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Plasmin induces the formation of multicellular spheroids of breast cancer cells
Cancer Letters 117 (1997) 51–56
Plasmin induces the formation of multicellular spheroids of breast cancer cells Myung H. Chun Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA Received 28 February 1997; received in revised form 20 March 1997; accepted 20 March 1997
Abstract MCF7 and ZR75-1 breast cancer cells grow as adherent monolayers in tissue culture. Treatment with the serum serine protease plasmin causes them to detach and to grow as floating multicellular spheroids. Two plasmin activators, urokinase plasminogen activator and streptokinase, induce the same growth pattern changes in the presence of plasminogen. Serum contains also plasminogen activator inhibitors. Aged serum, deficient in plasminogen activator inhibitors, converts spontaneously monolayer breast cancer cells into multicellular spheroids which readily revert to monolayer growth after addition of fresh serum. Urokinase blocks the reversion. The formation of multicellular spheroids does not affect the proliferative rate of breast tumor cells but endows tumor cells with increased resistance to the chemotherapeutic drugs, doxorubicin and paclitaxel. 1997 Elsevier Science Ireland Ltd. Keywords: Chemotherapy; Monolayer; Spheroids; Plasmin; Urokinase
1. Introduction Breast carcinoma cells grow in situ as solid tumor masses [1,2]. The MCF7 and ZR75-1 cell lines, derived from breast carcinomas, grow in tissue culture in monolayers with an epithelial sheet-like morphology [3,4]. Attempts to establish carcinoma cell lines which grow in a three dimensional structure succeeded with a limited number of solid tumor cell lines when agarose or spinner culture technology was employed [5–14]. In these studies, tumor cells grow in spheroids slower than in the monolayer, package their DNA differently, generate more extracellular matrix, and the matrix contains proteoglycan and fibronectin which are not usually detected in the ECM of monolayer cells. Furthermore, monolayer cells and spheroids respond differently to chemotherapeutic
agents; generally spheroids are more drug-resistant than monolayer cells [5–14]. This report describes a new method to change the growth pattern of two breast cancer cell lines from adherent monolayer to multicellular spheroids. Using the method, monolayer and spheroid tumor cells are compared in regard to their growth rate and their sensitivity to chemotherapeutic drugs.
2. Materials and methods 2.1. Cells Breast carcinoma cell lines, MCF7 cells (3, ATCC HTB22) and ZR75-1 cells (4, ATCC CRL 1500) were obtained from the American Type Culture Collection
0304-3835/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0213-9
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M.H. Chun / Cancer Letters 117 (1997) 51–56
(ATCC), Rockville, MD. The cell lines were maintained as adherent monolayer according to the ATCC instruction except that 3% fresh human serum was used instead of fetal calf serum.
3. Results 3.1. Monolayer growth of human breast cancer cells in vitro depends on the presence of labile serum factors
2.2. Treatment with chemotherapeutic drugs Adherent monolayer MCF7 cells were trypsinized, plated with 3000 cells in 0.1 ml RPMI 1640 supplemented with 200 mg/ml streptomycin, 200 IU/ ml penicillin and 300 mg/ml l-glutamine per well in flat bottom 96-well microtiter plates, and cultured for 3 days with 3% aged human serum (outdated human serum) to obtain big multicellular spheroids or with 3% fresh human serum (human serum frozen immediately after preparation and thawed on the day of the experiment) to obtain adherent monolayer cells. Cells were treated with doxorubicin or paclitaxel(taxol) in the concentration indicated in the figure legends and their survival was measured in the thymidine uptake assay [15] or in the (3-(4,5-dimethylthiazol-1-yl)-2,5-dophenyltetrazolium bromide; thyazolyl blue (MTT) assay [16]. For the thymidine uptake assay, cells were treated with doxorubicin for 2 days, pulsed with [3H]thymidine (100 nCi/ml) for 4 or 16 h, and the uptake of [3H]thymidine was measured as described [15]. For the MTT assay, cells were treated first with the drugs for 2 days, washed once, and incubated with medium containing 1% fresh human serum for 24 h to revert spheroids to monolayer growth before the MTT assay was performed as described [16].
MCF7 and ZR75-1 breast carcinoma cells grow in epithelioid monolayers when cultured in the presence of fetal calf serum or in the presence of fresh human serum. Supplementing the culture medium with an outdated source of human serum, it was noted that breast cancer cells detached themselves from the culture dish and formed solid multicellular spheroids (Fig. 1). The spheroids remained intact after treatment with trypsin, EDTA or collagenase (not shown). The addition of fresh serum to these cultures reverts spheroids into adherent monolayers within 24 h (not shown). 3.2. Growth in spheroids does not affect the tumor cell proliferative activity The growth of tumor cells in spheroids has previously been induced in spinner as well as in agarose culture [5–14]. It was noted that tumor cells prolifer-
2.3. Reagents RPMI 1640, penicillin, streptomycin and l-glutamine were obtained from Gibco, Long Island, NY. Aged human serum is the outdated human serum obtained from Gibco. Fresh human serum was obtained from healthy volunteers, both male and female, of the New York Medical College, NY. Urokinase was obtained from Abbott Laboratories, North Chicago, IL. Doxorubicin was obtained from Adria Laboratories, Columbus, OH. Taxol was obtained from Bristol-Myers Squibb, Wallingford, CT. Plasmin and MTT were purchased from Sigma, St. Louis, MO. [3H]Thymidine was obtained from Dupont NEN, Boston, MA.
Fig. 1. Serum factors control the growth pattern of breast carcinoma cell lines. Monolayer MCF7 cells (A,B) or ZR75-1 cells (C,D) were trypsinized, plated (3000 cells in 0.1 ml) in flat bottom 96-well plates, and cultured for 3 days with either 3% fresh (A,C) or aged (B,D) human serum. Magnification: × 400.
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M.H. Chun / Cancer Letters 117 (1997) 51–56 Table 1
Proliferative activity of breast tumor cells growing as monolayers or as spheroids Cell number × 1000
Monolayer Spheroids
Fig. 2. Breast tumor cells growing in spheroids exhibit increased resistance to chemotherapeutic drugs. MCF7 cells (3000) were cultured with either 3% aged (X) or fresh (W) human serum for 3 days and treated with doxorubicin or taxol in concentrations indicated. The thymidine uptake assay was performed in (A) and the MTT assay was performed in (B). The assays were performed in triplicates. The error bars indicate the standard deviation.
ate less vigorously in these aggregates as compared to monolayer cultures [6–14]. We examined whether spontaneous formation of tumor cell spheroids in cultures supplemented with aged human serum affects the proliferative activity of tumor cells. This was not found to be the case. The uptake of [3H]thymidine by monolayer and spheroid cells was similar in magnitude (Figs. 2, and 4). Furthermore, the number of cells in monolayer and spheroid cultures was comparable (Table 1). The data indicate that the growth capacity of tumor cells is not compromised by the spheroid formation. 3.3. Multicellular tumor cell spheroids are less sensitive to chemotherapeutic drugs than tumor cell monolayers
Day 4
Day 7
Day 10
780 (110) 980 (200)
2000 (270) 2150 (360)
4450 (650) 4600 (930)
200 000 MCF7 cells in 1 ml were cultured with either 3% aged human serum to obtain spheroids or 3% fresh human serum to obtain monolayer cells, treated with 1% fresh human serum for 24 h, trypsinized and counted using a hemocytometer. The data from four cultures were averaged and expressed with standard deviation. The average of the samples is not different at the 0.02 level calculated as described [33].
prevented the clumping of tumor cells in vitro. Screening serum proteases, we tested plasmin, a serine protease. An unexpected observation was made. Rather than dissociating tumor cell spheroids and enabling them to form monolayers, plasmin caused, on the contrary, the strong conversion of tumor cell monolayers into floating spheroids (not shown). Plasmin occurs in serum in its inactive form, plasminogen [17–21]. The activation of plasminogen into plasmin is positively controlled by plasminogen activators and negatively controlled by plasminogen activator inhibitors [17–21]. One plasminogen activator which can be purified from urine and which is referred to as urokinase is known to bind to breast tumor cells that express specific receptors for it [22–26]. MCF7 breast tumor cells growing in monolayer cultures
Having established that spontaneous formation of multicellular spheroids does not affect the proliferation of MCF7 cells, we asked whether tumor cell spheroids and monolayers are equally sensitive to chemotherapeutic drugs. Experiments described in Fig. 2 demonstrate that MCF7 cells increase their resistance to doxorubicin and to taxol when they form spheroids. 3.4. The serum serine protease plasmin converts tumor cell monolayers into spheroids We were interested to identify serum factors which
Fig. 3. Urokinase induces the formation of tumor cell spheroids. MCF7 cells (10 000/0.1 ml) were incubated with 3% fresh human serum for 24 h to allow them to form monolayers and subsequently treated with 10 ml of either RPMI 1640 medium (A) or 50 U/ml urokinase (B) for 24 h. Magnification: × 400.
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M.H. Chun / Cancer Letters 117 (1997) 51–56
Fig. 4. The change in growth pattern alters the response of MCF7 cells to doxorubicin. (A) MCF7 monolayers (W) or spheroids (X) established as described in Fig. 1 were treated with doxorubicin for 3 days and their proliferation was measured in the thymidine uptake assay. (B) MCF7 spheroids established as described in Fig. 1 were treated for 24 h with either 1% fresh human serum (W) to revert them to monolayers or outdated serum (X) to maintain spheroids, treated with doxorubicin for 2 days, washed once, and treated with 1% fresh human serum for 24 h before the MTT assay was performed.
were treated with urokinase. In the presence of 50 units/ml urokinase, monolayer cells began to retract within 2 h and formed solid tumor cell spheroids within 24 h of incubation (Fig. 3). As the tumor cells changed their morphology and formed multicellular spheroids, they simultaneously enhanced their resistance against the chemotherapeutic drug doxorubicin (Fig. 4A). Reverting tumor spheroids to monolayer cells enabled the tumor cells to regain their susceptibility to doxorubicin (Fig. 4B).
4. Discussion Epithelial breast cells grow in a sheet-like structure lining the breast duct and the ductules [1,2]. Malignant transformation enables breast cells to ignore the morphogenic principles which guide the organization of the gland and thereby facilitates the growth of tumors in amorphous cell masses [1,2]. Previous attempts to mimic the clustered in vivo growth of breast tumor cells in vitro succeeded in some experiments using agarose or spinner culture systems [5–14]. The obtained multicellular spheroids exhibited reduced proliferative activity and were readily dissociated by trypsin treatment [6,11,12]. The experiments reported here demonstrate a new procedure for growing breast tumor cells in
spheroids with undiminished proliferative activity and resistance to dissociation by trypsin or collagenase. Our experiments implicate the serine protease plasmin as a factor controlling the growth pattern of breast tumor cells. Plasminogen, the precursor of plasmin, is converted to plasmin on the cell surface by plasminogen activators bound to cell membrane receptors [18– 21,25,26]. The urokinase-type plasminogen activator is of special interest in this context because malignant breast cells express the receptors for urokinase [22]. Urokinase is unstable and exhibits low enzymatic activity in its free form but becomes highly active and stable upon binding to membrane receptors [17–21,26–29]. When breast cells become malignant, plasmin is activated on their membrane and their morphology is reported to change from sheet-like structures to multicellular masses. A potential important consequence of the formation of spheroids may be the protection of micrometastatic breast cancer cells against chemotherapeutic drugs. Micrometastatic breast cancer cells are detected as multicell clusters in patients [30]. Postoperative adjuvant chemotherapies, which are applied to eliminate micrometastatic tumor cells, are moderately effective in beast cancer [31,32]. Considering the reports together with the findings described here, one may speculate that through the activation of plasmin on their membrane, micrometastatic tumor cells form multicell clusters and thus manage to shield themselves from the action of chemotherapeutic drugs, thereby impeding chemotherapies and raising therapeutic drug doses to prohibitively high levels. The presented study describes an experimental procedure to restore the full susceptibility of tumor cells to chemotherapeutic drugs by reverting tumor cell spheroids into monolayers. Compromising the protective shield may be a reasonable target of new therapeutic designs for postoperative adjuvant therapies for early breast cancer.
References [1] J.R. Harris, M. Morrow, G. Bonadonna, Cancer of the breast, in: V. Devita Jr., S. Hellman, S. Rosenberg (Eds.), Cancer Principles and Practice of Oncology, J.B. Lippincott, Philadelphia, PA, 1993, 4th ed., pp. 1264–1331.
M.H. Chun / Cancer Letters 117 (1997) 51–56 [2] R.N.M. McSwann, K. Whaley (Eds.), Muir’s Textbook of Pathology, Edward Arnold, London, 1993, 13th ed., pp. 13–22. [3] H.D. Soule, J. Vazquez, A. Long, M.A. Brennan, Human cell line from a pleural effusion derived from a breast carcinoma, J. Natl. Cancer Inst. 51 (1973) 1409–1416. [4] L.W. Engel, N.A. Young, T.S. Tralka, M.E. Lippman, S.J. O’Brien, M.J. Joyce, Establishment and characterization of three new continuous cell lines derived from human breast carcinomas, Cancer Res. 38 (1978) 3352–3358. [5] R.E. Durand, Flow cytometry studies of intracellular adriamycin in multicell spheroid in vitro, Cancer Res. 41 (1981) 3495–3498. [6] C. Erlichman, D. Vidgen, Cytotoxicity of adriamycin in MGH-U1 cells grown as monolayer cultures, spheroids, and xenografts in immune-deprived mice, Cancer Res. 44 (1984) 5369–5375. [7] B. Glimelius, B. Norling, T. Nederman, J. Carllsson, Extracellular matrices in multicellular spheroids of human glioma origin: increased incorporation of proteoglycans and fibronectin as compared to monolayer cell cultures, AMPIS 96 (1988) 433–444. [8] A. Gorlach, P. Herter, H. Hentschel, P.J. Frosch, H. Acker, Effects of nIFN b and rIFN r on growth and morphology of two human melanoma cell lines: comparison between twoand three-dimensional culture, Int. J. Cancer 56 (1994) 249– 261. [9] T. Nederman, Effects of vinblastin and 5-flurouracin on human glioma and thyroid cancer cell monolayers and spheroids, Cancer Res. 44 (1984) 254–258. [10] R.M. Sutherland, J.A. McCredie, W.R. Inch, Growth of multicell spheroids in tissue culture as a model of nodular carcinoma, J. Natl. Cancer Inst. 46 (1971) 113–120. [11] R.M. Sutherland, Cell and environmental interactions in tumor microregion: the multicell spheroid model, Science 240 (1988) 177–184. [12] S. Walenta, A. Bredel, U. Karbach, L. Kunz,, W.Vollrath, eller-KlieserMu, Interrelationship among morphology, metabolism, and proliferation of tumor cells in monolayer and spheroid culture, Adv. Exp. Med. Biol. 248 (1989) 847– 853. [13] G.W. West, R. Weichselbaum, J.B. Little, Limited penetration of methotrexate into human osteosarcoma spheroids as a proposed model for solid tumor resistance to adjuvant chemotherapy, Cancer Res. 40 (1980) 3665–3668. [14] J.M. Yuhas, A.P. Li, A.O. Martinez, A.J. Ladman, A simplified method for production and growth of multicellular spheroids, Cancer Res. 37 (1977) 3639–3643. [15] S. Pollack, A. Micali, W.E. Enker, D.W. Kinne, A. Geller, H.F. Oettgen, M.K. Hoffmann, Endotoxin-induced in vitro release of interleukin-1 by cancer patients’ monocytes: relation to stage of disease, Int. J. Cancer 15 (1983) 733– 736. [16] T. Mosmann, Rapid colometric assay for cellular growth and survival: application to proliferation and cytotoxic assays, J. Immunol. Methods 65 (1983) 55–63. [17] F. Bachmann, The plasminogen-plasmin enzyme system in
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hemostasis and thrombosis, in: R.W. Colman, J. Hirsh, V.J. Marder, E.W. Salzman (Eds.), Basic Principles and Clinical Practice, J.B. Lippincott, Philadelphia, PA, 1994, 3rd ed., pp. 1592–1622. F. Blasi, J.D. Vassali, K. Dano, Urokinase-type plasminogen activator: proenzyme, receptor, and inhibitors, J. Cell Biol. 104 (1987) 801–804. D. Collen, H.R. Lijnen, P.A. Todd, K.L. Goa, Tissue-type plasminogen activator. A review of its pharmacology and therapeutic use as a thrombolytic agent, Drugs 38 (1989) 346–388. F. Fazioli, F. Blasi, Urokinase-type plasminogen activator and its receptor: new targets for anti-metastatic therapy? Trends Pharmacol. Sci. 15 (1994) 25–29. O. Saksela, D.B. Rifkin, Cell-associated plasminogen activation: regulation and physiological functions, Annu. Rev. Cell Biol. 4 (1988) 93–126. E. Bianchi, R.L. Cohen, A.T. Thor, R.F. Todd III, I.F. Mizukami, D.A. Lawrence, B.M. Ljung, M.A. Shuman, H.S. Smith, The urokinase receptor is expressed in invasive breast cancer but not in normal breast tissue, Cancer Res. 54 (1994) 861–866. S.S. Husain, V. Curewich, B. Lipinski, Purification and partial characterization of single-chain molecular weight-form of urokinase from human urine, Arch. Biochem. Biophys. 220 (1983) 31–38. H. Sumi, K. Robbins, A functionally active heavy chain derived from human high molecular weight urokinase, J. Biol. Chem. 258 (1983) 8014–8019. V. Ellis, C. Pyke, J. Eriksen, H. Solberg, K. Dano, The urokinase receptor involvement in cell surface proteolysis and cancer invasion, Ann. N. Y. Acad. Sci. 667 (1992) 13– 31. A. Roldan, M.V. Cubellis, M.T. Masucci, N. Beherendt, L.R. Lund, K. Dano, E. Appella, F. Blasi, Cloning and expression of the receptors for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent protease, EMBO J. 9 (1990) 467–474. V. Ellis, M.F. Scully, V.V. Kakkar, Plasminogen activation initiated by single-chain urokinase in functional isolation. Potentiation by U937 monocytes, J. Biol. Chem. 264 (1989) 2185–2188. M. Kohler, S. Sen, C. Miyashita, Half-life of single chain urokinase-type plasminogen activator (scu-PA) and twochain urokinase-type plasminogen activator (tcu-PA) in patients with acute myocardial infraction, Thromb. Res. 62 (1991) 75–81. N. Manchanda, B.S. Schwartz, Single chain urokinase: augmentation of enzymatic activity upon binding to monocytes, J. Biol. Chem. 266 (1991) 14580–14584. R.J. Cote, P.P. Rosen, M.L. Lesser, L.J. Old, M.P. Osborne, Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow micrometastases, J. Clin. Oncol. 9 (1991) 1749–1884. Early Breast Cancer Trialists’ Collaborative Group, Systemic treatment of early breast cancer by hormonal, cytotoxic, or immunotherapy, Lancet 339 (1992) 1–15.
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[32] Early Breast Cancer Trialists’ Collaborative Group Systemic treatment of early breast cancer by hormonal, cytotoxic, or immunotherapy. Lancet, 339 (1992) 71–86.
[33] B.H. Munro, E. Batten, Statistical Methods for Health Care Research, J.B. Lippincott, Philadelphia, PA, 1993, pp. 99– 105.
Plasmin induces the formation of multicellular spheroids of breast cancer cells Myung H. Chun Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA Received 28 February 1997; received in revised form 20 March 1997; accepted 20 March 1997
Abstract MCF7 and ZR75-1 breast cancer cells grow as adherent monolayers in tissue culture. Treatment with the serum serine protease plasmin causes them to detach and to grow as floating multicellular spheroids. Two plasmin activators, urokinase plasminogen activator and streptokinase, induce the same growth pattern changes in the presence of plasminogen. Serum contains also plasminogen activator inhibitors. Aged serum, deficient in plasminogen activator inhibitors, converts spontaneously monolayer breast cancer cells into multicellular spheroids which readily revert to monolayer growth after addition of fresh serum. Urokinase blocks the reversion. The formation of multicellular spheroids does not affect the proliferative rate of breast tumor cells but endows tumor cells with increased resistance to the chemotherapeutic drugs, doxorubicin and paclitaxel. 1997 Elsevier Science Ireland Ltd. Keywords: Chemotherapy; Monolayer; Spheroids; Plasmin; Urokinase
1. Introduction Breast carcinoma cells grow in situ as solid tumor masses [1,2]. The MCF7 and ZR75-1 cell lines, derived from breast carcinomas, grow in tissue culture in monolayers with an epithelial sheet-like morphology [3,4]. Attempts to establish carcinoma cell lines which grow in a three dimensional structure succeeded with a limited number of solid tumor cell lines when agarose or spinner culture technology was employed [5–14]. In these studies, tumor cells grow in spheroids slower than in the monolayer, package their DNA differently, generate more extracellular matrix, and the matrix contains proteoglycan and fibronectin which are not usually detected in the ECM of monolayer cells. Furthermore, monolayer cells and spheroids respond differently to chemotherapeutic
agents; generally spheroids are more drug-resistant than monolayer cells [5–14]. This report describes a new method to change the growth pattern of two breast cancer cell lines from adherent monolayer to multicellular spheroids. Using the method, monolayer and spheroid tumor cells are compared in regard to their growth rate and their sensitivity to chemotherapeutic drugs.
2. Materials and methods 2.1. Cells Breast carcinoma cell lines, MCF7 cells (3, ATCC HTB22) and ZR75-1 cells (4, ATCC CRL 1500) were obtained from the American Type Culture Collection
0304-3835/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0213-9
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M.H. Chun / Cancer Letters 117 (1997) 51–56
(ATCC), Rockville, MD. The cell lines were maintained as adherent monolayer according to the ATCC instruction except that 3% fresh human serum was used instead of fetal calf serum.
3. Results 3.1. Monolayer growth of human breast cancer cells in vitro depends on the presence of labile serum factors
2.2. Treatment with chemotherapeutic drugs Adherent monolayer MCF7 cells were trypsinized, plated with 3000 cells in 0.1 ml RPMI 1640 supplemented with 200 mg/ml streptomycin, 200 IU/ ml penicillin and 300 mg/ml l-glutamine per well in flat bottom 96-well microtiter plates, and cultured for 3 days with 3% aged human serum (outdated human serum) to obtain big multicellular spheroids or with 3% fresh human serum (human serum frozen immediately after preparation and thawed on the day of the experiment) to obtain adherent monolayer cells. Cells were treated with doxorubicin or paclitaxel(taxol) in the concentration indicated in the figure legends and their survival was measured in the thymidine uptake assay [15] or in the (3-(4,5-dimethylthiazol-1-yl)-2,5-dophenyltetrazolium bromide; thyazolyl blue (MTT) assay [16]. For the thymidine uptake assay, cells were treated with doxorubicin for 2 days, pulsed with [3H]thymidine (100 nCi/ml) for 4 or 16 h, and the uptake of [3H]thymidine was measured as described [15]. For the MTT assay, cells were treated first with the drugs for 2 days, washed once, and incubated with medium containing 1% fresh human serum for 24 h to revert spheroids to monolayer growth before the MTT assay was performed as described [16].
MCF7 and ZR75-1 breast carcinoma cells grow in epithelioid monolayers when cultured in the presence of fetal calf serum or in the presence of fresh human serum. Supplementing the culture medium with an outdated source of human serum, it was noted that breast cancer cells detached themselves from the culture dish and formed solid multicellular spheroids (Fig. 1). The spheroids remained intact after treatment with trypsin, EDTA or collagenase (not shown). The addition of fresh serum to these cultures reverts spheroids into adherent monolayers within 24 h (not shown). 3.2. Growth in spheroids does not affect the tumor cell proliferative activity The growth of tumor cells in spheroids has previously been induced in spinner as well as in agarose culture [5–14]. It was noted that tumor cells prolifer-
2.3. Reagents RPMI 1640, penicillin, streptomycin and l-glutamine were obtained from Gibco, Long Island, NY. Aged human serum is the outdated human serum obtained from Gibco. Fresh human serum was obtained from healthy volunteers, both male and female, of the New York Medical College, NY. Urokinase was obtained from Abbott Laboratories, North Chicago, IL. Doxorubicin was obtained from Adria Laboratories, Columbus, OH. Taxol was obtained from Bristol-Myers Squibb, Wallingford, CT. Plasmin and MTT were purchased from Sigma, St. Louis, MO. [3H]Thymidine was obtained from Dupont NEN, Boston, MA.
Fig. 1. Serum factors control the growth pattern of breast carcinoma cell lines. Monolayer MCF7 cells (A,B) or ZR75-1 cells (C,D) were trypsinized, plated (3000 cells in 0.1 ml) in flat bottom 96-well plates, and cultured for 3 days with either 3% fresh (A,C) or aged (B,D) human serum. Magnification: × 400.
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M.H. Chun / Cancer Letters 117 (1997) 51–56 Table 1
Proliferative activity of breast tumor cells growing as monolayers or as spheroids Cell number × 1000
Monolayer Spheroids
Fig. 2. Breast tumor cells growing in spheroids exhibit increased resistance to chemotherapeutic drugs. MCF7 cells (3000) were cultured with either 3% aged (X) or fresh (W) human serum for 3 days and treated with doxorubicin or taxol in concentrations indicated. The thymidine uptake assay was performed in (A) and the MTT assay was performed in (B). The assays were performed in triplicates. The error bars indicate the standard deviation.
ate less vigorously in these aggregates as compared to monolayer cultures [6–14]. We examined whether spontaneous formation of tumor cell spheroids in cultures supplemented with aged human serum affects the proliferative activity of tumor cells. This was not found to be the case. The uptake of [3H]thymidine by monolayer and spheroid cells was similar in magnitude (Figs. 2, and 4). Furthermore, the number of cells in monolayer and spheroid cultures was comparable (Table 1). The data indicate that the growth capacity of tumor cells is not compromised by the spheroid formation. 3.3. Multicellular tumor cell spheroids are less sensitive to chemotherapeutic drugs than tumor cell monolayers
Day 4
Day 7
Day 10
780 (110) 980 (200)
2000 (270) 2150 (360)
4450 (650) 4600 (930)
200 000 MCF7 cells in 1 ml were cultured with either 3% aged human serum to obtain spheroids or 3% fresh human serum to obtain monolayer cells, treated with 1% fresh human serum for 24 h, trypsinized and counted using a hemocytometer. The data from four cultures were averaged and expressed with standard deviation. The average of the samples is not different at the 0.02 level calculated as described [33].
prevented the clumping of tumor cells in vitro. Screening serum proteases, we tested plasmin, a serine protease. An unexpected observation was made. Rather than dissociating tumor cell spheroids and enabling them to form monolayers, plasmin caused, on the contrary, the strong conversion of tumor cell monolayers into floating spheroids (not shown). Plasmin occurs in serum in its inactive form, plasminogen [17–21]. The activation of plasminogen into plasmin is positively controlled by plasminogen activators and negatively controlled by plasminogen activator inhibitors [17–21]. One plasminogen activator which can be purified from urine and which is referred to as urokinase is known to bind to breast tumor cells that express specific receptors for it [22–26]. MCF7 breast tumor cells growing in monolayer cultures
Having established that spontaneous formation of multicellular spheroids does not affect the proliferation of MCF7 cells, we asked whether tumor cell spheroids and monolayers are equally sensitive to chemotherapeutic drugs. Experiments described in Fig. 2 demonstrate that MCF7 cells increase their resistance to doxorubicin and to taxol when they form spheroids. 3.4. The serum serine protease plasmin converts tumor cell monolayers into spheroids We were interested to identify serum factors which
Fig. 3. Urokinase induces the formation of tumor cell spheroids. MCF7 cells (10 000/0.1 ml) were incubated with 3% fresh human serum for 24 h to allow them to form monolayers and subsequently treated with 10 ml of either RPMI 1640 medium (A) or 50 U/ml urokinase (B) for 24 h. Magnification: × 400.
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Fig. 4. The change in growth pattern alters the response of MCF7 cells to doxorubicin. (A) MCF7 monolayers (W) or spheroids (X) established as described in Fig. 1 were treated with doxorubicin for 3 days and their proliferation was measured in the thymidine uptake assay. (B) MCF7 spheroids established as described in Fig. 1 were treated for 24 h with either 1% fresh human serum (W) to revert them to monolayers or outdated serum (X) to maintain spheroids, treated with doxorubicin for 2 days, washed once, and treated with 1% fresh human serum for 24 h before the MTT assay was performed.
were treated with urokinase. In the presence of 50 units/ml urokinase, monolayer cells began to retract within 2 h and formed solid tumor cell spheroids within 24 h of incubation (Fig. 3). As the tumor cells changed their morphology and formed multicellular spheroids, they simultaneously enhanced their resistance against the chemotherapeutic drug doxorubicin (Fig. 4A). Reverting tumor spheroids to monolayer cells enabled the tumor cells to regain their susceptibility to doxorubicin (Fig. 4B).
4. Discussion Epithelial breast cells grow in a sheet-like structure lining the breast duct and the ductules [1,2]. Malignant transformation enables breast cells to ignore the morphogenic principles which guide the organization of the gland and thereby facilitates the growth of tumors in amorphous cell masses [1,2]. Previous attempts to mimic the clustered in vivo growth of breast tumor cells in vitro succeeded in some experiments using agarose or spinner culture systems [5–14]. The obtained multicellular spheroids exhibited reduced proliferative activity and were readily dissociated by trypsin treatment [6,11,12]. The experiments reported here demonstrate a new procedure for growing breast tumor cells in
spheroids with undiminished proliferative activity and resistance to dissociation by trypsin or collagenase. Our experiments implicate the serine protease plasmin as a factor controlling the growth pattern of breast tumor cells. Plasminogen, the precursor of plasmin, is converted to plasmin on the cell surface by plasminogen activators bound to cell membrane receptors [18– 21,25,26]. The urokinase-type plasminogen activator is of special interest in this context because malignant breast cells express the receptors for urokinase [22]. Urokinase is unstable and exhibits low enzymatic activity in its free form but becomes highly active and stable upon binding to membrane receptors [17–21,26–29]. When breast cells become malignant, plasmin is activated on their membrane and their morphology is reported to change from sheet-like structures to multicellular masses. A potential important consequence of the formation of spheroids may be the protection of micrometastatic breast cancer cells against chemotherapeutic drugs. Micrometastatic breast cancer cells are detected as multicell clusters in patients [30]. Postoperative adjuvant chemotherapies, which are applied to eliminate micrometastatic tumor cells, are moderately effective in beast cancer [31,32]. Considering the reports together with the findings described here, one may speculate that through the activation of plasmin on their membrane, micrometastatic tumor cells form multicell clusters and thus manage to shield themselves from the action of chemotherapeutic drugs, thereby impeding chemotherapies and raising therapeutic drug doses to prohibitively high levels. The presented study describes an experimental procedure to restore the full susceptibility of tumor cells to chemotherapeutic drugs by reverting tumor cell spheroids into monolayers. Compromising the protective shield may be a reasonable target of new therapeutic designs for postoperative adjuvant therapies for early breast cancer.
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