: ductal carcinomas were positive for CK 14 (43). Thus, this 146-156. 1990. correlation remains a controversial issue, particularly in studies 12. Dulbecco, R., Allen, W. R., Bologna, M.. and Bowman. M. Marker evolution on human tumors. A new designation of "adenomyoepithe- during the development of the rat mammary gland: stem cells identified by lioma" has recently been suggested for human mammary tu markers and the role of myoepithelial cells. Cancer Res., 46: 2449-2456, 1986. mors containing primarily spindle cells that exhibit a subset of 13. Rudland, P. S. Stem cells and the development of mammary cancers in experimental rats and humans. Cancer Metastasis Rev., 6: 55-83, 1987. CK markers characteristic for the basal/myoepithelial lineage 14. Papotti, M., Gugliota, P.. Euscbi. V., and Bussolati, G. Immunohistochcm- (44), some of which exhibit metastatic potential (45-48). ical analysis of benign and malignant papillary lesions of the breast. Am. J. The results of the present study indicate that the acquisition Surg. Pathol.. 7:451-461, 1983. 15. Altmannsberger, M.. Dirk. T.. Droese, M., Weber. K.. and Osborn, M. of metastatic potential does not necessarily correlate with the Keratin polypeptide distribution in benign and malignant breast tumors: disappearance of CK markers for the basal/myoepithelial line subdivision of ductal carcinomas using monoclonal antibodies. Virchows Arch. B Cell Pathol., 51: 265-275. 1986. age in the rat mammary tumor system. The dominant CK 16. Gusterson, B. A., Warburton, M. J., Mitchell, D., Ellison, M., Neville, A. phenotype present in the most metastatic of the three CK- M., and Rudland, P. S. Distribution of myoepithelial cells and basement positive cell lines, MTLn3, was CK 14* and Vim*, both of membrane proteins in normal breast and malignant breast diseases. Cancer Res., 42: 4763-4770, 1982. which distinguish basal/myoepithelial from luminal cells in 17. Dairkec. S. H., Hyung. B. M.. Smith. H., and Hackett. A. G. Immunolocal- normal mammary gland. This CK phenotype proved to be ization of a human basal epithelium specific keratin in benign and malignant mixed, however, matching neither the normal luminal nor breast disease. Breast Cancer Res. Treat-, 10: 11-20. 1987. 18. Jarasch, E-D., Nagle. R. B., Kaufmann, M., Mauer, C, and BÃ³cker,W. J. basal/myoepithelial CK phenotypes, since CK 14 and Vim were Differential diagnosis of benign epithelial proliferation and carcinomas of coexpressed with CKs 7, 8, and 19. The dominant MTLn3 the breast using antibodies to cytokeratins. Hum. Pathol., 19:276-289,1988. 19. Dunnington, D. J., Kim, U., Hughes, C. M.. Monaghan. P.. Ormerod, E. J., tumor phenotype was distinguished by the negligible levels of and Rudland. P. S. Loss of myoepithelial cell characteristics in metastasizing expression of CKs 5 and 18, which have been detected in normal rat mammary tumors relative to their nonmetastasizing counterparts. J. Nati. cells coexpressing determinants of both cell lineages (putative Cancer Inst., 72: 455-466. 1984. 20. Lichtner, R. B., Julian, J. A.. Glasser. S. R.. and Nicolson, G. L. Character stem cells). Thus, the MTLn3 cellular phenotype matched ization of cytokeratins expressed in metastatic rat mammary adenocarcino- neither the stem cell, luminal lineage, nor the basal/myoepithe mas cells. Cancer Res., 49: 104-111, 1989. lial lineage. Retention of a subset of determinants characteristic 21. Neri, A., Welch, D. R., Kawaguchi. T., and Nicolson, G. L. Development and biologic properties of malignant cell sublines and clones of a sponta of an intermediate developmental stage for mammary epithelia neously metastasizing rat mammary adenocarcinoma. J. Nati. Cancer Inst., may ultimately define the malignant phenotype of the 13762NF 68: 507-517, 1982. 22. Segaloff. A. Hormones and breast cancer. Recent Prog. Horm. Res., 22: 351- tumors. 379. 1966. 23. Neri, A., and Nicolson, G. L. 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R. B. Lichtner, J. A. Julian, S. M. North, et al.

Cancer Res 1991;51:5943-5950.

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