Induction of Experimental Bone Metastasis in. Mice by Transfection of Integrin a4f31 into. Tumor Cells. Nariaki Matsuura,*Wilma Puzon-McLaughlin,t. Atsushi Irie ...
Amnerican journal of Patholqgy, Vol. 148, No. 1, january 1996 Copyrgbt C) Amner/can Society for Investigative Pathology
Short Communication Induction of Experimental Bone Metastasis in Mice by Transfection of Integrin a4f31 into Tumor Cells
Nariaki Matsuura,*Wilma Puzon-McLaughlin,t Atsushi Irie,t Yoshihiro Morikawa,* Kennichi Kakudo,* and Yoshikazu Takadat From the Department of Pathologv II,* Wakayama Medical School, Wakayama, Japan, and the Department of Vascuilar Biology,t 7Te Scripps Research Institute, La Jolla, Calfobrnia
Cell adhesion receptors (eg, integrins and CD44) play an important role in invasion and metastasis during tumor progression. The increase in integrin a4,81 expression on primary melanomas has been reported to signfircantly correlate with the development of metastases. a4,(31 is a cell surface heterodimer that mediates cell-cell and cell-extracelular matrix interactions through adhesion to vascular cell adhesion molecule (VCAM)-1 and to the IIICS region offibronectin. To test the effects of a4161 expression on tumor ceUl metastasis, Chinese hamster ovary ceUs were transfected with human a4 cDNA. Whereas ax4negative Chinese hamster ovary cells developed only pulmonary metastasis, a4-positive Chinese hamster ovary cells developed bone and pulmonary metastasis in 3 to 4 weeks when injected intravenously into nude mice. Bone metastasis was inhibited by antibody against a4 or VCAM-1. Expression of a3 (31, a6131, or acVI81 did not induce bone metastasis. Expression of a4f81 also induced bone metastasis in K562 human erythroleukemia ceUs injected into SCID mice. These resuls demonstrate that a4f81 can induce tumor
cel trafficking to bone, probably via interaction with VCAM-1 that is constitutively expressed on bone marrow stromal cells. (Am J Pathol 1996,
148:55-61)
Cell adhesion receptors (eg, integrins and CD44) play an important role in invasion and metastasis during tumor progression.` Integrin a4131 is a cell surface heterodimer that mediates cell-cell and cellextracellular matrix interactions through adhesion to vascular cell adhesion molecule (VCAM)-1 and to the IIICS region of fibronectin.F6 a4f1 has been implicated in lymphocyte trafficking through its adhesion to high endothelial venules of Peyer's patches7 and in leukocyte/endothelial cell interaction in the pathogenesis of atherosclerosis8 and experimental allergic encephalitis.9 The increase in a4f31 expression on primary melanomas significantly has been reported to correlate with the development of metastases.10 The adhesion of several human tumors (eg, melanoma and osteosarcoma) to activated endothelium in vitro is also mediated by an interaction between a4f31 and VCAM-1.1112 Vascular endothelial cells express VCAM-1 when they are activated by cytokines.13,14 VCAM-1 is constitutively expressed on the bone marrow stromal cells and follicular dendritic cells of lymph nodes.15,16 Here we developed Chinese hamster ovary (CHO) or K562 cells expressing a4031 integrin to examine the specific role of a4f31 in tumor cell behavior in vivo. In this paper we describe that the expression of a4f31 integrin induced the bone metastasis of tumor cells. Supported by National Institutes of Health grants GM47157 and GM49899 to Y. T. and a grant from the Ministry of Education, Science and Culture, and Special Coordination Funds of the Science and Technology Agency of the Japanese Government to N. M. Accepted for publication September 12, 1995. Address reprint requests to Dr. Yoshikazu Takada, Department of Vascular Biology, VB-1, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92038. Nariaki Matsuura's present address is Department of Pathology II, Osaka University Medical School, Osaka 565, Japan.
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Materials and Methods
37°C for 1 hour. Bound cells were quantified by assaying endogenous phosphatase activity.25
Monoclonal Antibodies (MAbs) 7E2 (anti-hamster 131) and PB1 (anti-hamster a5)17'18 obtained from R. L. Juliano (University of North Carolina, Chapel Hill, NC), 8A2 (anti-human 131)19 from N. Kovach and J. Harlan (University of Washington, Seattle, WA), AlA5 (anti-human 131)2° and B5G10 (anti-human a4)21 from M. E. Hemler (DanaFarber Cancer Institute, Boston, MA), and SG/73 (anti-human a4)29 and KH/72 (anti-human a5, K. Miyake, unpublished) from K. Miyake (Saga Medical School, Saga, Japan). M/K-2.7 (mouse VCAM-1)15 was obtained from ATCC. were
Preparation of a4-CHO and a4f31 -CHO Cells Human a4 cDNA (4.5 kb)22 and human ,13 cDNA clone B3 (2.9 kb)23 were subcloned into the Xbal site of the pBJ-1 vector with a SRa promoter.24 CHO-Ki cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heatinactivated fetal calf serum at 37°C in a 5% CO2 incubator. A total of 10 jig of the a4 pBJ-1 cDNA was transfected into CHO cells (8 x 106 cells) with 1 jig of pCDneo DNA by electroporation. Transfected cells were maintained for 2 days in the above medium and then transferred to the medium supplemented with 700 jig/ml G418 (Gibco, Grand Island, NY). After 10 to 14 days, resulting colonies were harvested and cells expressing human a4 were collected by single-cell sorting with B-5G10 in FACStar (Becton Dickinson, Mountain View, CA). 131 cDNA in pBJ-1 (10 jig) was transfected into clonal a4-CHO cells (clone A2) with pCD-hygromycin (1 ,ug) by electroporation and clonal a4131 CHO cells were obtained as described above except that 500 ,tg/ml hygromycin and A1A5 were used instead of G418 and B5G10.
Adhesion Assay Immulon-2 96 well plate (Dynatech Laboratories, Chantilly, VA) was incubated with 100 ,tl per well of recombinant soluble VCAM-1 (10 jig/ml), CS-1 peptide (50 p.g/ml), or fibronectin (10 ,ug/ml) in phosphate-buffered saline (PBS; 140 mM NaCI, 10 mM Na-phosphate, pH 7.4) at 40C overnight and then 1 % bovine serum albumin (crystal, Calbiochem, San Diego, CA) for 1 hour at room temperature and washed with PBS. a41l1-CHO cells (105 cells per well in 100 ,ul of DMEM) were added and incubated at
In Vivo Metastasis Assay Tumor cells (106 cells per mouse) were injected intravenously into the tail vein of 4-week-old female nude or SCID mice (Clea, Shizuoka, Japan) that had been raised under pathogen-free conditions. Care of mice was in accordance with institutional guidelines. Mice were sacrificed by deep anesthesia with ether. Organs were removed and examined for metastatic foci macroscopically with dissecting microscope and microscopically after fixation in 10% (v/v) formalin and staining.
Other Methods Flow cytometric analysis and immunoprecipitation of surface 1251-labeled cell extracts with specific MAbs were performed as described.23 The x2 test26 was used to determine the significance of the in vivo metastasis experiments.
Results and Discussion To define the role of a4l31 in tumor cell behavior in vivo, we developed clonal CHO cells expressing human a4 (designated a4-CHO), or both a4 and (31 (designated a44p1-CHO). CHO cell lines expressing the neomycin resistance gene were used as controls (CHO-neo). Parent CHO cells express mostly a5p1 as (31 integrins; ,B3 integrins are not detectable.23'27 Upon flow cytometric analysis (Figure 1A), human a4 was expressed homogeneously on a4- or a411 -CHO cells but not on CHO cells. The endogenous hamster a5 level was comparable in all CHO cell lines used. Immunoprecipitation detected human a4 (150 kd and its 80- and 70-kd fragments) on a4-CHO cells and human a4 and 131 (110 kd) on a4p1l-CHO cells (Figure 1 B). Both CHO and a411 -CHO cells adhered well to fibronectin but not to collagen. The recombinant a4131 on a4p1l-CHO cells is functional with high avidity for VCAM-1 but low avidity for CS-1 peptide (Figure 2).28 a4-CHO cells adhere to VCAM-1 but not to CS-1; a human a4/hamster (1 hybrid cannot be activated by anti-human 13.128 CHO cells did not bind to either CS-1 or VCAM-1, whereas a4p1l-CHO cells adhered to VCAM-1 well but to CS-1 only after activation with MAb. CHO-neo cells showed ligandbinding properties essentially identical to parent CHO cells. There was no difference among all the CHO lines used in the morphology and the growth
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