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[CANCER RESEARCH 44, 3398-3402, August 1984] Identification of a Chemoattractant for Fibroblasts Produced by Human Breast Carcinoma Cell Lines Wayne E. Gleiber1 and Elliott Schiffmann Laboratory of Developmental Biology ano Anomalies, National Institute of Dental Research, NIH, Bethesda, Maryland 20205 ABSTRACT human breast tumor cell line (11), as well as three other human breast tumor cell lines, secretes a potent Chemoattractant for Serum-free, conditioned media from the human breast tumor fibroblasts which appears to be a high-molecular-weight protein. cell line ZR-75-1 and three other breast tumor cell lines were each found to contain a potent Chemoattractant for fibroblasts. MATERIALS AND METHODS The Chemoattractant activity was characterized and found to reside in a high-molecular-weight (M, > 100,000) protein. This Materials activity was stable to heating to 56°and from pH 3 to 11, but it L-Glutamine, penicillin, streptomycin, and fetal bovine serum were was sensitive to trypsin and pepsin treatment and to reduction. obtained from Grand Island Biological Co. Bovine insulin, L-thyroxine, When crude attractant was placed in vivo in a slowly releasing human transferrin, 17/i-estradiol, BSA, aprotinin, iodoacetic acid, gelatin pump, a fibrous tissue mass that formed at the releasing site from swine skin, 2-mercaptoethanol, pepsin, and soybean trypsin inhib within 11 days was 3- to 5-fold larger than was that observed itor were obtained from Sigma Chemical Co. PDGF and fibronectin were with pumps containing control medium. It is likely that the pro from Collaborative Research, Inc. DNase, RNase, and trypsin were from duction of a Chemoattractant for fibroblasts by breast tumor cells Worthington Biochemical Corp. Dithiothreitol was from Schwarz/Mann. might contribute to the fibrotic involvement that is common in Formylmethionylleucylphenylalanine was obtained from Peninsula Labo breast carcinomas. ratories. Cell Lines INTRODUCTION Human embryonic skin fibroblasts CRL-1475 and 3T3 murine embry Fibrosis occurs in many diseases in which chronic inflammation onic fibroblasts were obtained from the American Type Culture Collec tion. Human breast tumor cell lines, ZR-75-1, MCF-7, T-47D, and ZR- is found, such as rheumatoid arthritis, tuberculosis, and liver 75-31 and human renal, Ewing's sarcoma, and mucoepidermoid tumor cirrhosis. In addition, fibrotic involvement is associated with some cell lines were obtained from Dr. Lance A. Liotta (National Cancer tumors. Fibrosis is associated with greater than 80% of human Institute, NIH). Murine melanoma BL/6 cells were obtained from Dr. breast carcinomas (24), especially the infiltrating duct carcinoma, Victor P. Terranova (National Institute of Dental Research, NIH). Cells which is characterized by a dense fibrous stroma (6). This fibrotic were grown in 75-sq cm tissue culture flasks (Falcon Plastics), incubated tissue may be formed as an attempt by the host at wound repair, at 37°in a 5% CO2 humidified atmosphere. The growth medium was since the area around the tumor is damaged by its growth (22, changed every 3 to 4 days, and cells were split at a ratio of 1:4 every 1 25). This damage causes an influx of inflammatory cells which to 3 weeks, depending upon the growth rate of the cell line. release mediators that attract fibroblasts from the surrounding Media tissue. As in wound repair, the fibroblasts synthesize new con nective tissue matrix components (20). Growth Medium. All cell lines were grown in DMEM with glucose (4.5 Alternatively, it may be possible that factors other than those g/liter) (NIH medium unit) with additions of 2 mw t-glutamine, 100 related to inflammation may be involved, such as factors pro penicillin (100 units/ml), streptomycin (100 fig/ml), and 7% (v/v) fetal duced by the tumor itself. Previous studies have demonstrated bovine serum. that fibroblasts can be attracted to various soluble factors by Defined Medium. DMEM containing high glucose was supplemented with insulin (3 ^g/ml), thyroxine (7.0 ng/ml), transferrin (1 ¿ig/ml),17/3- chemotaxis, the directed migration of cells toward an attractant (14, 17). Lymphokines (7, 11), fibronectin (12, 19, 23), PDGF,2 estradiol (2.7 ng/ml), L-glutamine, penicillin, and streptomycin. (9,18) complement-derived factors (15), and collagen fragments Conditioned Medium. Procedures for the isolation and characteriza tion of the activity required a defined medium without serum. Serum (4,13) have been found to be attractants for fibroblasts. Each of contains other chemotactic factors for fibroblasts, such as fibronectin, these components could be present at the site of a tumor, due complement factors, and PDGF. Cells were cultured in serum-containing to the inflammation and injury of normal tissue, and each could growth medium following passage to provide a rapid growth of cells. contribute to the formation of fibrotic tissue around the tumor. When the cultures were confluent, the growth medium was removed, We have investigated the possibility that breast tumor cells and the flasks were rinsed twice with phosphate-buffered saline. A produce a fibroblast Chemoattractant. Here we have used cell serum-free defined medium (10 ml) was added to the flasks, and the lines derived from human breast carcinomas (5, 21), grown in cells were incubated for 2 days. The supplements added to the defined serum-free defined medium (1-3). We find that the ZR-75-1 medium (insulin, thyroxine, transferrin, and estradiol), while not essential for production of the attractant, are beneficial for the continued growth of the cells. The hormones lack chemotactic activity and appeared to 1Support is provided by a USPHS National Research Service Award have no effect upon the chemotactic response. The CM was collected (3F32DE05299-03). and centrifuged at 10,000 x g for 10 min, and the supernatant fraction 2 The abbreviations used are: PDGF, platelet-derived growth factor(s); DMEM, was stored at -20°. Production of CM was repeated 3 to 5 times before Dulbecco's modified Eagle's medium; CM, conditioned medium (or media); BSA, bovine serum albumin. cells were split into new flasks. The concentration of protein in dialyzed Received August 8, 1983; accepted March 30, 1984. CM ranged from 70 to 3398 CANCER RESEARCH VOL. 44 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1984 American Association for Cancer Research. Tumor-produced Fibroblast Chemoattractant Chemotaxis Assay BSA (1.5 mg/ml). Indsions were made at the lower right or left flank of lightly anesthetized C57BL/6 mice (2 pumps/animal), and the pumps Chemotaxis assays on fibroblasts were performed in modified Boyden were inserted head first to insure that the releasing end of each pump chambers as described by Posttethwaite ef al. (14). Nudeopore filters was distal to the incision. Each incision site was closed with a single 9- (Neuroprobe Corporation) with 8-¿imdiameter pores were treated with mm stainless steel Autodip (Clay-Adams). On Days 4, 6,9, and 11 after 0.087 M acetic acid at 50°for 20 min, washed twice in distilled water, insertion of the pumps, the sites of implantation on each animal were placed in a gelatin solution (25 pg/ml) at 100° for 60 min, and dried opened, and any fibrous tissue mass which formed was excised, overnight at 37°.The test material in DMEM containing 0.1% BSA was weighed, and fixed in a 10% formaldehyde solution. placed in the lower well of the Boyden chamber, and a treated Nudeopore filter was placed over this. Cells freshly trypsinized from confluent cultures were washed first with DMEM containing soybean trypsin RESULTS inhibitor and then with DMEM containing 0.1% BSA; they were finally resuspended in DMEM plus BSA at a concentration of 3 x 105 cells/ml. Chemotactic Activity from ZR-75-1 CM for Fibroblasts. A A 0.2-ml aliquot of this cell suspension was added to the upper chamber. potent Chemoattractant for human embryonic, adolescent, and The chambers were incubated for 4 hr at 37°in a 5% CO2 humidified adult skin fibroblasts, as well as human lung and mouse skin atmosphere. The filters were then removed, fixed, and stained in Diff- fibroblasts (data not shown), was found in CM from the ZR-75- Quik staining solutions (Harleco), rinsed in water, and placed on micro 1 human breast tumor cell line. This activity was concentration scope slides, such that cells migrating through the filter and attached to dependent, with an ED50(that amount of material which produced the underside of the filter were against the surface of the glass. We did a half-maximal response) between dilutions of 1:8 and 1:32, not observe any cells detached from the filter and present in the lower depending on the preparation (Chart 1); the concentration of chamber. The cells on the upper surface of the membrane were wiped away with a wet cotton-tipped applicator, and the number of cells protein ranged from 70 to 100 ¿«g/ml.Whencompared to the migrating to the lower surface of the membrane was determined by chemotactic activity of PDGF (18) or fibronectin (12, 19, 23), known attractants for fibroblasts, ZR-75-1 CM induced a 2-fold counting the number of stained nuclei in randomly selected microscopic fields encompassing an area of 1 sq mm (Vi? of the total area of greater number of cells to migrate. Attempts to concentrate the migration). Nonstimulated migration of cells, determined in control cham medium resulted in the formation of a precipitate with a loss of bers, with defined medium as the attractant, accounted for less then some activity from the soluble fraction. Therefore, it was not 10% of the maximal response and was subtracted from all values. All possible to measure an increase, if any, in chemotactic activity assays were performed in duplicate.

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