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Effect of Thrombin Treatment of Tumor Cells on Adhesion of Tumor Cells to Platelets in Vitro and Tumor Metastasis in Vivo1

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Effect of Thrombin Treatment of Tumor Cells on Adhesion of Tumor Cells to Platelets in Vitro and Tumor Metastasis in Vivo1 [CANCER RESEARCH 52. 3267-3272, June 15. 1992] Effect of Thrombin Treatment of Tumor Cells on Adhesion of Tumor Cells to Platelets in Vitro and Tumor Metastasis in Vivo1 Mary Lynn R. Nierodzik, Francis Kajumo, and Simon Karpatkin2 New York University Medical School, New York, New York 10016 [M. L. R. N., F. K., S. K.J, and Department of Veterans Affairs Medical Center, New York, New York 10010 ¡M.L. R. N.¡ ABSTRACT liunit concentrations of thrombin results in a 2- to 5-fold enhancement of adhesion to 6 different tumor cell lines from 3 Seven different tumor cell lines (human melanoma SK MEL 28; different species in vitro; infusion of thrombin in vivo results in hamster melanoma HM29; murine melanomas B16F10 and amelanotic melanoma B16a; human colon carcinoma 11("IX:murine colon carcinoma a 4- to 413-fold enhancement in pulmonary metastasis with CT26; and murine Lewis lung carcinoma) were treated with thrombin at two different tumor cell lines (5). 0.5-1 unit/ml and examined for their ability to bind to adherent platelets; Because of the marked effect of thrombin on platelet adhe HM29 was studied for its ability to bind to fibronectin and von Willebrand siveness to tumor cells in vitro and tumor metastasis in vivo, as factor; <T2<>.»1611.B16F10, and B16a were studied for their ability to well as the requirement of active thrombin on the platelet form pulmonary metastasis after i.v. injection of thrombin-treated tumor surface, we elected to determine whether tumor cells could be cells; CT26 was studied for its ability to grow s.c. Five of 7 thrombin- directly activated by thrombin to enhance their adhesiveness treated tumor cell lines increased their adhesion to adherent platelets 2- and metastatic potential. Such proved to be the case with 5 of to 3-fold. HM29 increased its adherence to fibronectin and von Wille- brand factor 2- to 3-fold. CT26, B16F1, B16F10, and B16a increased 7 different tumor cell lines from 3 different species. The present experimental pulmonary metastasis 10- to 156-fold. Thrombin-treated report documents our observations on the effect of thrombin CT26 cells demonstrated 2-fold greater growth in vivoafter s.c. injection. treatment of tumor cells on their enhanced adhesion to plate The mechanism of enhanced adhesion of thrombin-treated tumor cells to lets, fibronectin, and von Willebrand factor in vitro and their platelets required the platelet integrin GPHb-GPIIIa since it could be enhanced metastasis in vivo. inhibited by agents known to block adhesion of ligands to CPIIb-GPIIIa (monoclonal antibody 10E5, tetrapeptide RGDS, disintegrin Albolabrin); as well as a "GPIIb-GPIIIa-like" structure on tumor cells since it could MATERIALS AND METHODS be inhibited by treatment of thrombin-treated tumor cells with 10E5 and Tumor Cell Lines and Tissue Culture Media. CT26, /V-nitroso-;V- RGDS. The thrombin effect on tumor cells was optimum at l h of methylurethane-induced mouse undifferentiated colon carcinoma cells incubation with thrombin, did not require active thrombin on the tumor were obtained through the courtesy of Dr. M. H. Goldrosen, Roswell cell surface, and did not require protein synthesis (not inhibited by Park Memorial Institute, Buffalo, NY. HCT8 spontaneous human cycloheximide). Thus, thrombin-treated tumor cells markedly enhance colon adenocarcinoma cells were obtained through the courtesy of Dr. pulmonary metastasis. It is suggested that this may be secondary to E. Cadman, Yale University Medical School, New Haven, CT. HM29 thrombin-induced enhanced adhesion as well as growth of tumor cells. hamster malignant melanoma cells were obtained through the courtesy of Dr. Jean-Claude Bystryn, New York University Medical Center. INTRODUCTION Lewis lung carcinoma and SK-Mel-28 human melanoma cells were obtained from American Type Culture Collection, Rockville, MD. B16 The requirement of platelets for experimental as well as amelanotic mouse melanoma cells were obtained through the courtesy spontaneous tumor metastasis is well established in the murine of Dr. C. Maniglia, Yale University Medical School. B16F1 and system (1,2). Thrombocytopenia results in an 85-95% inhibi B16F10 mouse melanoma cells were obtained through the courtesy of Dr. I. J. Fidler, University of Texas, M. D. Anderson Cancer Center, tion of pulmonary metastasis (2). We have recently demon Houston, TX, and grown in minimal essential media supplemented strated that it is the adhesive function of platelets via the platelet with 5% fetal bovine serum, 2% glutamine, 2% penicillin-streptomycin GPHb-GPIIIa adhesive ligand receptor that is important for (GIBCO, Grand Island, NY), and 1% sodium pyruvate (100 mivi), 1% tumor platelet interaction in vitro and tumor metastasis in vivo vitamins (lOOx) and 1% nonessential amino acids (Sigma, St. Louis, (3). Agents which inhibit binding of GPHb-GPIIIa to fibronec MO). All other cell lines were maintained and grown in tissue culture tin or von Willebrand factor such as monoclonal antibody 10E5, as described previously (2-4). tetrapeptides RGDS, fibrinogen decapeptide LGGAKQAGDV, Anti-PIatelet-adhesive Protein Receptor and Adhesive Protein Re inhibit adhesion of tumor cells to platelets in vitro; 10E5 and agents. Monoclonal antibodies 10E5 and 6D1 were provided through RGDS inhibit metastasis in vivo (3,4). Antibodies which inhibit the courtesy of Dr. Barry Coller, State University of New York, Stony Brook, NY. 10E5 binds to the GPHb-GPIIIa complex and inhibits the binding of fibronectin or von Willebrand factor to platelets inhibit binding of tumor cells to platelets in vitro; anti-von binding of fibrinogen, fibronectin, and von Willebrand factor to acti vated platelets (6); 6DI, used as a control monoclonal antibody, binds Willebrand factor antibody inhibits metastasis in vivo in the to GPIb and inhibits ristocetin-induced platelet aggregation (7). Mon- absence of thrombocytopenia (3). ospecific anti-human fibronectin antibody was raised in rabbits immu Of particular interest is our recent observation on the role of nized with human fibronectin purified by Sepharose 4B gel filtration thrombin in enhancing platelet tumor interaction in vitro and and gelatin-Sepharose 4B affinity chromatography. The details of fi metastasis in vivo. Treatment of adherent platelets with mil- bronectin purification and antibody production have been described previously (3). Human von Willebrand factor was purified by the Received7/11/91;accepted4/6/92. method of Thorell and Blomback (8). RGDS was kindly synthesized 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 for us by Dr. David Schlesinger, New York University Medical Center, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and purified by gel filtration on a Sephadex G-25 column. Amino acid ' Supported by Grant HL-13336-21 of the National Lung and Blood Institute, composition and sequence were verified by amino acid analyzer. Grant CH-59789 of the American Cancer Society, and a Department of Veterans GRGES, the control peptide for RGDS was obtained from Peninsula Affairs Research Advisory Group Grant. Presented at the Thirty-second Annual Meeting of the American Society of Hematology. Boston, MA, December 1, Laboratories, Inc., Belmont, CA. Albolabrin, the disintegrin from viper 1990. venom is a potent RGDS-containing peptide isolated from Trimeresu- 2To whom requests for reprints should be addressed. rus albolabrin which inhibits metastasis (9), kindly supplied by Dr. 3267 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1992 American Association for Cancer Research. THROMBIN-TREATED TUMOR CELLS ENHANCE METASTASIS Stefan Niewiarowski, Temple University Medical School, Philadelphia, assuming a sphere. The mean nodule volume for each mouse was PA. calculated by dividing the total nodule volume for each group of mice Thrombin and Anti-Thrombin Reagents. Human thrombin (3000 NIH units/mg) was purchased from Sigma, DAPA,1 a potent, highly specific by the number of mice in each group. Tumor mass was calculated by multiplying the mean nodule number by the mean nodule volume per competitive inhibitor of thrombin (10) was a gift of Dr. Michael lung. Nesheim, Queens University, Ontario, Canada. Preparation of Platelets. Human platelets were separated from cit- Microscopic examination was performed in some experiments. The rated platelet-rich plasma (0.38% final concentration) on a 10 and 20% fixed lungs were embedded with paraffin in a unicassette (Miles Labo discontinuous Stractan gradient (arabino-galactan polymer) dissolved ratories, Inc., Elkhart, IN), and three coronal slices, 3 ^m thick, were in an isotonic balance salt solution, as described in detail, previously made through the lung. The pathological specimen was stained with (3, 4). hematoxylin-eosin and the largest slice was enumerated for number In Vitro Platelet-Tumor Cell Adhesion Assay. Platelets (3 x IO7) and surface area of tumor foci. Surface area of each metastatic focus were applied to 96-well flat bottomed microtitre plates (Falcon Labware was calculated with a micrometer by multiplying the largest two diam 3072, Oxnard. CA) for I h at room temperature to develop a platelet eters at right angles to each other. lawn as described previously (3, 4). Platelets were then treated with Statistics. Paired two-tailed t tests (or one-tailed, when indicated) buffer, thrombin, DAPA, or their combination and incubated overnight were used for statistical evaluations. at 4°C.Plates were then incubated with 0.01 M PBS plus 1% BSA for l h at 37'C, washed once with the above, and three times with PBS- BSA plus 0.9 mM CaCl;, 0.9 rriMMgCl2 before the addition of 1 x 10s RESULTS naive or thrombin-treated tumor cells in PBS-BSA-Ca-Mg for l h at 37°C.In some experiments, blocked and washed platelets were treated Comparison of Effect of Thrombin Treatment of HM29 Tumor with various monoclonal antibodies and inhibitors prior to the addition Cells versus Thrombin Treatment of Platelets on Adhesion of of tumor cells.
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