[CANCER RESEARCH 38, 4327-4339. November 1978]

Human Breast Carcinoma Cells in Continuous Culture: A Review

Linda W. Engel and Nathaniel A. Young

Laboratory of Pathology, National Cancer Institute. NIH. Bethesda, Maryland 20014

Abstract

A comprehensive listing of putative human breast carcinoma cell lines and the extent to which each has beencharacterized is presented. Criteria used to certify thehuman, mammary, and malignant origin of a cell lineinclude: (a) a reliable histopathological diagnosis; (b)interspecies specificity established by human karyotype,isoenzyme profiles, and/or cell surface antigenicity; (c)intraspecies specificity, demonstrated by genetic evidence of a unique, human donor distinct from other cellsincluding HeLa cells; and (d) organ specificity, supportedby morphological evidence of epithelial structure andsecretory activity, and especially by the expression ofdifferentiated functions; these include presence of receptors for sex steroid hormones, hormone responsiveness,and production of milk proteins, fatty acids, or milk-specific antigens.

Of the 47 cell lines for which data are here reported, 22have been shown to be derived from human, non-HeLadonors and to have epithelial morphology as revealed bylight or electron microscopy. Differentiated function hasbeen recorded for 19 cell lines. Additional human breastcancer cell lines have been reported, but characterizationof some of these has been insufficient to judge thelegitimacy of their pedigrees. For others mammary originis questionable. Six purported breast cell lines are inreality HeLa cells, and one is of nonhuman origin.

Introduction

Human breast tumor cell lines are needed for multidisci-plinary research in breast cancer. However, there are fewwell-characterized cell lines derived from human mammarycarcinomas. Reports of attempts to culture breast cellsappeared as early as 1937 (19), but it was not until 1958that Lasfargues and Ozzello reported the first successfullong-term culture of a breast tumor (47).

Technical difficulties in achieving continuous cultures ofthese tumors have been well documented (15, 18, 31, 44,78, 79, 92, 97, 111-113). First, numbers of malignant cellsprovided by tumor samples may not be great, and theirviability is often low. In addition, most breast tissue resected from cancer patients consists of stroma and supporting cells, with tumor cells comprising only a minority ofthe population. Abundant fibroblasts tend to outgrow theless prolific epithelial cells.

No uniformly successful method exists. Some investigators have attempted to recreate the in vivo milieu by providing connective tissue substrates such as mucopolysaccha-rides or collagen (47, 84,112,113). Others have focused onnutritional requirements (15, 31, 44, 47, 111, 115). Hormones especially may play a role, with serum additivescontributing variable and unknown amounts of steroids (24,

44), while the optimal dose response may lie within adefined range (54).

Attempts to culture breast cancer cells have apparentlybeen successful more often with cells from malignanteffusions than with cells from solid tumors (15-17, 23,108).' Effusions have the advantage of providing large

numbers of dissociated, viable tumor cells with little or nocontamination by fibroblasts. Several additional factorshave been suggested as contributing to success (23). Theseinclude: (a) seeding cells at extremely high densities because their growth may be dependent on their concentration; (b) serially decanting supernatants over a period ofseveral days to enrich cultures for malignant cells, whichattach more slowly than do other cell types such as meso-thelial cells and fibroblasts; (c) allowing prolonged periodsof dormancy; and (d) making initial passages at low splitratios.

Since the establishment of the BT-20 cell line in 1958, 15additional putative breast cancer cell lines from primarytumors have been established, and a greater number of celllines have been derived from solid tumors and pleural orascitic effusions of patients with metastatic disease. Theusefulness, however, of any purported breast cancer cellline as an in vitro model system rests on its pedigree and itscharacterization. Cell lines reported to date have beencharacterized to varying degrees. Furthermore, many spurious reports have appeared of cells that were not human orwere human but not of breast origin.

The present review attempts to provide a comprehensivelist of all human breast cancer cell lines established to date,both published and unpublished, and the extent to whichthey have been characterized. The data are derived frompublished reports, supplemented where possible by personal communication with those investigators who werewilling to permit incorporation of their unpublished data inthis review. We recognize that inevitably the data may notbe complete or comparable from one laboratory to anotherbecause of differences in methodology. A total of 57 designations for putative breast cell lines is listed. These refer to47 presumably distinct cell lines which are grouped according to the type of donor tissue from which they were derived(Table 1). Included are 16 lines derived from primary tumors, 4 lines from solid métastases,5 lines from asciticfluids, and 22 lines from pleural effusions.

For each cell line data have been provided when availablefor the following categories of information: (a) reference:unless otherwise indicated all data relating to a cell linewere obtained from this primary source. In cases in whichadditional specific information was obtained elsewhere, thereference(s) are provided in parentheses beneath the data

1 L. W. Engel, N. A. Young, T. S. Tralka and M. E. Lippman. Establishment and Characterization of a Progesterone-responsive Human BreastCancer Cell Line, manuscript in preparation.

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L. W. Engel and N. A. Young

Table1—¿�Continuedwhich

human,non-HeLaoriginandepithelialmorphologychromosomestudiesorphenotypeanalysisestablishes5;B,brainmetastasis;AG,adrenalglandmetastasis;A,noma;SC,solidcancerwithscirrhousregions;IOCS,

noma;ILC,infiltratinglobularcarcinoma.nspecified.

'Nelson-Rees(73);unique,presenceofbandedmarker).è*

ì lìÕ25*,celllinesforwhichhistopathologicaldiagnosesestablishbreastcancerandlightorelectronmicroscopyhavebeendemonstrated;"*,evidenceprovided

que,non-HeLaorigin.

Tissuesource:O,originaltumor;L,lungmetastasis;LN,lymphnodemetas

itesfluid;P,pleuraleffusion.

Race:C,Caucasian;I,AmericanIndian;B,Black.

Histopathologicdiagnosis:IDC,infiltratingductalcarcinoma;AC,adenoc;(ratingductalcarcinoma,mucin-producing,signet-ringtype;MC,medullaryc

Species:H,human;R,rat.

Morphology:E,epithelial;Sp,spindle-shaped;S,spherical.

R.Nordquist,personalcommunication.

Animals:N,nudemice;I,immunosuppressedmice;R,rat;H,hamster;M,mieHeLa,presenceofmarkerchromosomescharacteristicofHeLacells,criteriomosomespresumablydistinctfromotherknowncells,criteriaofNelson-Rees

E.Miller,personalcommunication.

Oncofetalantigen(BOFA)positive(32).E.Lasfargues,personalcommunication.

LDH:H,human;R,rat.1

>,E- H« •¿�£•%.•*-Ê-*-•¿�_Q3 rc C uelli

inBraziltodeterminewhetherBTM-1cellshavebeen.

ocrDuetoMycoplasmacontamination,BT-410cellsareunavailablefordistributioM.Parshley,personalcommunication;wehavebeenunabletocontactDr.MaC

0¿>O

CO.c

>CD

COCDQ.R.

PalilloandM.Story,personalcommunication.aI.

Benjamin,personalcommunication.»ddition

toinfiltratingductalcarcinoma.acCarcinosarcoma

isquestionedbecauseofsarcomatousappearanceofstroma

G.Seman,personalcommunication;SH-2andSH-3cellsareidentical.

A.Leibovitz,personalcommunication.i.

«~the

samespecimen.oM.

Lippman,personalcommunication;EvsaEandEvsaTcellswerederivedfr

T.S.Tralka,personalcommunication.a

-om

whichtheLevandSalcultureswerederivedwere

liveni.v.injectionsinthetailveinwith106cells.•^

togE.Beth,personalcommunication;multiplealiquotsoftheoriginaleffusion

gnedRomannumeralssequentially,e.g.,LevI,II,III.

L.Liotta,personalcommunication;pulmonarymetastasisobservedin0/10mia

u>HCOCDike

particlesdetected(60).

scellswithXXandcellswithXYcomplement(2)to

toZR-75-27

andZR-75-30cellswerederivedfromthesamepatient(23).734BandMCF-7cellswerederivedfromthesamespecimen(102).

"H.Soule,personalcommunication.

''Reexpressionoftheoriginaltumorpatterninspongeculture(95);Oncornavir''Produceslymphomatoustumorbutnotcarcinoma.Chromosomeanalysisre\

''I.Keydar,personalcommunication.a

e* c ^ t, t:COsoCo£Corao

co^5Q>C

HI4,4)

4330 CANCER RESEARCH VOL. 38

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Breast Carcinoma Cells

for that characteristic; (b) patient characteristics: age andrace; (c) histopathological diagnosis; (d) evidence of inter-and intraspecies specificity: karyology (including species,modal number of chromosomes, and trypsin-Giemsa banding patterns), isoenzyme profiles, and HLA antigens; (e)morphology: structure as demonstrated by light and electron microscopy; (f) growth characteristics: split-ratio,doubling time, and tumorigenicity; (g) differentiated functions: morphological evidence of secretory activity, presence of hormone receptors, hormone responsiveness, andsynthesis of casein, a-lactalbumin, fatty acids, or milk-

specific antigens (Table 2).

Histopathologic Diagnoses

Cell lines for which histopathological diagnoses are provided are indicated in Table 1. Pathological data wereunavailable to us for the CaMal, MDA-MB-253, MDA-MB-309, MDA-MB-331, MDA-MB-390, MDA-MB-416, MDA-MB-431, and MDA-MB-436 cell lines. The diagnosis for theCaMa cell line, "solid cancer with scirrhous regions," is not

a currently accepted diagnostic term but probably indicatesmedullary carcinoma. The AlAb cell line was derived froma poorly differentiated adenocarcinoma in the lung forwhich a primary site in breast was postulated but uncertain(90).

Among the 37 cell lines for which histopathological dataestablish breast cancer, infiltrating ductal carcinoma is themost frequent nosological designation. The Evsa E andEvsa T cell lines (from the same specimen) were derivedfrom a mucin-producing, signet-ring cell carcinoma, anunusual variant of infiltrating ductal carcinoma. The MDA-MB-330 cell line was derived from an infiltrating lobularcarcinoma, while MDA-MB-157 cells were derived from amedullary carcinoma of the breast. Some additional tumorsof origin carry a diagnosis of adenocarcinoma of the breast,but these diagnoses offer no information as to specific celltype.

Tumorigenicity

Available data concerning growth of cell lines in nudemice, in other animals, and in soft agar are provided inTable 1. Success in heterotransplantation of solid breastcancers in nude mice has been variable despite their malignancy (35). In contrast, success has been reported in 20 of24 breast cancer cell lines transplanted into nude mice and8 of 9 lines cultivated in soft agar (Table 1). Published dataare insufficient to evaluate the correspondence betweenhistopathology of the original tumors in vivo with that of theheterotransplanted tumor cells in mice, but such data whenavailable may prove helpful in supporting specific humanmammary origin of cells maintained in continuous culture.

Interspecies Specificity

Interspecies specificity, or human origin of a cell line,may be established by several methods: (a) cell surfaceantigenicity determined by membrane immunofluorescencewhen reacted with species-specific antisera (100); (b) anal

ysis of isoenzyme electrophoretic mobility patterns forLDH2 or other enzymes (76); and (c) most commonly, anal

ysis of chromosome preparations. Detection of 28S rRNAmay provide additional evidence supportive of humanorigin (101).

Those cell lines for which cytogenetic studies were performed have all been shown to possess human chromosomes with the exception of the HBC cell line. Cell membrane immunofluorescence, isoenzyme mobility pattern forG6PD, LDH, and karyotype of HBC cells were all shown tobe compatible with the rat (Rattus norvegicus) (72).

Human LDH further confirms species origin for BT-20,BT-410, BT-474, BT-483, HBT-3, Hs578T, 734B, MCF-7, andT47-D cells (Refs. 3, 37, and 46; I. Keydar, E. Lasfargues,and H. Soûle,personal communication). For the MCF-7 cellline, cell surface antigenicity specific for humans, and RNAwith molecular weight characteristic of human rRNA furtherattest to their human origin (102).

Intraspecies Specificity

Since Gartler (34) proposed widespread HeLa cell contamination of other established cell lines based on isoen-zyme analysis, this particular ¡ntraspeciescontaminationhas received widespread attention. More recently, Nelson-Rees and Flandermeyer (71) have defined HeLa cultures ascells that meet the following criteria: (a) type A (fast)mobility for G6PD (the X-linked G6PD A phenotype occursin about 25% of Negro women while it is not seen inCaucasians; HeLa cells, derived from a Negro patient, aretype A); (b) phosphoglucomutase alíele1 at locus 1 and atlocus 3; (c) absence of a Y-chromosome by fluorescen*staining; and (d) possession of a complex of trypsin-Giemsa-banded marker chromosomes present in knownHeLa cells. Since laboratory contamination is a potentialproblem for even the most meticulous worker and since anumber of alleged breast cancer cell lines have been shownto be HeLa cells (70-73), investigators are now obliged toprovide evidence that cultures are of unique human originor, at a minimum, that they were derived from a donordistinct from HeLa cells.

Of the 47 cell lines here listed, 6 are in reality HeLa cellsby the criteria of chromosome markers and G6PD mobility:HBT-3 and its sublines G-11 and HBT-E (16c); HBT-39 andHBT-39B (clone 6); BrCa5, SH-2, SH-3, and the EICo celllines (69-73). SH-2 and SH-3 cells are identical (G. Seman,personal communication) as are the EICo, HBT-39B, andHBT-3 cell lines (W. Nelson-Rees, personal communication). Cell lines that have been identified as non-HeLa onthe basis of their possession of G6PD type B are listed inTable 1 (Refs. 7, 23, 30, 37, 46, 75, and 102; E. Lasfargues,A. Leibovitz, and I. Keydar, personal communication). Inaddition, unique karyotypes that are distinct from HeLacells by trypsin-Giemsa banding techniques have beendemonstrated for the MCF-7 and 734B cell lines and for BT-20, BT-474, BT-483, T-47D, Hs578T, MDA-MB-157, AlAb,SK-Br-3, ZR-75-1, ZR-75-27, ZR-75-30, and ZR-75-31 cells

2 The abbreviations used are: LDH, lactic dehydrogenase; G6PD, glucose-6-phosphate dehydrogenase; BCTF, Breast Cancer Task Force.

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L. W. Engel and N. A. Young

(Refs. 23, 37, 46, and 74; I. Keydar, personal communication).1

Laboratory contamination with human cells other thanHeLa has also been noted. For example, while the SW-527cell line, which exhibits 9% polyploidy, and the SW-613 cellline, which exhibits 20% polyploidy, have a karyotype distinct from HeLa cells, they each have a modal number of 60chromosomes and identical trypsin-Giemsa-banded chromosome markers (W. Nelson-Rees, personal communication) and identical isozyme patterns (29). Pending furtherstudies to determine their origin, it is requested that thesecells not be referred to as breast cell lines (A. Leibovitz,personal communication).

Using a modification of the enzyme typing earier described by Povey ef al. (89), O'Brien et al. (71)3 have

reported a system that can further discriminate betweenhuman, non-HeLa donors. Electrophoretic resolution of 8human gene-enzyme systems that are polymorphic andexpressed in cultured cells produces a virtually uniquegenetic signature. Since the specific frequency of eachenzyme genotype within human populations is known, theprobability of genetic identity by chance alone between 2cell lines can be calculated as the multiplicative product ofthe individual probabilities. Enzyme analysis has been reported for the EICo, ZR-75-1, ZR-75-27, ZR-75-30, and ZR-75-31 cell lines (23)'-3 ZR-75-27 and ZR-75-30 cells were

derived from the same patient and, although they possessunique karyotypes distinct from one another, have identicalallozyme phenotypes (23). Typing of human histocompati-bility (HLA) antigens, which may also be useful for identification of cultured cells lines (26), has been performed forthe BT-20, MCF-7, MDA-MB-134, MDA-MB-157, and MDA-MB-231 cell lines (Table 1).

Morphology

Cell lines for which light or electron microscopy hasrevealed characteristically epithelial morphology are indicated in Table 1. Cell lines SK-Br-2 III, or Sal III, SK-Br-1 III,or Lev III, and ZR-75-31 (floating subculture) each havespherical morphology by light microscopy. ZR-75-31 cells,however, reveal typical epithelial features when examinedby electron microscopy. Data on the ultrastructure of SK-Br-1 and SK-Br-2 are unavailable to us. For the Lev III cellline, an origin other than female breast is likely because itproduced lymphomatous infiltrates of primitive cells resembling reticulum cells when heterotransplanted into immu-nosuppressed mice (2). Furthermore, a portion of the cellpopulation possesses a Y-chromosome (2).

Minimum Criteria for Establishment of Human MammaryOrigin

Establishment of human, non-HeLa origin and demonstration of epithelial morphology by light or electronmicroscopy is relatively straightforward. These criteriamight therefore serve as a minimal requirement for estab-

3 S. J. O'Brien, J. E. Shannon, and M. H. Gail. A Molecular Approach to

the identification and individualizaron of Human and Animal Cells in CultureIsozyme and Allozyme Genetic Signature, manuscript in preparation.

lishing that a putative breast cancer cell line is of humanmammary origin. Cell lines for which a reliable diagnosis ofbreast cancer is available and that meet these criteria areidentified by a single asterisk in Table 1. Additional geneticevidence provided by chromosome studies or phenotypeanalysis may be required to establish a unique, non-HeLaorigin and has been obtained for those cell lines identifiedby a double asterisk.

Several points regarding these cell lines may be of interest. Although 734B cells were the parental culture fromwhich the MCF-7 cell line was derived, after 25 serialpassages only the latter designation was retained. For theMDA-MB-134 and MDA-MB-175 cell lines, 4 and 6 multiplecultures, respectively, were derived from pleural effusionscollected serially from each patient. Roman numerals wereused to designate sequential subcultures, e.g., MDA-MB-134 VII. Care should be taken to distinguish for whichsubculture data are provided, since cultures sequentiallyderived from the same patient may have different biologicalproperties (23).

A normal diploid myoepithelial cell line, Hs57Bst, wasisolated from the same human breast tissue as the aneu-ploid mammary epithelial cell line, Hs578T (37). These celllines, therefore, provide a useful model for comparison ofnormal and neoplastic breast tissue.

ZR-75-27 and ZR-75-30 cells, although derived respectively from a pleural effusion and ascitic fluid from the samepatient and although phenotypically identical, have stabledifferences in their karyotypes, with respect to both modalnumber and trypsin-Giemsa-banded markers. In addition,lymphoblastoid cells, for which the presence of Epstein-Barr virus nuclear antigen was demonstrated by indirectimmunofluorescence, were separated from early passagesof ZR-75-30 cells and have been established in pure cultureseparate from the epithelial cells (23). ZR-75-31 cells existas both floating and attached subcultures.1

Cloning has been claimed for a number of cell lines, butmethodology on which to establish the validity of theseclaims has not been reported. Breast cancer cells established in our laboratory have proved to be extremely densitydependent in their growth, and we have had no success inestablishing subcultures from single cells.

Organ Specificity (Differentiated Function)

No single criterion affords conclusive proof of mammaryorigin. However, evidence strongly supportive of organspecificity may be provided by morphological studies andespecially by biochemical data indicating that the cells arehormonally responsive or synthesize proteins characteristicof breast epithelium. In practice the demonstration of thesefunctions is often complex and not always reproduciblefrom one laboratory to another or even within the samelaboratory.

Morphological Studies. Buehring and Hackett (13) reportthat ultrastructure of cells in culture may reflect the tissueof origin, since specific cell organelles characteristic oftheir in vivo tissue may be retained after long-term growthin vitro. They listed 3 structural markers that may serve toidentify mammary epithelial cells: (a) desmosomes; (b)tonofibrils; and (c) intracytoplasmic ductlike vacuoles.

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While desmosomes and tonofibrils may be exhibited byendothelial and mesothelial as well as epithelial cells,intracytoplasmic ductlike vacuoles are structures frequentlyfound in breast tumor cells and occasionally in mammaryepithelial cells in vivo (4, 13, 97, 104, 109). The presence ofdesmosomes, tonofibrils, and intracytoplasmic ductlikevacuoles therefore suggests that the cells originated frommammary epithelium, although an origin from secretoryepithelium of other tissues is also theoretically possible.

The reliability of ultrastructural criteria of human breasttumor cell lines is suggested by data reported by Buehringand Hackett (13). While they observed desmosomes, tonofibrils, and intracytoplasmic vacuoles in BT-20, 734B, andMDA-MB-157 cells, they failed to observe significant numbers of these structures in cell lines HBT-3 and AIAb. HBT-3cells have been shown to be HeLa cell contaminants by thecriteria of characteristic chromosome markers and G6PDtype A; in addition, they resemble HeLa cells ultrastruc-turally (12, 13, 33, 110). The AIAb cell line may have beenderived from lung tissue since the tumor of origin was alung tumor in a patient with previously documented breastcarcinoma, and the pathological diagnosis was uncertain(90).

Cell lines that contain desmosomes, tonofibrils, and intracytoplasmic vacuoles (Table 2) include BOT-2, BT-20,BT-474, BT-483, Evsa E, Hs578T, 734B, MCF-7, MDA-MB-157, T-47D and ZR-75-1, ZR-75-27, ZR-75-30 and ZR-75-31(Refs. 1, 13, 23, 37, 46, 83, 93, and 115; I. Keydar, T. S.Tralka, and R. Nordquist, personal communication).1 Inaddition, evidence of secretory activity has been observedfor BT-474, BT-483, Hs578T, and MaTu cells and for the ZR-75-1, ZR-75-27, ZR-75-30, and ZR-75-31 cells lines (23, 37,46, 114).'

A chromosome marker involving the distal segment of thelong arm of chromosome 1, designated 1q, has beenreported by Jones Cruciger ef a/. (41) in cell lines andexfoliated cells derived from malignant effusions in patientswith breast cancer. The marker was observed in cells fromall 9 subjects examined. However, the significance of theseobservations is unclear because data are not provided forcells not originating from human breast cancer. Moreover,one of the 9 specimens, the SH-3 cell line, has beenreported to be HeLa (71). Presently available data thereforeappear to indicate that the 1q marker is not specific forbreast cancer cells.

Biochemical Studies

a-Lactalbumin Synthesis. u-Lactalbumin, a protein believed to be specifically synthesized by functionally differentiated mammary epithelial cells (86), has been consideredby some workers as the sine qua non of mammary origin.The protein has been identified as the B protein of lactosesynthetase, which catalyzes the formation of lactose fromUDP-galactose and glucose (Equation A) (9, 10, 22). The Aprotein of lactose synthetase is a galactosyltransferase, andin the absence of a-lactalbumin catalyzes the second reaction (Equation B) (8).

UDP-galactose + glucoseLactose synthetase. Mn"

UDP-galactose + A/-acetylglucosamineGalactosyltransferase, Mn", N.

Lactose synthetase !A protein (galactosyltransferase)B protein (a-lactalbumin)

U[)p (A)

UDP (B)

Galactosyltransferase has been isolated from most tissues including breast (61). a-Lactalbumin functions as a"specifier" protein, in that it lowers the apparent Km for

glucose, thus making it a physiologically active substratefor the galactosyltransferase (10, 66-68). It is this reactionthat is believed to be organ specific and results in lactosesynthesis.

Two techniques are available for detection of «-lactalbu-min. The first is an enzymatic assay in which incorporationof [14C]galacotse from UDP-[14C]galactose into lactose is

measured. In the presence of a saturating amount of the Aprotein, increased activity may be attributed to the presenceof a-lactalbumin. One limitation of the enzymatic method isthe ability of galactosyltransferase itself, under certainassay conditions, to catalyze formation of limited amountsof lactose (67). a-Lactalbumin synthesis as measured byenzymatic assays performed by B. Vonderhaar, has beenrecorded for a number of purported breast cell lines (48,49, 51, 52, 54, 55, 56, 58, 80). She reports at this time, however, that the presence of enzymatically active «-lactalbu-min can be attributed with certainty only to MCF-7 cells.These cells have been tested on several occasions, undervarious growth conditions, and the product was confirmedas lactose by Chromatographie techniques. Levels of lactose formed were variable. Reports of a-lactalbumin production for other cell lines, including HT-39 and G-11,which are HeLa strains, were based on single assays thatmeasured very low levels, and the significance of theseresults is questioned because of the limitations in the specificity of the method detailed above. AIAb cells were mistakenly reported as having been assayed (B. Vonderhaar,personal communication). Enzymatic assay for ZR-75-1cells (performed by G. Bolán) measured 0.2 to 0.45 pmola-lactalbumin per /¿gprotein (M. Lippman, personal communication).

The second method, radioimmunoassay, offers the advantages of greater sensitivity and superior specificity (42,43, 91 , 96). Results of radioimmunoassays on breast cancercells in continuous culture have been variable. Generally,only low levels of «-lactalbumin have been detected, andeven within a single laboratory repeated assays on a singlecell line have not been consistently positive. Rose andMcGrath (91) reported that the amount of a-lactalbuminsynthesized by MCF-7 cells, nourished by medium containing insulin, corresponded to 200 ng/mg of soluble cellprotein in 20 x 106 cells. Kleinberg ef a/., measured «-lactalbumin in homogenates of Evsa T cells on repeatedoccasions and for MCF-7 and BT-20 cells on a singleoccasion; but repeated assays on additional subcultures,as well as for the BT-474, SW-527, SW-613, ZR-75-1 , ZR-75-27, ZR-75-30, and ZR-73-31 cell lines, were negative, evenwhen prolactin was added to cultures at a concentration of1 M9/ml (Fiefs. 23 and 43; D. Kleinberg, personal communication).1

Recently, Schultz and Ebner (96) reported 0.3 ng a-

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lactalbumin per mg of cell protein for the MDA-MB-157 andMDA-MB-231 cell lines and 3.1 ng a-lactalbumin per mgprotein for the MCF-7 cell line. In contrast, their assaysdetected <0.1 ng a-lactalbumin per mg of protein for theBT-20 and MDA-MB-134 VII cell lines. Assays performed oncultures submitted by several investigators and stored inthe BCTF Cell Bank were negative for SW-613, BT-474,MDA-MB-330, and T-47D cells. In some instances theseassays were performed on prolactin-stimulated cells. Incontrast, BT-20 and MDA-MB-231 cells were negative initially, but prolactin-stimulated cultures were consistentlypositive for BT-20 cells and variably positive for MDA-MB-231 cells at a later date. MCF-7 cells were positive on bothoccasions. These assays were performed by H. Esber (personal communication). Negative results are also recordedin the BCTF Cell Bank Inventory for BT-483, Hs578T, MDA-MB-134, MDA-MB-157, MDA-MB-175, MDA-MB-361, andMDA-MB-415 cells. These assays were performed by K.Ebner (personal communication).

Additonal reports (51, 81) have described a-lactalbuminmeasured by radioimmunoassay for MCF-7, ZR-75-1, EvsaT, Evsa E, MDA-MB-231, HT-39, and AlAb cells (referred toin these reports as 496). While these reports are confirmedfor MCF-7 and Evsa T cells, reexamination of the datareveals that assays on ZR-75-1 cells were negative and thatthose for the Evsa E, MDA-MB-231, HT-39, and AlAb celllines were mistakenly reported as having been performed(M. Lippman, personal communication). In summary, whileconflicting results have been reported for a number ofputative breast cell lines, only for MCF-7, BT-20, and MDA-MB-231 cells has detection of this protein been confirmedindependently in several laboratories.

The reasons for inconsistent demonstration of a-lactalbumin synthesis by breast cancer cell lines are not clear: (a)the majority of breast carcinomas in continuous culture areof duct cell origin, while other cell types, such as epithelialcells originating in the mammary lobules, may be thesecretors of milk-specific proteins; (b) breast cells in culture may lose differentiated function either as a result ofmalignant transformation or as a result of in vitro cultureconditions. Conversely, cells in culture may acquire functions following long-term culture as a result of derepressionof genes not normally expressed; (c) synthesis of a-lactalbumin may be dependent upon some other factor, such asprolactin. However, all reports to date have failed to showan effect clearly attributable to prolactin stimulation (Refs.43, 91, and 96; H. Esber, personal communication); (d)differing assay conditions with respect to variables such ascell mass, methods of solubilizing cell extracts, and passage level of the cell lines may each affect results; (e) sincethe levels of a-lactalbumin measured are often at the limitsof detection of the assays, results may be positive only inoccasional experiments.

Recent data suggest that synthesis of a-lactalbumin isnot asine qua non criterion of mammary origin. Kleinberger al. (43) reported that, while a-lactalbumin was detectedby radioimmunoassay in 48.5% of histologically normalbreast tissues, only 20% of breast cancer tissues werepositive. These data suggest that some breast tumors maylose their capacity to synthesize a-lactalbumin. Moreover,a-lactalbumin has been detected in some non-breast tis

sues, including amniotic fluid, cord blood, uterus, andserum of males as well as females, suggesting the possibility of extramammary sources of this protein.

Casein Synthesis. Casein has been detected in T-47Dcells by immunofluorescence with the use of antisera directed against human casein (I. Keydar, personal communication). The BCTF Cell Bank (7) reports casein synthesisfor MCF-7 and Hs578T cells. For the Hs578T cell line, theclaim of casein production was based on the observationby electron microscopy of aggregates of protein granuleswith the periodicity and organization of casein (37). In theabsence of biochemical or immunological data, it is difficultto evaluate the significance of this observation. The claimfor MCF-7 is apparently mistaken (C. McGrath, personalcommunication). There are as yet no reports of caseinidentified by radioimmunoassay in any alleged humanbreast cancer cell line (63).

As in the case of a-lactalbumin, a lack of breast specif ¡cityfor casein production is supported by the demonstrationthat serum casein levels were elevated in about 50% ofsamples from patients with carcinoma of the colon as wellas in 10% of samples from patients with carcinoma of thebreast (63).

Other Milk-specific Antigens. Ceriani ef al. (20) recentlydescribed an immunofluorescence technique in which rabbit antibodies against components of the human milk fatglobule bind specifically both to normal human breastepithelial cells and to cell lines derived from breast carcinomas. Antigens of human mammary epithelium have beendetected on the surfaces of 734B, BT-20, Hs578T, MCF-7,and MDA-MB-157 cells, and this technique may provevaluable as a method to identify mammary epithelial cells.

Hormone Receptors, Hormone Responsiveness. Thepresence of receptors for and biological responses toseveral hormones known to influence the growth and development of the breast strongly supports the origin of anestablished cell line from mammary epithelium. Assays forhormone receptors are becoming widely available andcould provide additional markers for comparisons betweenestablished breast cancer cell lines and the original tumorsfrom which they were derived (55, 57).

Eight of 22 cell lines have been shown to contain high-affinity estrogen-specific receptors (Table 2). However, thetrue number of cell lines containing estrogen receptors mayactually be higher, because Horwitz ef al. (40) report thatestrogen receptor, when present, is usually localized in thenucleus, often occupied by hormone; assays that examinecytoplasmic content of estrogen only may therefore havefailed to detect the presence of this protein. Assays for arange of steroid hormone receptors detected the presenceof receptors for sex steroids in 10 of 22 cell lines tested.While the distribution of types of receptors among themvaries, the data seem to suggest that the presence of sexsteroid hormone receptors in human breast cancer cells inculture is common. Data on hormonal receptors may therefore be useful both in supporting mammary origin and inidentifying their potential usefulness for in vitro modelsystems of hormonal responsiveness.

Biological responses to steroid hormones have beenstudied in a few cell lines only (Table 2). The methods havebeen described in detail (52). Briefly, incorporation of

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radiolabeled thymidine into DMA is measured in cultures towhich physiological concentrations of hormones have beenadded after preincubation in serum-free medium or mediumcontaining serum treated with charcoal-dextran. This"leaching" period allows previously occupied receptor sites

to be freed of steroids introduced in serum additives to themedia in which the cells were grown.

Evsa T cells fail to resond to any of the hormones (48, 49,51, 57, 58, 81), while MDA-MB-231 cells are inhibited byglucocorticoid (48, 49, 51, 57, 58, 81). MCF-7 cells respond

positively to estrogen (51, 52, 54, 56, 58, 81) despite failureto demonstrate a corresponding increase in growth in vitroin response to this hormone (39, 52, 54). They have,however, been shown to be estrogen dependent in athymicmice (C. McGrath, personal communication). In addition toestrogen, MCF-7 cells respond positively to androgen (51,

53, 55, 81, 116) and are inhibited by glucocorticoid (48, 49,51, 57, 58, 81). Androgen responsiveness, however, may bemediated by the estrogen receptor (116). ZR-75-1 cells are

stimulated by estrogen, show no response to progesterone,and are inhibited by androgen and glucocorticoid (23, 49,56, 57, 58, 81). The specificity of stimulation of MCF-7 andZR-75-1 cells by estradiol is reinforced by the inhibitory

effect of the antiestrogen, tamoxifen, and the ability ofestradiol to reverse this effect (23, 52, 54, 56, 58). For ZR-75-27 cells, significant increases above control were dem

onstrated for estrogen, for progesterone and, to a lesserdegree, for dihydrotestosterone (23). ZR-75-30 cells re

spond minimally to progesterone only (23), while greaterresponses to this hormone have been demonstrated for ZR-75-31 cells.1 The ZR-75-31 cell line may therefore be the

only one yet available for the in vitro study of progesteroneresponsiveness in human breast cancer.

Several reasons may explain failure to demonstrate hormonal responsiveness despite the presence of the corresponding receptors: (a) the cells may, in fact, be nonresponsive; (b) variation from experiment to experiment ischaracteristic, and maximal stimulation (or inhibition) mayrange from as little as 20% to more than 100%; (c) results ofindividual experiments in which low levels of response havebeen recorded may fail to achieve statistical significance.These results may be dismissed as negative by those whoanticipate dramatic departures from control levels. Poolingdata from repeated experiments, however, may providenumbers large enough to demonstrate a significant response; (d) responsiveness may depend on passage level;(e) the extent of stimulation in experiments may be limitedby intolerance of the cells to serum-free conditions and

failure to leach cells adequately of hormone already boundto receptor sites.

Although a requirement for insulin by the human mammary gland has not been clearly defined, insulin is essentialfor growth and development of the rodent mammary gland(106). The Evsa T, MCF-7, MDA-MB-175, and ZR-75-1 cell

lines have been studied for the presence of insulin receptor,and each has been shown to possess specific, high-affinity

insulin receptors. In addition, 2 of the 4 breast cancer celllines tested, MCF-7 and ZR-75-1, respond to physiological

concentrations of insulin with enhanced macromolecularsynthesis, growth, and the lactational function of fat synthesis (64, 82). While MDA-MB-231 and Evsa T cells contain

insulin receptor, they fail to respond to this hormone.

Current Status

The data reported here are at variance with the idea thatthere are few breast cancer cell lines in continuous culture.In fact, 47 cell lines have been established over a period of20 years, 36 of these wihin the last 5 years. We have beenable to verify that 46 of the cell lines are currently maintained either by serial passages or in the frozen state.

No single criterion provides conclusive evidence of human mammary origin. Therefore, the breast cancer celllines most useful for research are those that have been themost thoroughly characterized. Twenty-one of the 47 puta

tive breast cell lines listed here meet minimal criteria ofhuman mammary origin, and 13 of these cell lines havebeen shown to be derived from genetically distinct donors.Differentiated function has been demonstrated for 19 of the32 cell lines studied for morphological and/or biochemicalevidence. In the majority of cases biochemical data, inaddition to morphological data, support mammary origin.Instead of attempts to develop new breast cancer cell lines,research efforts might now more profitably be directedtoward more thorough characterization of existing candidate human breast cancer cell lines for which only limiteddata are presently available.

Acknowledgments

The authors wish to express their appreciation to the many investigatorswho cooperated by verifying data and providing unpublished observations:R. Bassin, I. Benjamin, E. Beth, G. Cannon, K. Ebner, H. Esber, A. Hackett,K. Horwitz, l. Keydar, D. Kleinberg, E. Lasfargues, A. Leibovitz, L. Liotta. M.Lippman, C. McGrath, E. Miller. M. Monaco, R. Nordquist, S. O'Brien. C. K.

Osborne, M. Parshley, E. Plata. R. Palillo. G. Seman, H. Soûle,M. Story, T.S. Tralka, and B. Vonderhaar.

We are also grateful to G. Cannon, J. Fogh, P. Cullino, and J. Taylor forreading the manuscript and for their helpful suggestions. We wish especiallyto thank W. Nelson-Rees, S. O'Brien, B. Vonderhaar, and D. Kleinberg for

critical review of the manuscript and the contributions that they made towardits accuracy and completeness. A special note of thanks is extended to BettyPachine and Alice Rohan for their unstinting efforts in typing of the manuscript.

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