A Human Fibrosarcoma Inhibits Systemic Angiogenesis and the Growth of Experimental Metastases Via Thrombospondin-1

Proc. Natl. Acad. Sci. USA Vol. 95, pp. 6343–6348, May 1998 Medical Sciences A human fibrosarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1 OLGA V. VOLPERT*, JACK LAWLER†, AND NOE¨L P. BOUCK*‡ *Department of Microbiology-Immunology and R. H. Lurie Cancer Center, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611; and †Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115 Edited by M. Judah Folkman, Harvard Medical School, Brookline, MA, and approved March 23, 1998 (received for review November 10, 1997) ABSTRACT Concomitant tumor resistance refers to the see ref. 8 and citations in refs. 1 and 2). One such case is that ability of some large primary tumors to hold smaller tumors of the patient from whom the widely used human fibrosarcoma in check, preventing their progressive growth. Here, we dem- line HT1080 was derived and who died from widespread onstrate this phenomenon with a human tumor growing in a metastases within 3 mo of removal of the primary tumor (9). nude mouse and show that it is caused by secretion by the In addition, an occasional human tumor cell line or primary tumor of the inhibitor of angiogenesis, thrombospondin-1. explant is able to drive nude mice in which it is growing into When growing subcutaneously, the human fibrosarcoma line an antiangiogenic state in which the animals become unable to HT1080 induced concomitant tumor resistance, preventing mount a corneal angiogenic response that is induced in the growth of experimental B16yF10 melanoma metastases in tumor-free controls (5, 6). The molecules generated by these the lung. Resistance was due to the production by the tumor human tumors that are responsible for this effect have not been cells themselves of high levels of thrombospondin-1, which was identified. present at inhibitory levels in the plasma of tumor-bearing The HT1080 line is unusual both in that it was derived from animals who become unable to mount an angiogenic response a patient whose history is compatible with concomitant tumor in their corneas. Animals carrying tumors formed by anti- resistance and in that, unlike most sarcomas that have inacti- sense-derived subclones of HT1080 that secreted low or no vated the p53 tumor suppressor gene (10–12), it retains thrombospondin had weak or no ability to control the growth wild-type p53 activity (13). In fibroblasts, wild-type p53 sup- of lung metastases. Although purified human platelet throm- ports the production of thrombospondin-1 (TSP-1) (14, 15), a bospondin-1 had no effect on the growth of melanoma cells in potent inhibitor of angiogenesis (16, 17). As might be predicted vitro, when injected into mice it was able to halt the growth of for a fibroblast-derived tumor line that retains wild-type p53, their experimental metastases, providing clear evidence of the HT1080 cells secrete relatively high levels of TSP-1. Here, we efficacy of thrombospondin-1 as an anti-tumor agent. show that this tumor-derived TSP-1 is active and can drive tumor-bearing animals into an antiangiogenic state that blocks Concomitant tumor resistance is an unusual phenomenon the growth of experimental metastases. Exogenous TSP-1 also wherein animals harboring large primary tumors are resistant blocked the growth of metastases. This work identifies a to the growth of distant smaller tumors (reviewed in refs. 1 and molecule produced directly by a human tumor that is respon- 2). This resistance depends on circulating factors and disap- sible for concomitant tumor resistance and provides direct pears upon surgical resection of the primary tumor. In stun- evidence for the anti-tumor efficacy of the antiangiogenic ning advances over the past several years, O’Reilly and Folk- agent TSP-1. man (3–7) have shown that, in syngeneic rodent systems, the circulating factors responsible for such resistance are inhibitors MATERIALS AND METHODS of angiogenesis. Two of these have been identified: angiostatin and endostatin. In these model systems, primary tumors Cell Culture. Human fibrosarcoma cell line HT1080 (Amer- secrete inducers of angiogenesis and also generate inhibitors of ican Type Culture Collection) and pigmented mouse mela- angiogenesis. In the microenvironment of the large primary noma line B16 F10P (a kind gift from P. Polverini, University tumor, the inducers are apparently sufficient to overcome the of Michigan) were maintained in DMEM with 10% fetal calf effects of the inhibitors because the new vessels essential for serum. A second highly metastatic but unpigmented strain of progressive tumor growth are present. However, when induc- the B16 F10 cell line (18, 19) was kindly provided by I. J. Fidler ers and inhibitors are shed from the tumor bed into the (University of Texas M. D. Anderson Cancer Center, Hous- circulation, levels of the more labile inducers fall off rapidly ton) and maintained in MEM supplemented with glutamine, whereas levels of the more stable inhibitors rise, creating a nonessential amino acids, and 10% fetal calf serum. Bovine systemic antiangiogenic environment that prevents small nests adrenal capillary endothelial cells, BP10T8, a kind gift of J. of metastatic cells from inducing neovascularization. As a Folkman (Children’s Hospital, Harvard Medical School, Bos- ton) were maintained in DMEM supplemented with 10% result, these incipient tumors remain small and dormant. On m y removal of the primary tumor, inhibitor levels fall and the donor calf serum, 2 mM glutamine, and 100 g ml endothelial previously dormant metastases expand vigorously. cell mitogen (R & D Systems) and used at passage 14 or 15. All Concomitant tumor resistance likely also occurs in human of the cells were incubated at 37°C at 7% CO2. Antisense Clones. cancer patients. There are a variety of anecdotal clinical The central fragment of human TSP-1 reports in the literature describing the fast and disastrous cDNA (nucleotides 883-2572) was amplified by PCR by using primers with HindIII linkers, 59-ggg agctt CCGCAACTAAC- development of what must have been preexisting metastases TAC-39 (left) and 59-ggg agctt GTACTGGCAGTT- following surgical removal of a primary tumor, (for example, GTCCG-39 (right), and cloned into the polylinker of pRC The publication costs of this article were defrayed in part by page charge This paper was submitted directly (Track II) to the Proceedings office. payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: TSP-1, thrombospondin-1; bFGF, basic fibroblast accordance with 18 U.S.C. §1734 solely to indicate this fact. growth factor; VEGF, vascular endothelial growth factor. © 1998 by The National Academy of Sciences 0027-8424y98y956343-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: n-bouck@ PNAS is available online at http:yywww.pnas.org. nwu.edu. 6343 Downloaded by guest on September 25, 2021 6344 Medical Sciences: Volpert et al. Proc. Natl. Acad. Sci. USA 95 (1998) CMVneo (Invitrogen) in reverse orientation. HT1080 fibro- of human TSP present was estimated from Western analysis by sarcoma cells were transfected with plasmid expressing anti- using the human-specific antibody MA-1. sense TSP-1 were selected in G418, and screened individually To measure the growth of metastases, B16 F10 mouse for the loss of TSP-1 secretion by Western blotting of serum- melanoma cells were grown to 80% confluence, harvested, and free conditioned media. Vascular endothelial growth factor resuspended at 106 (for unpigmented cells) or 107 (for pig- (VEGF) and basic fibroblast growth factor (bFGF) were mented cells) cellsyml in PBS, and 0.1 ml was injected into the measured in serum-free conditioned media by using commer- mouse tail vein using a 27-gauge needle. After 14 days for cial kits (R & D Systems). TSP-1 was quantitated using pigmented cells or 21 days in the case of unpigmented cells, Western blots with purified human platelet TSP-1 as a stan- animals were killed and lungs weighed and fixed overnight in dard. formalin for pigmented cells or Bouin’s solution for the Western Analysis. Seven to 10 mg of total protein from unpigmented cells, and metastases on the surface of the lung conditioned media, or 40 mg from mouse plasma, was resus- were measured using an ocular micrometer and counted. To pended in loading buffer containing 7.5% b-mercaptoethanol, test the effect of preexisting tumors on metastasis, tail vein boiled 3–5 min, run on 6% SDSyPAGE and blotted to injections were performed on animals harboring primary Hybond-N membrane (Amersham). The membrane was tumors that had reached a diameter of '5 mm. probed with anti-thrombospondin mAbs A4.1 (16) or MA-1 (20), developed with an enhanced chemiluminescence kit RESULTS (Amersham) and exposed to x-ray film for 1–20 s. Thrombospondin. TSP-1 was purified from human platelets HT1080 Secreted Active Antiangiogenic Throm- as described (21, 22) by heparin affinity column with subse- bospondin-1. In cells of the fibroblastic lineage, the wild-type quent sizing chromatography on Sephacryl-200 (Pharmacia) p53 tumor suppressor gene supports the secretion of high and subjected to batch chromatography with gelatin Sepharose levels of antiangiogenic thrombospondin-1 that prevents the to eliminate possible fibronectin. Protein purity was assessed cells from inducing angiogenesis (14, 15). As such cells by 7.5% SDSyPAGE, and no contaminating factor H was progress toward tumorigenicity in vitro, wild-type p53 is inac- detected (23). Contamination with transforming growth fac- tivated and such inactivation is essential for the development tor-b that can copurify with thrombospondin was measured of an angiogenic phenotype (27). The HT1080 human fibro- (24) and found to be ,0.1 pgymg protein. Batch chromatog- sarcoma cell line is atypical in that it has retained wild-type p53 raphy with polymyxin-B beads (Sigma) was performed to and, as expected, secretes high levels of thrombospondin.

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