Breast Cancer Research and Treatment 17: 121-129, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.

Report

Cultured cell lines from human breast cancer biopsies and xenografts

Walter M. Lewko, Rupa Vaghmar, Diane Hubbard, Marthanna Moore, Yun-Ju He, Lee Chang, Salah Husseini, Karen Wallwork, Gary B. Thurman, and Robert K. Oldham

Biotherapeutics Inc., Tumor Cell Biology Section, Franklin, Tennessee 37064, USA

Key words: breast cancer, tissue culture, xenograft, immunochemistry, extracellular matrix Summary Eighty-five breast cancer specimens were processed as part of a program in tumor acquisition, propagation, and preservation for biotherapy. Nine long-term culture cell lines were developed. Four cell lines were from solid tumor metastases, two lines were from pleural fluid specimens, and three were from xenograft tumors grown in nude mice. Two of the xenograft-derived cell lines were from biopsies which produced tumor cell lines as well. Success in establishing cultures did not correlate with the viability of the biopsy received. Poor tumor cell attachment to culture plastic was the most common problem. For certain specimens, attachment and growth were enhanced on collagen and extracellular matrix substrates. Collagen was beneficial in the development of one cell line. The cell lines were characterized and each of the lines contained more nuclear DNA than found in normal cells. Four of five lines tested were tumorigenic in nude mice. Five of nine were clonogenic in soft agar. Each of the cell lines tested reacted with at least two anti-tumor monoclonal antibodies. Xenograft and biopsy-derived cell lines from the same tumor were similar in their characteristics. While breast cancers are indeed difficult to establish and propagate in culture, the use of xenografts and special substrates appears to be beneficial in the development of cell lines from some tumors.

Introduction A tumor acquisition, propagation, and preservation (TAPP) program was set up to provide the tumor cells required for development and testing of biological therapies. These therapies include active specific immunization [1], development of drugantibody conjugates [2], toxin-antibody conjugates [3], and tumor-derived activated cells [4]. Patients with advanced metastatic disease who have failed standard therapies are candidates for biological therapy. Expansion of tumor tissue is an important goal of the TAPP program, particularly for breast

cancer patients for whom the available biopsy material is usually small. Several methods have been described for the short-term growth of normal and neoplastic breast tissue in culture. However, success in long-term propagation has been limited. Only about 40 breast cancer cell lines have been described in the literature, the majority of which were derived from pleural effusions. Only four cell lines have been documented from solid metastatic tumors [5-9]. In this paper, we describe the development and characteristics of several new breast cancer cell lines including four lines from solid tumor metastases. We

Address for offprints: W.M, Lewko, 6913 Southern Woods Drive, Brentwood, TN 37027, USA

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also describe efforts to enhance success in the development of long-term cell lines by the use of xenografts as a source of cells for culture and the use of various culture substrates for increased attachment and growth. Propagation and properties of an uncommon form of breast cancer, cystosarcoma phylloides, are presented in a separate report [10].

Materials and methods

Tumor transport Solid tumors were transported to the laboratory in sterile RPMI 1640 medium (Gibco, Grand Island, NY) with 10% heat-inactivated fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT), penicillin (200 U/ml), and streptomycin (200/zg/ml). Specimens were packed in an insulated case with frozen reusable ice packs to keep them cool. Heparin (Elkin-Sinn, Cherry Hill, N J) was added to pleural and ascites fluid specimens, and the fluids were shipped at ambient temperature in an insulated case.

Processing Specimens were processed for cell culture, xenograft development, immunohistochemistry, and cryopreservation. A small part of the specimen was quick-frozen in HistoPrep Embedding Media (Fisher Scientific, Orangeburg, NY) to prepare blocks for sectioning. The rest of the tumor was finely minced using crossed scalpels. Mechanically released cells in the culture medium were collected after allowing the heavier tumor pieces to settle. Cells were counted by microscopic analysis using a hemacytometer. Viable cells were determined by the trypan blue dye exclusion technique. Mechanically prepared cells were plated at 1 × 106 cells per T25 flask in 5 ml of growth medium. Part of the remaining tumor mince was treated with collagenase (type II, Worthington, Freehold, NJ) using 3125 units per 10 ml medium per gram tissue. The

medium contained 5% FBS. The tissue dispersion was carried out for 70 minutes at 37° C with stirring. Deoxyribonuclease (Worthington, Freehold, N J) was added for the last ten minutes of the incubation at 100 units/ml. The dispersed cells were then passed through a 60 micron mesh nylon filter. Part of the enzymatically prepared cells were plated at 1 × 106 cells per T25 flask in 5 ml growth medium. Explant cultures were prepared by placing five 1 mm 3 pieces of minced tumor tissue onto the surface of T25 flasks. The pieces were allowed to adhere for two minutes, after which growth medium was carefully added to the flask so as not to dislodge the explants. Growth medium was composed of RPMI 1640 with 10% FBS, penicillin (100 U/ml), and streptomycin (100t~g/ml). Cultures were incubated at 37° C with 5% COz in air. Two weeks after plating, antibiotics were no longer used. Culture medium was routinely replaced when acidified by metabolic activity. Certain breast cancer cultures appeared to fare better when left in a relatively acidic (approximate pH6.7) medium. At passage, the 10% FBS medium was switched to RPM11640 containing 4% FBS and 6% Hyclone supplemented calf bovine serum (CBS). Control studies indicated breast cell lines grew well in medium supplemented with this serum combination. Cultures that proliferated were expanded into T75 and T150 flasks. Breast cell lines generally grew better when cultured at a relatively high density. Therefore, care was taken to replate and maintain the cultures at a density of 4 × 104 cells/cm2 or greater. For substrate studies, collagen-coated flasks were prepared by layering sterile collagen solution (2-3 mg/ml, Vitrogen, Collagen Corp., Palo Alto, CA) onto the surface of tissue culture flasks, After two minutes, the excess solution was removed. The flasks were allowed to dry overnight [11]. The dried film of collagen was rinsed once with RPMI 1640 medium prior to plating the cells. For other studies, ECM-coated flasks were purchased from Accurate Chemical Co. (Westbury, NY), and Matrigel was purchased from Collaborative Research (Newton, MA).

Cultured breast cancer cell lines Fibroblast control Fibroblasts were routinely controlled by differential trypsinization and adherence [12]. Earl-Clay Ultraclone Growth Chambers (Earl-Clay Laboratories, Novato, CA) were used to control fibroblast growth in certain cultures. These chambers are composed of a polymer in the form of a small bowl-shaped soft gel with a molecular exclusion size of 106 daltons. Chambers (0.3 ml) containing 100,000 cells were placed in culture dishes with growth medium. The chamber surface does not permit attachment. Tumor cells are maintained or grow in an attachment-indifferent manner, while normal attachment-dependent cells are not maintained.

Xenografts Anesthetized female athymic nude mice (outbred animals of BALB/C background, Harlan Sprague Dawley, Indianapolis, IN) were subcutaneously implanted with 0.1 g minced tumor. Xenografts developed over a 3-6 month period. Harvested xenografts were prepared for cell culture as described above for biopsies. Xenograft tumors were confirmed by gross inspection at autopsy. Most of the tumors were fixed, sectioned, and confirmed microscopically. Control immunohistochemical studies using specific antibodies to mouse and human MHC antigens showed that cell preparations, both enzymatic and mechanical, contained human tumor cells and a variable number of normal nude mouse cells. Xenografts were reimplanted into one or more nude mice to continue propagation. Estrogen supplementation was tested to determine whether xenograft success could be improved [21]. Eighteen tumor specimens were implanted in estrogenized as well as normal mice. The estrogenized mice received s.c. implants of estradiol in the form of a pellet (for nude mice, Innovative Research of America, Toledo, Ohio, one per month) or estradiol valerate (Steraloids, Wilton, NH., 25t~g/mouse in 0.1ml sterile sesame oil, once weekly). In control studies, cultured MCF-7 breast cancer cells formed xenograft tumors in estroge-

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nized mice [21] but not in control animals. Of the 18 tumors tested, 2 xenografts grew in an estrogendependent manner; 2 additional xenografts were estrogen-independent for growth. One of the two estrogen-dependent xenografts gave rise to a cultured cell line (BRXBr7X). The other estrogendependent tumor failed to grow in culture and did not respond to media containing additional estradiol.

Characterization of cell lines Doubling times were estimated from the time-topassage given a 1 : 4 split ratio. Immunocytochemical procedures were performed on cultured cells, either dispersed in PBS containing 0.02% EDTA and then sedimented onto slides using a cytospin (Shannon), or grown on Lab Tek chamber slides. Slides were fixed with cold acetone. Bound antibody was detected using the avidin-biotin-peroxidase complex method [13]. Antibodies to the human H L A (W6-32) served as positive controls. The antibody panel included rabbit antibodies to cytokeratin and vimentin (Amersham Searle Corp., Arlington Heights, IL), human type I collagen (Pel Freeze, Brown Deer, WI), and type IV collagen (Dr. Heinz Furthmayer, Yale University). Anti-tumor monoclonal antibodies included BABrl and BABr3 prepared against membranes of breast cancer cells, BTCol prepared against colon tumor xenograft cells, and BTMe3 and BTMe7 prepared against cultured melanoma cells [13]. These antibodies are relatively specific in their recognition of tumor cells versus normal cells. There is some cross reactivity between tumor types

[13]. Tumorigenicity of cultured cells was assessed by implanting nude mice with 10 million viable trypsin-EDTA-dispersed cells, suspended in 0.2 ml of phosphate buffered saline. Cultured cells were considered tumorigenic if a tumor exceeding 200 mm 3 developed within 3 months. Presence of tumors was confirmed by gross inspection at autopsy. Clonogenicity of cultured cells was determined in soft agar [14]. One thousand viable single cells

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were plated in triplicate. After a 21 day incubation period, colonies containing more than 30 cells were counted. Cultures were considered clonogenic if more than 0.5% of the plated cells formed colonies. Cultured fibroblasts from several sources were not clonogenic in this assay. DNA content of nuclei prepared from cultured cells was analyzed by flow cytometry using the propidium iodide method [15]. Peripheral blood lymphocytes served as reference for the calculation of D N A index. Mean values were compared using Student's tTest. Differences were considered significant if p -< 0.05. Correlations between groups were determined by regression analysis using a linear model [16]. Correlation was considered significant if p -< 0.05.

Results

Characteristics of biopsy specimens Eighty-five breast carcinomas were acquired as part of the TAPP program. Seventy-one solid tumor masses included ten primary breast cancers, five recurrences at the site of surgery, and 56 metastatic masses. The average weight of specimens received was 5.4 g (Table 1). Half of the specimens were smaller than 2 g. Metastases from skin lesions were significantly smaller compared to primary lesions; otherwise there were no remarkable differ-

ences in the size of specimens comparing the primary with lymph node, liver, skin, and miscellaneous sites. An average of 3.4 million viable cells were released mechanically per gram (Table 1). Half of the specimens released less than 1 million cells/g. Viability of mechanically released cells was 15% (Table 1). Tumor size was plotted vs viability to determine whether increased breast tumor specimen size was associated with poor viability, perhaps due to necrosis. There was no significant decrease in viability with size (p = 0.53). Tumor size was plotted vs cell yield per gram to determine whether yield changed (decreased) as tumor size increased. The analysis showed no significant change in yield with size (p = 0.180). The viability of enzymatically prepared cells was 80%, with a range of 4-99%. An average of 5 million viable cells was released per gram tissue (Table 1). Viabilities of enzymatically derived cells were plotted against those of mechanically prepared cells to determine whether there was a direct relationship between the viabilities of these two cell preparations (Table 1). The correlation was significant (p = 0.008). Fourteen of the breast cancer biopsies were fluid specimens, four ascites and ten pleural effusions (Table 2). The average volume of ascites fluid, approximately 3 liters, was larger than that of effusion specimens, 900 ml. Average cell viabilities of the two fluid specimens were essentially the same, 85%.

Table 1. Solid breast tumor specimens received in the TAPP program Parameter Size (g)a Viabilityb mechanical cells (%) enzymatic cells (%) Yield ° mechanical cells (x 10-6/g) enzymatic cells (x 10-6/g)

Mean

S.D.

n

5.4

11.4

71

17 26

71 37

5.7 12

71 37

15 80 3.4 5.1

Median 2.0 10 93 1.0 1.7

Range 0.1-71 0-77 4--99 0-25 0-72

a Specimens trimmed of normal and necrotic tissue. b Linear regression analysis was carried out on the enzymatically prepared cell viability (EV) vs non-enzymaticallyprepared cell viability (NV). n = 30, NV = 1.29EV + 63.5, R = 0.48. The correlation was significant, p = 0.008. °Yield of viable cells.

Cultured breast cancer cell lines Primary culture Successful long term cultures of solid tumors were derived from the mechanical cell preparations plated from 4 solid tumors and three xenografts. No cell lines were developed from cultures of enzymatically treated cells or explants. The viabilities of those tumor biopsies that were successfully established in culture were compared with viabilities of biopsies that were unsuccessful. For mechanically prepared cells from solid tumors, the viability (% __+_SEM) of the successful biopsies was 10.3% + 3.3% vs 16.8% + 2.5% for unsuccessful biopsies. For enzymatically prepared cells, the viability was 79.3% + 13.7% vs 81.7% + 24.3%. For fluid specimens, the biopsy viability for two successful cultures was 46% and 87%, compared to 94.8% + 1.7% for 8 unsuccessful specimens. Success in cell line development did not appear to be related to higher biopsy viability. Certain culture substrates, including Matrigel, Extracellular Matrix, and dried type I collagen were tested to determine whether these would enhance attachment of certain breast cancer cells which did not attach well on tissue culture plastic (Table 3). No benefit was observed for 4 specimens repeated on Matrigel. One of 5 specimens repeated on ECM attached and grew well initially, but this culture could not be successfully passaged. Four of 12 specimens replated on dried type I collagen attached and proliferated. One of these cultures underwent passage and developed into cell line Table 2. Breast cancer specimens received as ascites and pleural effusion samplesa

Fluid volume (ml) Ascites Pleural Viable cells (x f0 -6) Ascites Pleural Viability (%) Ascites Pleural

Mean

Median

Range

3088 907

3750 750

800-4050 125-2350

213 64

145 28

19-542 1-352

85 87

88 93

56-99 46-99

a Ascites specimens, n = 4; pleural effusion specimens, n = 10.

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BRXBr6X. After the third passage, the collagen substrate was no longer required for propagation. Fibroblast outgrowth is frequently a problem in the development of tumor cell lines. Generally, fibroblasts were controlled by differential trypsinization and attachment. For one specimen (BRXBr3), mechanically prepared cells were cultured in Earl Clay Ultraclone growth chambers. Fibroblast growth was inhibited while the tumor cells slowly expanded. After two weeks, the residual cells were replated in culture wells in 0.1ml of acidified (pH 6.7) medium. Cell attachment for this line appeared to be pH responsive; after seven days at pH 7.4, 10% of cells attached, while at pH 6.7, 70% attached.

Cultures from xenografts Xenograft tumors developed for 12 of 58 (21%) breast cancers implanted in nude mice. Three culture cell lines (BRXBrlX, BRXBr6X, and BRXBr7X) developed from xenografts. Original biopsies of two of these tumors also developed cell lines (BRXBrl and BRXBrT). Cells prepared from a biopsy and the xenograft from the same biopsy generally behaved alike in primary culture. This is illustrated by similarity of responses on substrates shown in Table 4. Where the biopsy cells attached well on a given substrate, generally the xenograft cells did likewise. One

Table 3. Influence of culture substrate on the attachment and growth of breast cancer cells Substrate

n"

Attachment growthb

No effect

Matrigel Extracellular matrix Dried type I collagen

4 5 12

0 1 4c

4 4 8

aTotal number of patient culture specimens tested. Each exhibited little or no attachment (< 1%) of viable cells when plated 7 days on standard tissue culture plastic surfaces. bWhen replated, cultures which exhibited good attachment (> 10%) and growth (two-fold colony expansion), over 30 days in culture. One of these developed into a cell line, BRXBr6X.

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specimen (G) did not follow this general trend. The reason for this specimen's behavior did not appear to be due to difference in viabilities.

Characteristics of cell lines The cell lines and their characteristics are listed in Table 5. Doubling times of 3-6 days were typical. One biopsy and xenograft culture pair exhibited a long doubling time, 30 days. Each of the cell lines were aneuploid with DNA contents ranging from 1.3-2.3 times the DNA in normal cells. Three cell lines analyzed had an excess number of chromosomes. Four of 5 cell lines tested were tumorigenic in nude mice. Five of 9 cell lines were clonogenic in soft agar (Table 5). Immunochemical reactivities are shown in Table 6. MCF-7 cells were included for comparison. Each of the cell lines, including those of xenograft origin, showed reactivity with antibodies to the human major histocompatibility locus. Each of the cell lines tested reacted with at least two anti-tumor antibodies. Most of the cell lines reacted well with antibodies induced against breast tumors. Two reacted well with the anti-colon tumor antibody, and three cell lines reacted weakly with the anti-melanoma antibodies.

Viabilities of the tumor cells varied considerably between different biopsies. The viability of mechanically prepared cells was usually lower than that of enzymatically prepared cells. It is probable that more cells were damaged during mechanical mincing. The apparent higher viability of enzymatic preparations may be due in part to lysis of dead cells during the enzyme procedure. We have observed the elimination of dead cells in mechanical cell preparations treated with enzyme, resulting in an apparent increase in viability. Higher viability was not a distinguishing characteristic of the successfully cultured biopsies. It has been suggested that high viability was a factor in the good culture success rate for pleural fluid specimens [7]. Clearly, a population of viable biopsy cells, capable of outgrowth, is required for success. However, we have not found overall specimen cellular viability to be a particularly good predictor of successful cell line development for breast or several other types of cancer. The reason for the low success rate of breast tumors in culture when compared to other major tumor types, is not certain. Breast tissue is composed of several different types of cells, and the Table 4. Attachment and growth response of biopsy and xenograft tumor cells cultured on various substratesa Specimen

Culture plastic Extracellular matrix

Collagen

Biopsy Xeno.

Biopsy Xeno.

Discussion

Successful cultures typically developed from mechanical cell preparations. The reason for the lack of success using enzymatic and explant cultures is not clear. The collagenase procedure appears to be a relatively gentle technique for cell dissociation with little cell mortality. Control studies we have carried out on antigens recognized by the antitumor antibodies indicate that the dispersion technique used generally spares surface antigens. Nonetheless, enzymatically prepared cells did not appear to plate as well as mechanically prepared cells. Fibroblast outgrowth, a problem observed to some extent in culture from each of the types of cell preparations, tended to be less with the mechanical cell preparations.

A

.

B C D E F G H I

+ +b +b

.

.

+__ +b +b

Biopsy Xeno.

ND

.

ND ND ND ND ND +

+ -

ND ND

+b

+ ND ND

a Approximately 1 million viable cells were plated on T25 culture vessels; ' - ' indicates no attachment and growth; ' + ' indicates attachment with little or no growth;' + ' indicates attachment and colony expansion over a 7-30 day culture period. b Cultures which developed into cell lines were B R X B r l and B R X B r l X for specimen H, BRXBr7 and BRXBr7X for I, and BRXBr6X for specimen F.

Cultured breast cancer cell lines

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T a b l e 5. Breast cancer cell lines

Cell line"

Patient age (years)

Cultured cell source

Doubling time (d)

DNA index b

Anti-tumor MoAb reactive c,d

Tumor forms Clonogenic in nude in soft agar miced

BRXBrl BRXBrlX BRXBr2 BRXBr3 BRXBr4 BRXBr5 BRXBr6X BRXBr7 BRXBr7X

NA d NA 54 37 30 53 31 49 49

Pleural effusion Xenografl pleural eft. Skin nodule Chest wall Pleural effusion Supraclavicular node Xenograft lymph node met. Neck mass Xenograft neck mass met.

3-4 3-4 5-6 6 4-5 3-4 3-5 30 30

1.8 (68) 1.6 1.5 1.4 2.3 (82) 1.5 1.3 (78) 1.6 1.6

2/5 2/4 NA 3/4

+ + NA +

+ + -

2/5

-

+

3/3 2/5 2/5 2/5

NA + NA NA

+ + -

aThe suffix 'X' refers to lines developed from xenograft tumors. b Numbers in ( ) , chromosome numbers determined on 3 cell lines. cReactive antibodies/number antibodies tested. d NA means data not available.

interaction of these cells appears to be critical for the hormonal regulation of growth. Disruption of tissue during processing conceivably breaks communication between responsive cells. Restoration of these signals or reconstitution of a more natural tissue architecture may be important during expansion. Clearly, the right conditions have not been obtained for the majority of breast cancers cultured. A common problem encountered in culturing breast cancer is the failure of initial attachment. We have tested various plating substrates in an attempt to stimulate and maintain attachment and

long-term growth. Four of eight cultures tested had better attachment to collagen-coated flasks compared with standard culture ware. On ECM, 1 of 5 specimens tested attached better. On Matrigel, none of the 4 specimens tested responded. It was interesting that for carcinoma cells, the latter two basement membrane derived materials did not produce better attachment and growth than stromal collagen. Epithelial cells are separated from the stromal compartment by a basement membrane. It has been shown that the synthesis of basement membrane collagen is involved in the growth of normal mammary cells [17] and the growth of cer-

T a b l e 6. Immunocytochemical characterization of breast cancer cell lines

Sample

BRXBrl BRXBrlX BRXBr3 BRXBr4 BRXBr5 BRXBr6X BRXBr7 BRXBr7X MCF7

Antitumor antibodies W6-32

EMA

BABrl

BABr3

BTCol

BTMe3

BTMe7

Cytokeratin

Collagen I

3 2 3 3 2 2 2 1 3

3 3 2 2 3 0 0 0 3

3 2 4 4 2 0 0 1 4

1 1 4 3 3 0 0 0 2

0 0 3 0 2 0 0 0 0

0 0 ND 0 ND 2 l 0 0

0 ND 0 0 ND 2 1 1 0

3 2 2 3 ND 0 0 0 4

0 1 0 0 ND 3 3 3 2

1Antibody reactivity was scored on a scale of 0-4, 0 being nonreactive and 4 strongly reactive.

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W M L e w k o et al.

tain mammary tumor epithelial cells [18] as well. However, there is some evidence using a rat model system that breast cancer cells may exhibit a relaxed preference for type IV collagen over stromal collagen compared to normal mammary cells [19]. Collagen substrate benefitted initial attachment in the development of one cell line (BRXBr6X). For breast cancer, we did not observe any case in which dependence on special substrates continued in the cell line. This had been the case in other cancer cell lines, in particular, colorectal cancer [20]. It is possible that breast tumor cells may benefit from a more complex substrate, perhaps an extracellular matrix derived from adenocarcinoma cells or specifically from breast cancer cells. This is currently being evaluated. Aneuploidy, tumorigenicity in nude mice, and clonogenicity in soft agar are generally considered to be characteristics of tumor cells. Each of the cell lines tested were positive for one or more of these characteristics. Only 5 of the 9 cultures tested were clonogenic. Lack of clonogenicity may have been due to relatively long doubling times for some of these cell lines. Cells derived from biopsies and the xenografts of those biopsies had similar characteristics. In primary culture patient biopsy and xenograft cells exhibited similarities in plating, comparing culture plastic and various substrates (Table 4). Cell fines derived from related biopsies and xenografts were very similar in morphology, doubling time, D N A content, and the expression of certain antigens (Tables 5 and 6). Similarity between biopsy and xenograft cell lines has also been observed for other types of cancers [10, 20]. Certainly, changes may occur, particularly with multiple passages. However, within the first several passages needed to generate the 200-400 million cells useful in biological therapy, cultures and xenografts appeared to be relatively stable. BRXBr6X and the biopsy-xenograft cell line pair BRXBr7 and BRXBr7X were different from the other 6 cell lines. The pathologies of the original biopsies, and derived xenografts, were carcinomas. However, the cells in these cultures contained processes. They did not grow in a cobblestone-like pattern, characteristic of the other breast cancer

cultures. By immunocytochemical staining, these cell lines exhibited little or no reactivity with antibodies to breast tumor, cytokeratin, or epithelial membrane antigen. These cells did react weakly with antimelanoma antibodies and with antibodies to vimentin and type I collagen. While these cells have certain fibroblast-like characteristics, they do not appear to be fibroblasts. Two of the lines were derived from xenografts. Normal human fibroblasts do not grow in nude mice. DNA content was aneuploid. BRXBr6X was clearly clonogenic in agar and tumorigenic in nude mice. Weak reactivity of BRXBr6X with antibody to type IV collagen and strong reactivity of all three lines with antibodies to laminin suggest the possibility that these cells may be of myoepithelial cell origin. Cell lines prepared for patients have been used successfully to prepare and test biological reagents. Cell fines BRXBrl, BRXBr2, and BRXBr4 were used for the induction and screening of monoclonal antibodies for use in antibody-drug conjugate therapy. Cell lines BRXBr5, BRXBr6X, and BRXBr7 have been utilized in the induction and characterization of tumor-derived activated cells (TDAC) [4]. For this therapeutic approach, tumor mince was cultured in medium containing IL-2. Lymphocytes which grew out of the tumor mince generally killed the patient's tumor cells in vitro specifically. As the tumor cells in the lymphocyte cultures were killed, tumor cells were added back to stimulate the cell-killing specificity and expansion of the lymphocytes. Cultured tumor cells have served this purpose [4] for TDAC of breast cancer, and for several other types of cancer, such that cultured cells may replace the requirement for biopsy tumor cells in the development of TDAC cultures. As a result of successful tumor cell culture, the possibility of TDAC therapy can be extended to a greater number of cancer patients.

Acknowledgements Flow cytometry was carried out by Cytometry Associates, Franklin, TN. We are indebted to Drs. S-K Liao, James Maleckar, Charles Montgomery, John Yannelli, and Ramrao Gangavalli for their

Cultured breast cancer cell lines

valuable consultation. We appreciate the assistance of Ms. Deborah Plant and Marie Sweeney in the preparation of this manuscript.

11.

12.

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