ICANCER RESEARCH57. 1590-1596. AprIl IS. 19971

ABSTRACT

The goal of this study was to isolate and expand tumor cells in culturethat closely resemble invasive cells in primary breast carcinoma tissue.Based on the hypothesis that invasive tumor cells are released more

readily upon digestion with connective tissue-degrading enzymes becausethey are not confined within a basement membrane, we have designed anovel procedure for their isolation. Using this method, we have successfully expanded In culture aneusomic tumor cells from several primarybreast tumors. Twenty nine of 44 (66%) specimens processed yieldedproliferative and passageable cultures of up to 2 x i07 celia. The originaltumor tissue and cultures derived therefrom were compared for aneusomy and the abnormal expression of the erb-B2, p53, and bcl-2 geneproducts. Remarkable similarities were observed. However, some intratumor heterogeneity In chromosome content was found between touchpreparations and cultured cells. Overexpression oferb-B2 was observed inthe vast majority of cases (16 of 20), suggesting that this phenotype maybe important for dysregulated proliferation in vitro.

The simple and rapid method described in this report could enableroutine expansion of primary breast tumors and provide adequate awnhers of viable cells for studying and manipulating their functional characteristics.

INTRODUCTION

Difficulties in routine isolation and culture of primary breast tumorcells have resulted in the lack of appropriate model systems forstudying the various stages of breast cancer progression. The commonmisconception that malignant cells proliferate more rapidly than nonmalignant cells has led to the adoption of dissociation procedures andcell culture media that are optimized for the growth of nonmalignantepithelium. Paradoxically, in the case of primary breast tumors knownto be relatively slow-growing in vivo (I), the use of conventionalprocedures designed for normal breast epithelial cell culture results inthe rapid overgrowth of tumor cells by diploid epithelium (2). It ispossible that these diploid cells may represent nonmalignant epithehum interspersed within the tumor tissue. Alternately, as postulatedpreviously (3), the slow evolutionary process in breast cancer mayresult in the coexistence of the originally diploid neoplastic cells andvarious aneuploid subsets within the primary lesion.

Previously used approaches of breast tumor tissue dissociation haveincluded: (a) mechanical disaggregation; or (b) complete degradationof connective tissue by enzymatic digestion. During mechanical mincing, large numbers of tumor cells “spillout―of the tissue. The vastmajority of such loosely aggregated cells that comprise the “spillage―are aneuploid; yet they are found to be nonviable and do not undergocell attachment or proliferation (4, 5).

Enzymatic dissociation is more widely used for the derivation ofpure epithelial cell populations from malignant and nonmalignant

Received 10/30/96; accepted 2/I 7/97.The costs of publication of this article were defrayed in pan by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

â€T̃his work is supported by NIH Grant lROl-CA66998, NIH Program Project Grant

1P01 -CA44768, and Specialized Programs of Research Excellence Grant 2P50-CA58207.2 To whom requests for reprints should be addressed, at Geraldine Brush Cancer

Research Institute, 2330 Clay Street, San Francisco, CA 941 IS. Phone: (415) 561-1653;Fax: (415) 561-1390; E-mail: shanaz@cooper.cpmc.org.

breast tissue. A mixture of connective tissue-degrading enzymes disrupts the stroma in which the breast epithelium lies embedded. Thus,upon completion of stromal degradation, reduction mammoplastytissue can be separated into a mesenchymal fraction consisting mostlyof fibroblasts and epithelial cell clumps termed “organoids.―Theseorganoids require additional protease digestion to yield individualcells, presumably due to the presence of intercellular tight junctionsbetween epithelial cells. When digested in this manner, tumor tissuealso yields organoids that can provide rapidly proliferating epithelialcultures, but these display a relatively normal chromosomal profile

(6).We hypothesize that nests or sheets of invasive malignant cells are

more susceptible to conventional enzymatic dissociation because ofabnormal deposition of basement membrane proteins (7—9).Thus,during long-term digestion (required for the isolation of organoids),these populations may be lost to degradation. Consequently, epithelialcells from the organoids cultured under routine conditions generallydo not display the malignant characteristics of the original tumortissue (10).

We have identified conditions that allow partial stromal degradation and thus facilitate the separation of nests of invasive tumor cellsfrom nonmalignant epithelium. Furthermore, in each case, we havedirectly compared the expression of a number of tumor phenotypes inthe original tissue with the cell populations that have emerged inculture. We demonstrate here that proliferating epithelial culturesderived in this manner from breast carcinoma tissue express severalcharacteristic phenotypes of the original tumor specimen.

MATERIALS AND METHODS

Tissue Specimens. Fresh surgical discard tissue from 44 cases of pathologically confirmed infiltrating primary breast carcinoma was obtained frompatients who had undergone surgery at the University of California, SanFrancisco, and at the California Pacific Medical Center, San Francisco. Twenty

cases of breast tissue peripheral to carcinoma and 7 cases of reduction mam

moplasty with no known malignancy were also processed. In all cases, aportion of the tissue was snap-frozen in liquid nitrogen for immunostaining.

Partial Enzymatic Degradation. An overall discussion of the method ispresented in “Results.―Details are as follows. Fresh tissue pieces in thefollowing categories: (a) primary breast tumor; (b) peripheral to carcinoma;

and (c) reduction mammoplasty, were first minced mechanically with scalpelsand scissors. For nonmalignant tissue in categories (b) and (c) above, adiposematerial was scraped off prior to mincing. Minced tissue was suspended incentrifuge tubes in a mixture consisting of collagenase type 1 at 200 units/mIand hyaluronidase at 100 units/mI (Sigma Chemical Co., St. Louis, MO) inF-12 medium supplemented with 10% FCS, penicillin, streptomycin, Fungizone, and gentamicin sulfate. The tubes were incubated at 37°Cand turned ona tube rotator for 1—6h until the suspension medium became turbid. Thedigested material was filtered through a 5 l-@m-pore nylon mesh. The filtrate

was centrifuged, and the cell pellet was plated in replicate dishes in lowcalcium (0.06 mM) MCDB 170 medium containing 2% FCS (UCSF CellCulture Facility, San Francisco, CA) and supplemented with insulin, epidermalgrowth factor, hydrocortisone (Sigma), and bovine pituitary extract (HammondCell Technology, Alameda, CA). Contaminating fibroblasts were removed bydifferential trypsinization, as described previously (6). Upon reaching subconfluence, cells were passaged by routine trypsinization procedures. Nonmalig

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Partial Enzymatic Degradation of Stroma Allows Enrichment and Expansion ofPrimary Breast Tumor Cells1

ShanazH. Dairkee,2Eileen C. Paulo,PedroTraquina,Dan H. Moore,Britt-MarieLjung,and HeleneS. SmithGeraldine Brush Cancer Research institute. California Pacific Medical Center, San Francisco, California 94115 [S. H. D., E. C. P., P. T., D. H. M., H. S. 5.1, and Department ofPathology. University of California. San Francisco, C'al:fornia 94143 fB-M. LI

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I

.—@

@ jc

PRIMARY BREAST TUMOR CELL CULTURE

Tissue

1-6hrs.

Fig. 1. Partial enzymatic degradation of breast tissue. Tissuedissociation procedure for enrichment of primary breast tumorcells. The most important methodological modifications in thisscheme are: (a) optimal period of enzymatic digestion; and (b)platingand expansionof the “filtrate―fractioninsteadof the“organoid―fraction.

.—.@ Cryopreserve

us ue TumorEpitheilum

CuftureFiltrate

@:‘@@ •@ ç@r\

M y,@ ‘@@ ‘@_,.@ ‘@

@@ . f.@.@.‘@1@ ‘1:J@1@

. _p_@ ..@rç@ .A@@':@1@

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.a'Fig. 2. Growth and morphology of tumor culture derived by partial enzymatic degradation of primary breast tumor tissue. a, cells 7 days after seeding. Note the presence of both

fibroblastsandepithelialcellsin the culture.b, cultureof pureepithelialcellsat secondpassage.c, expressionof cytokeratin19in tumorcellsat secondpassage.

nant peripheral tissue was digested for the same duration as the accompanyingtumor, whereas all reduction mammoplasty tissue was digested for 4 h.

Immunofluorescence.Primaryculturedisheswere trypsinizedwhensufficient cell numbers were achieved. An aliquot of the trypsinized culture wasplated on sterile microscope slides. Upon reaching subconfluence, slides werefixed in Orthopermeafix (Ortho Diagnostics, Raritan, NJ) for 40 mm. Eachslide was divided into chambers using a PAP pen (The Binding Site, SanDiego, CA) for indirect immunolocalization with mouse monoclonal antibodies to the gene products of p53, erb-B2 (Santa Cruz Biotechnology, Inc., Santa

Cruz, CA), bcl-2 (Dako, Carpinteria, CA), or no primary antibody control.Biotinylated antimouse secondary antibody (Vector Laboratories, Burlingame,

CA), followed by fluoresceinated-ultra avidin (Leinco, St. Louis, MO), wereused for signal amplification and detection. Cryostat sections of the originaltumor tissue were tested simultaneously with the cultured cells within the sameexperiment. Nonmalignant epithelium within the section served as normalbaseline control. Breast cancer cell lines, BT474 and T47D, cultured inDME + 10%FCS were used as positive controls for aberrant expression of thecellular phenotypes mentioned above. Expression of luminal epithelium-asso

ciated cytokeratins 18 and 19 was examined with monospecific antibodies(Progen, Heidelberg, Germany).

FISH? FISH analysis of tumor tissue was done on touch preparationsobtained by touching freshly sliced tissue to clean slides for capturing looselyadherent tumor cells. Details of fixation, in situ hybridization, immunostaining,and scoring of signals were described previously (11). Cultured cells weredirectly analyzed as adherent monolayers on microscope slides after fixationwith Carnoys. Pericentromeric probes to chromosomes I, 3, and 17 (kindlyprovided by Dr. Fred Waldman, University of California, San Francisco) wereused to detect numerical chromosome changes. Epithelial cultures expandedfrom reduction mammoplasty-derived organoids served as normal, diploid

controls. Fluorescence was visualized and recorded using a Zeiss Axioskopmicroscope and an Optronics CCD camera.

3 The abbreviation used is: FISH, fluorescence in situ hybridization.

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RESULTS

Tumor Dissociation Procedure

In contrast to methods of tissue dissociation reported in the literature, in the protocol described here, most importantly, we have optimized overall time of exposure to stroma-degrading enzymes for eachtumor. In this procedure, referred to as “partialenzymatic degradation,―instead of a long duration (usually 24 h or more to yieldorganoids), enzymatic dissociation was monitored closely after initiating digestion of minced tissue. Digestion was terminated when grossmacroscopic inspection displayed a relatively turbid suspension. Consequently, the time in enzyme mix ranged from 1—6h. The cellsuspension obtained upon filtration contained a dissociated fractionthat was reduced to small clusters or single cells by the partialenzymatic degradation of the collagenous matrix of the connectivetissue.

Another important methodological modification was the use ofcells in the “filtrate―fraction for initiating cultures of tumor epithehum instead of the “organoid―fraction retained on the filter. Theprotocol outlined in Fig. 1 was followed for processing and harvestinga total of 44 primary breast tumors, 20 specimens of nonmalignanttissue peripheral to carcinoma, and 7 reduction mammoplasty specimens.

Characteristics of Primary Breast Tumor Cells Isolated byPartial Enzymatic Degradation

The primary tumor digest obtained by partial enzymatic degradation generally displayed a high level of cellularity. In addition toputative tumor cells, it was contaminated with lymphocytes andfibroblasts to varying degrees. The lymphocytes remained in suspension and rapidly degenerated and lysed. Upon attachment to the

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Table I Growth potential of breast epithelial cells isolated by partial enzymaticdegradation

Tumor tissue and peripheral tissue differ significantly at P 0.012, based on the t@statistics.

Extent of growthTumor tissue (N)Peripheral tissue(N)No

epithelial cells18% (8)50%(10)Primaryculture, not passageable16% (7)15%(3)Two

passages36% ( 16)35%(7)Threeto seven passages30% (13)0%

Table 3 Aneusomy in cultures derivedfrom primary twnor tissue by partiaIenzymaticdegradation%

aneusomy (1 or 3 copies)―Chromosome1317

ChromosomeNo. ofcopiesTPCCTPCCI

3

171

23—6

12

3—b12

3—61%

13%86%

NDa

ND9%

42%49%

>99%

>90%>90%

12%58%30%5%55%40%>90%

>90%

28%45%27%65%

30%5%

70%30%

>90%75%

20%5%

64%5%

31%

>90%a

ND,notdone.

PRIMARY BREAST TUMOR CELL CULTURE

resentative of the invasive cells in the original tumor, fixed culturesand tumor tissue were compared: (a) for the presence of aneusomy byFISH analysis with chromosome enumeration probes (summarized inTables 2 and 3); and (b) for the expression of the erb-B2, p53, andbc!-2 gene products by indirect immunofluorescence (summarized inTable 4). Similarly derived cultures from nonmalignant tissue peripheral to carcinoma, as well as from routinely derived reduction marsmoplasty organoids were also included in the comparison.

The following observations were made which demonstrate themalignant characteristics of tumor-derived cultures.

Aneusomy FISH with pericentromericprobes to chromosomes I,3, and 17 showed that numerical changes in these three chromosomeswere common in tumor-derived cultures but not in cells that grew outof nonmalignant tissue (Table 2 and 3). A total of 18 cultures wereexamined at second passage, which included all 13 cases that werepassageable three to seven times and 5 cases that were growth arrestedafter two passages. Comparisons were made between the originaltumor and cultures derived from these tumors in three cases whereoptimal touch preparations were available (Table 2). Overall, touchpreparations and cultures were similar, although some differenceswere also detected. For example, in specimen G274T, the majority ofcultured cells contained one to three copies of chromosome 1, whereasthe touch preparations displayed a large number of cells with morethan or equal to four copies (Figs. 3 and 4). Similarly, in specimenS162T, although the tissue contained a large population (30%) of cellswith greater than two copies of chromosome 3, this population wasnot observed in culture. In contrast, for specimen S175T, the culturewas more abnormal than the touch preparations because a third of thepopulation displayed more than or equal to three copies of chromosome 3, which was not present in the touch preparations (Table 2). Itis possible that these variations are due to the sampling of differentareas of the tumor for touch preparations and cell culture isolation.

culture dish, contaminating fibroblasts (Fig. 2a) were readily removedby differential trypsinization to yield pure epithelial populations (Fig.2b).

As summarized in Table 1, in 18% of the tumor specimens cultured,there were no detectable epithelial cells; only fibroblasts grew out. Inanother 16%, there was no appreciable growth of epithelial cells; onlysmall patches of <50 cells, which did not progress further, wereobserved during the first week of culture. The majority (66%) of thecultures displayed continuously proliferating colonies. The cells had acharacteristic epithelial morphology, although some heterogeneity inshape and size was seen. Immunostaining for the luminal epitheliumassociated cytokeratins I 8 and 19 demonstrated strong homogeneousreactivity (Fig. 2C).

The proliferation rate of the tumor cultures was remarkably slow,with doubling times ranging from 4 days to 2 to 3 weeks. Yet almostone-half of the specimens were proliferative beyond second passageand were subcultured three to seven times. These cases yielded arange of 3 X lO@to 2 X l0@cells, which enabled the further in-depthcharacterization described below.

In 10 of 20 cases of nonmalignant tissue peripheral to carcinomathat were subjected to partial enzymatic degradation, no epithelialcells grew out of the filtrate upon plating in culture. From theremaining cases, the yield of epithelial cells was minimal. Thus, whenthese cultures were initiated, the epithelial cells were at clonal density.Rapid proliferation of these single cells in MCDB 170 medium,known to support clonal growth, resulted in large colonies. However,proliferation is known to be arrested in unselected, nontransformedepithelial cultures after 50 population doublings. Therefore, unlike thetumor-derived cells, nonmalignant cultures, as shown in Table 1,could not be subcultured beyond passage 2 (approximately 50 population doublings). Tissue from 5 of 5 reduction mammoplasty specimens dissociated by the partial enzymatic degradation protocol onlyyielded fibroblast cultures. When the same specimens were processedby the routine long-term enzymatic dissociation method for isolatingorganoids, rapidly proliferating epithelial mass cultures were obtainedfrom the plated organoids, which could be passaged four to five times.As described below, fixed cultures of nonmalignant cells were usedfor comparison with tumor-derived cultures.

To ascertain that the epithelial cells cultured from primary tumortissue by the cell isolation protocol described above are indeed rep

Case no.S174T 5 47 28Sl8lT 5 5 84SI89T ND― 5 30S213T 30 ND 45G3O1T ND ND 47S22lT 58 ND NDG312T ND 42 25G302T 65 ND NDG325T ND ND 30G344T ND 6 27

aA@ of 100cellswasexaminedforeveryvalue.Aneusomyl6%issignificantlydifferent from diploid control cells derived from one reduction mammoplasty specimenand one case of tissue peripheral to carcinoma, G3OIP (5% aneusomy for all threechromosomes) by Fisher's Exact test at P < 0.01.

b ND, not done, due to inavailability of optimal preparations.

Table 2 Numerical changes in chromosome 1. 3, and 17 in primary breast tumor touch preparations (TP) and tumor-derived cell cultures (CC), analyzed by FISH

G274T SI62T S175T

TP CC

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Table 4 Expression of malignant phenotype in primary breast tumor tissueandculturesderived by partial enzymaticdegradationPhenotype

in tumortissue―No.

of cases erb-B2 p53bcl-2Primary

tumorConcordantcases― 18/205

+ ——4+ ++3— ——2— —+2+ +—1+ —+I— +—Discordant

casesc 2/201+(-) —-1+(—) +(—) +(—)

PRIMARY BREAST TUMOR CELL CULTURE

positive (Table 4). Concordant expression of the erb-B2 protein in theoriginal tumor and in the tumor-derived culture was seen in I2 of 14cases. Erb-B2-positive tumor tissue displayed a combination of punctate cytoplasmic and membrane localization of the staining, andrarely, membrane-associated reactivity was seen (Fig. Sb). Culturesderived from nonmalignant breast tissue were negative for the expression of erb-B2 protein (Fig. Sc).

In one case, discordant expression of erb-B2 was seen, where thetumor was positive although the culture was negative. However, FISHdata for this culture (S 174T) showed the presence of aneusomy forchromosomes 3 and 17 in a large population of cells (Table 3). Inaddition, another specimen was found to be positive in the originaltumor but negative in culture. Because this specimen was weaklypositive in vivo, it is possible that such low levels of expression maybe undetectable in cultured cells because of changes in antigen densityper unit area.

As summarized in Table 4, 40% (8 of 20) primary tumors displayedpositive cellular immunostaining for the p53 gene product. In sixcases, >50% of the tumor cells in the tissue were positively stained,whereas in two cases, a smaller proportion of positive cells was seen.Immunofluorescence was predominantly nuclear in seven of eightpositive tumors (Fig. Se), whereas cytoplasmic reactivity was seen inone case. As illustrated in Fig. Sf in seven of eight cases, tumorderived cultures displayed the same pattern of immunostaining as theoriginal tumor tissue. T47D breast carcinoma cells, included as positive controls in every assay, showed a strong nuclear pattern of p53immunostaining in over 75% of the culture (Fig. 5h). In contrast, incultures from eight of eight specimens of peripheral tissue and sevenof seven derived from reduction mammoplasty tissue, a low basallevel of cytoplasmic staining was observed in the majority of cells(Fig. Sg), whereas nuclear staining was sporadic (< 10%). One casedescribed above (S174T) for discordant expression of erb-B2 proteinwas also found to be discordant for p53 expression.

aImmunofluorescenceresultswererecordedaspositiveif 10%tumorcellswerestained in tissue sections and cell cultures.

b Identical immunostaining results were obtained for both original tumor tissue and

cultured cells.C Immunostaining on tumor tissue and derived culture did not match. Results shown in

parentheses represent cultured cells.

An additional 15 cases of tumor-derived cultures were examined, ofwhich 10 were aneusomic (summarized in Table 3). In these tumorcultures, a maximum of 27—84%of the population displayed aneusomy for one of the three probes examined. In contrast, five differentreduction mammoplasty cultures were 95% diploid for each chromosome. The differences between tumor-derived and reduction mammoplasty cultures were statistically significant by Fisher's Exact test(P < 0.01). Five of 18 tumor cultures showed no aneusomy forchromosomes 1, 3, and 17. However, each one of these culturesdisplayed other phenotypes of the tumor tissue (described below).

Expression of erb-B2, p53, and bcl-2. Immunofluorescent cell

preparations that were optimal for microscopic evaluation were available for 20 of 29 specimens that yielded passageable cultures. Seventy%of thecasesin thisgroupof primarybreasttumorswereerb-B2

Fig. 3. FISH analysis of breast carcinoma touch preparations andtumor-derived cultures at second passage with pericentromeric probe tochromosome I . a, touch preparation of original tumor tissue. Note thepresence of multiple signals. b, nuclei in a counterstained with 4,6-diamidino-2-phenylindole. c, trisomic nuclei from a second passage cultare derived from tumor shown in a. d@nuclei in c counterstained with4',6-diamidino-2-phenylindole.

•1

I.

S • •

. I.

tip.@ 4',

t: a b

C d

1593

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PRIMARY BREAST TUMOR CELL CULTURE

100

80

60

40

20

0 fl: U

C.)

C

0

a)a.

1 2 3 4 5 6

Chromosome 1 Copy Number

Fig. 4. Distribution of diploid versus aneusomic cells in touch preparations and cells cultured from a primary tumor specimen. Among thecultured population, note increased numbers of cells with one to threecopies of chromosome I and a concomitant decrease in the number ofcells with higher chromosome I copy number.

. touchprepU tumorcultureD diploidcontrol

Expression of the bcl-2 gene product was observed in 40% of thefrozen tumor specimens examined. In seven of eight cases, tumortissue and tumor-derived cultures were concordant (Table 4). Stainingof bcl-2 protein was cytoplasmic in tissue sections of bcl-2-positivetumors and in BT474 breast carcinoma cells, used as positive controls(Fig. 5, i and 1,respectively). The tumor cultures generally stained lessintensely than BT474 cells but showed similar cytoplasmic localization of bcl-2 protein (Fig. Sf). With the exception of mitotic figures,cultures derived from nonmalignant tissue were uniformly negative(Fig. 5k). In one case, the tumor tissue was positive but the derivedculture was negative. This result may reflect tumor heterogeneity forthis phenotype, because as shown in Table 3, this culture was foundto be aneusomic (S174T).

The 3 of 20 specimens that were noninformative, i.e., simultaneously negative for erb-B2, bcl-2, and p53 expression (Table 4),were found to be aneusomic for at least one of three chromosomeenumerator probes (Table 2, S162T; and Table 3, S221T and G302T).

Is a Specific Malignant Phenotype Important for Growth inCulture?

To determine whether primary tumors, which could be appreciablyexpanded in culture, represented a unique population of tumor cells orif this was a random occurrence, we compared expression of erb-B2,

PS3,andbcl-2 in these tumorswith those thatwere nonproliferativeorshowed minimal growth. All 15 cases that did not yield passageablecultures (Table 1) were compared with the 20 cases of proliferativetumors shown in Table 4. As summarized in Table 5, there was nosignificant difference between the two populations in the expressionof p53 and bcl-2. However, the frequency of erb-B2 overexpressionamong those tumors that were able to yield passageable cultures wassignificantly higher (P < 0.016). This finding suggests an importantrole for the erb-B2 protein in the maintenance of tumor cell growth invitro.

1594

DISCUSSION

We have demonstrated here that partial enzymatic degradation ofprimary tumor tissue enables the isolation of tumor cells that are moreaberrant than those obtained from tumor-derived organoids by conventional enzymatic dissociation procedures. The factors that simultaneously contribute toward the effectiveness of this improved procedure are: (a) the elimination of contaminating nonmalignantepithelium; and (b) the maintenance of tumor cell viability, which isfound to be lacking in mechanically dissociated tissue (4). Thus,breast tumor cells, known to be slow-growing (1), are provided acompetitive edge over rapidly dividing nonmalignant breast epithehum for proliferation in culture.

In a previous study, we have reported on modifications in thecellular microenvironment that enable the selective isolation of primary tumor cells from the organoid fraction of the enzymatic digest(12). However, the yield of proliferating tumor cells per gram oftumor tissue was substantially lower than reported here. In this study,we have demonstrated that relatively small pieces of tumor tissue(<0.5 g/specimen) upon expansion in culture yield up to 2 X lO@cells. Most importantly, the cultured cells closely resemble the tumorcells in vivo for a variety of aberrant, dysregulated cellular phenotypes. In some cultures, the vast majority of cells were aneusomic. Inothers, although some aneuploidy was seen, a large percentage ofdiploid cells were also present. It is possible that these apparentlydiploid cells harbored deletions of specific chromosomal bands asreported by Teixeira et a!. ( 13). Alternately, the cells that were diploidfor the specific probes used may be aneusomic for other chromosomesnot examined in this study and may indeed represent bonafide tumorcells. This possibility is supported by the presence of other malignantcharacteristics in these cultures also observed in the tumor tissue, suchas the aberrant expression of erb-B2, p53, and bcl-2 gene products.

Although, in rare instances (< 1%), mechanically dispersed primary

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@ I b

@-@-c@.

0

d,‘

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Side.

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Table 5 Comparison of malignant phenotype in primary breast tumorsproliferative or nonproliferative in culturethat

wereNo.

of positivespecimensPassageable

in culture erb-B2p53bcl-2Yes

14/20 8/20No 4/15 5/15pa 0.013 0.358/20

6/150.64a

Fisher's Exact test.

PRIMARY BREAST TUMOR CELL CULTURE

Fig. 5. Indirect immunolocalization in primary breast tumor tissue and tumor cultures at passage 2. a—d,anti-erbB2 immunofluorescence on: a, tumor section; b, tumor culture; c,nonmalignant culture; and d, BT474-positive control. Note membrane localization of antibody in tumor cells in vivo. Cultured tumor cells display a punctate cytoplasmic pattem alsoseen in the majority of erbB2-overexpressing BT474 cells, whereas some cells show membrane localization similar to the tumor tissue. Nonmalignant cells are unstained. e—h,anti-p53immunofluorescence on: e, tumor section;f tumor culture; g. nonmalignant culture; and h, T47D-positive control. Note strong nuclear fluorescence in tumor tissue as well as culturedprimary tumor cells and T47D cells, whereas nonmalignant cells show weak cytoplasmic staining. i—I,anti-bcl-2 immunostaining on: i, tumor section;j, tumor culture; k, nonmalignantculture; and I, BT474-positive control. Note strong cytoplasmic fluorescence of several large nests of tumor cells in the tissue. Cultured primary tumor cells and BT474 cells also showstrong cytoplasmic fluorescence, whereas in the nonmalignant culture, only mitotic figures are positive. Tumor-tissue shown in i was photographed at X200. Cells in all other panelsshown were photographed at X400.

tumor tissue can give rise to aneuploid established cell lines (14—19),yet in the vast majority of cases, it consists of cells that are neitherviable nor proliferative (4, 5). In most cases, at the time of derivationof these cell lines, characteristics of the original tumor tissue that maybe relevant to the ability for in vitro growth were not reported(14—16).Thus, specific properties that distinguish those tumor populations that maintain growth in culture from those that do not remainto be determined, and their identification may require the approachesdescribed in this study on several large series of primary tumors. Inthis regard, we have observed here that 70% of the tumor specimensthat proliferated in culture overexpressed erb-B2. A higher incidenceof erb-B2 overexpression has been reported in established breastcancer cell lines and xenografts than seen in primary breast tumors invivo (19, 20). Because the erb-B2 gene product is a putative growthfactor receptor, it is often speculated that its overexpression mayprovide some growth advantage to the tumor. This possibility issupported by the correlation between large tumor size and erb-B2gene amplification (21) and overexpression in breast cancer (22).Furthermore, such tumors are generally more aggressive and result ina poor prognosis for the patient (21). Our findings here provideevidence for the role of erb-B2 overexpression as an important pa

rameter for tumor growth under a variety of conditions, includinggrowth in culture.

In the specimens processed thus far, we have not observed cellimmortalization, a phenotype exemplified by cell lines establishedfrom specimens at late stages of widespread tumor dissemination andmetastasis. On the other hand, primary tumor cultures are slowgrowing and display a steady attrition rate, possibly by a variety ofmechanisms similar to those observed in histological sections. Thus,it is conceivable that some of the specimens in this study that did notdevelop into passageable cultures upon partial enzymatic degradationinitially had a low level of viable tumor cells. Those specimens thatwere proliferative in culture did not display the ability for indefinitegrowth or immortalization because this may require additional geneticinstability and selection within the tumor tissue. In this regard, ourdata suggest that overexpression of erb-B2 may be a phenotype that isnecessary but not sufficient for immortalization of primary breasttumor epithelium. Additional studies on erb-B2-overexpressing primary tumor cultures may identify key ligands and factors in the signaltransduction pathway mediated by erb-B2, the autocrine expression ofwhich may be necessary for immortalization in culture. Such information will indeed be useful in defining new approaches for blockinggrowth-promoting signals mediated through such factors.

Unlike the statistically significant correlation observed betweenerb-B2 overexpression and proliferation of tumor cells in culture,aberrant expression of the p53 and bcl-2 gene products did not appearto be associated with growth in vitro in this series of primary breasttumors. The p53 protein, a mediator of 0@ arrest, functions as acheckpoint for DNA repair, thereby directing unrepaired cells towardan apoptotic pathway. Because the wild-type protein has a shorthalf-life, the nuclear accumulation of high levels of protein, reflecting

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PRIMARYBREASTTUMOR CELL CULTURE

its stability, has been frequently correlated with the presence of afunctionally aberrant protein (23—25).Surprisingly, however, nuclearlocalization of the p53 protein in cultures of nonmalignant breastepithelium has been reported previously (26, 27). In our study, inchrect immunolocalization data have demonstrated that under identicalconditions of growth, nonmalignant cultures displayed sporadic nuclear positivity, as also observed by Gudas et a!. (28). In contrast, likeestablished tumor cell lines, primary tumor cultures derived fromp53-positive tumors were relatively homogeneous and consistent inthe expression of nuclear p53. As the ability to routinely expandprimary breast tumor cells to greater numbers is improved, it will bepossible to confirm these observations with additional techniques.Similarly, overexpression of the antiapoptotic bcl-2 protein, whichlocalizes to the outer mitochondrial membrane, nuclear envelope, andendoplasmic reticulum, is widely reported among primary breastcancers (29—31). In the normal breast in vivo, cyclical variation inprotein expression has been observed (32). Consistent with otherreports on cultured breast cells (33), we have found that the bcl-2protein is detectable only in mitotic nuclei of cultured normal breastepithelium. To our knowledge, this is the first report demonstratingthe maintenance of homogeneous bcl-2 expression in cultures derivedfrom bcl-2-positive primary breast tumors.

The development of methods that routinely and reliably allow thederivation of viable, proliferative cultures from primary tumors canhave a rapid and significant impact toward understanding tumorbiology. These cultures: (a) could provide representation of a muchwider spectrum of tumors, particularly in view of the widespreadheterogeneity of primary breast cancer; and (b) could enable in-depthanalyses of those critical stages in tumor progression that precedemetastatic dissemination. Furthermore, these viable cell populationscould serve in a more meaningful way toward studying the regulationof aberrant cell function directly related to tumorigenesis instead ofcharacteristics acquired through the genetic instability generated byyears of cell culture and passaging.

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1997;57:1590-1596. Cancer Res   Shanaz H. Dirkee, Eileen C. Paulo, Pedro Traquina, et al.   and Expansion of Primary Breast Tumor CellsPartial Enzymatic Degradation of Stroma Allows Enrichment

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