[CANCER RESEARCH 59, 5023–5029, October 1, 1999] Overexpression of Fibroblast Growth Factor 1 in MCF-7 Breast Cancer Cells Facilitates Tumor Cell Dissemination but Does Not Support the Development of Macrometastases in the Lungs or Lymph Nodes 1 Lurong Zhang, Samir Kharbanda, Sandra W. McLeskey, and Francis G. Kern 2 Lombardi Cancer Center [L. Z., S. K., S. W. M.], Department of Cell Biology [L. Z.], Department of Pharmacology [S. W. M.], and the School of Nursing [S. W. M.], Georgetown University Medical Center, Washington, D.C. 20007; and Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35255-5305 [F. G. K.] ABSTRACT Mice bearing primary tumors produced by LacZ-tagged MCF-7 human breast carcinoma cells transfected with fibroblast growth factor (FGF) 1 have frequent micrometastases, but macrometastases are not observed. i.v. injection of FGF-1-transfected tumor cells produced no pulmonary macrometastases, and removal of primary tumors resulted in the disap- pearance of spontaneous micrometastases. Thus, failure of micrometasta- ses to proliferate was not due to inhibitory factors released from the primary tumor, and the presence of the primary tumor is required for maintenance of the micrometastases. This indicates that the micrometas- tases result from continued seeding from the primary tumor balanced by clearance from the metastatic site. Tumor emboli trapped in the vessels of lungs and lymph nodes and single tumor cells observed in the pulmonary vein implied that FGF-1-overexpressing MCF-7 cells are deficient in their ability to extravasate. The frequency of tumor cells incorporating bro- modeoxyuridine was consistently lower in lung tissues when compared with primary tumors, indicating that disseminated tumor cells were un- able to maintain a high rate of proliferation. Increased angiogenesis resulting from FGF-1 production by the transfected cells with a concom- itant increased rate of intravasation into developing blood vessels may be the underlying determinant of spontaneous micrometastasis produced by these cells when compared with parental MCF-7 cells. INTRODUCTION Metastasis is the product of complicated interactions between tu- mor cells and stroma. Tumor cells must detach from the primary tumor, enter the circulation, extravasate at distant sites, and proliferate in the parenchyma of distant organs (1–3). Although a number of factors have been shown to be involved in this process, including proteases (4, 5), adhesion molecules (4, 6, 7), motility factors (3), and angiogenic factors (1, 8), the precise mechanism of the metastatic process remains unclear. However, a sufficient blood supply via tumor microvessels is critical for both primary tumor growth and secondary metastasis (1, 8, 9). Therefore, increased angiogenesis may increase opportunities for intravasation of tumor cells into the circu- lation (10, 11). A recent report (12) suggests that intravasation mediated by acti- vation of a plasmin-metalloproteinase cascade may be a rate-limiting step in the metastatic process. However, circulating tumor cells can commonly be found in patients with many types of solid tumors [Refs. 10 and 13–21 (reviewed in Ref. 13)]. Thus, the importance of intrav- asation as a rate-limiting step of metastasis is currently under debate. Likewise, recent studies seem to indicate that extravasation is a relatively common event and therefore perhaps not rate-limiting (5, 22). However, others have proposed that all sequential steps of me- tastasis are rate-limiting (1) and selective (23). Although the reasons for these controversies may have to do with the validity of the particular models of metastasis used, it is also possible that for different tumors, different steps of the metastatic process are either more or less important as rate-limiting steps. In previous studies, we have demonstrated that transfection of FGFs 3 can confer an antiestrogen-resistant and hormone-independent in vivo growth phenotype to MCF-7 breast carcinoma cells. In addi- tion, FGF-1 or FGF-4 overexpression confers a metastatic phenotype on these nonmetastatic and noninvasive cells (24 –26). The results of the current study indicate that overexpression of FGF-1 by MCF-7 cells can induce transient microdeposits of tumor cells in vessels of distant organs that do not seem to extravasate efficiently into the parenchyma and may fail to proliferate at the distant site. We submit that the increased dissemination of tumor cells is a result of enhanced intravasation via the large number of FGF-1-stimulated blood vessels within the primary tumors. Thus, this xenograft tumor model provides evidence that the processes of intravasation and extravasation can be distinct, requiring different attributes, and, together with control pa- rental MCF-7 cells, provides the opportunity to study these processes separately. MATERIALS AND METHODS Cell Lines. FGF-1 clone 18 is a clonal cell line of LacZ-transfected MCF-7 breast carcinoma cells retransfected with the cDNA for human FGF-1 (26). To isolate the clone 18-lung cell line, 10 million clone 18 tumor cells were injected into the mammary fat pads of ovariectomized nude mice treated with 17-estradiol (0.72 mg/pellet; 60-day release). After 40 days, 100 mg of lung tissue of each mouse were sterilely harvested, minced, and digested in 1 ml of a mixture of 800 g/ml collagenase (C-9891; Sigma, St. Louis, MO), 100 mg/ml bovine pancreatic protease (P-4630; Sigma), and 0.5 mg/ml bovine pancreatic DNase (DN-25; Sigma) in IMEM (Biofluids Inc., Rockville, MD) at 37°C for 1 h. After washing with IMEM, the disassociated cells were cultured in 10% fetal bovine serum-IMEM containing 500 g/ml G418 [the expression vector for FGF-1 confers G418 resistance (26)]. Two individual colonies surviving in G418-containing media were harvested and expanded as clonal cell lines. In this study, clone 2 was used. X-Gal Staining for -Galactosidase Activity in Tissues. Harvested lungs and lymph nodes were dissected free of surrounding fat and fixed and stained with X-gal, as described previously (25). After further fixation in 10% phos- phate-buffered formalin, the approximate number of foci of blue-stained met- astatic tumor cells present within the lymph node or lung was estimated with an Olympus SZH dissecting microscope. The following scoring system was used to score the degree of metastasis, based on the estimated counts: (a) -, no blue spots; (b) +, about 1–50 blue spots; (c) ++, about 50 –100 blue spots; (d) +++, about 100 –200 blue spots; and (e) ++++, 200 blue spots. Received 4/2/99; accepted 8/6/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grant CA50376 and the Adolph Weil Endowed Chair at Southern Research Institute (to F. G. K.); NIH Grant CA71545 and Department of Defense Grant DAMD BC97-1820 (to L. Z.); NIH Grant CA66154 and Department of Defense Grant DAMD 17-94-4173 (to S. W. M.). L. Z. was a Susan Komen research fellow. 2 To whom requests for reprints should be addressed, at Southern Research Institute, P. O.Box 55305, 2000 Ninth Avenue South, Birmingham, AL 35255-5305. Phone: (205) 581-2480; Fax: (205) 581-2877; E-mail: kern@sri.org. 3 The abbreviations used are: FGF, fibroblast growth factor; IMEM, improved minimal essential medium; X-gal, 5-bromo-4-chloro-3-indolyl -D-galactopyranoside; BrdUrd, bromodeoxyuridine; uPA, urokinase plasminogen activator; CAM, chorioallantoic mem- brane; MMP, matrix metalloproteinase; TUNEL, terminal deoxynucleotidyl transferase- mediated nick end labeling; PECAM-1, platelet/endothelial cell adhesion molecule 1; VEGF, vascular endothelial growth factor. 5023 on July 16, 2015. © 1999 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from