(‘LINICAI.

IMMUNOLOGY

AND

IMMUNOPATHOLDGY

60,

430-44!

( tY’)l)

Mesenchymal Specificity of Three New Monoclonal Antibodies Generated to the Osteosarcoma Cell Line 4444T FRANK BLOMMAERT,* Departments

of *Pathology

WILLIAM

and fPediatric Medicine.

F. CASSANO,~.' AND BYRON P. CROKER* Hematology-Onc.ology. Gainesville. Floridu

University

of Florida

College

of

32610

We have generated three murine monoclonal antibodies to the new human osteosarcoma ceil line 4444T. Analysis of their binding patterns to tumor cell lines, normal human tissues. and surgical tumor specimens indicates that the antibodies recognize a subset of human sarcomas and stromal tissues but fail to react with carcinomas or normal human epithelial tissue. These mesenchyme-specific monoclonal antibodies bind to antigens in the extracellular matrix. One antibody is specific in its binding to the muscularis arteriosus. We expect these antibodies to aid in the identification of sarcomas and to extend our knowledge of the components of the extracellular matrix and its interaction with tumors. ‘C 1991 Academic Preah. Inc. INTRODUCTION

Sarcomas, malignancies of mesodermal origin, arise in supportive and connective tissues. The relationship among the different sarcoma subtypes and normal cellular differentiation pathways of connective tissue is an area of active investigation. Antigenic heterogeneity of human sarcomas became apparent 20 years ago when intensive investigation of patients’ sera revealed antibody titers against their own tumors (1, 2). These early studies were hampered by low levels of antibody and by the lack of tumor specificity in these sera. With the advent of the hybridoma technology of Kohler and Milstein (3) in 1975, the generation of defined, monospecific antibodies became routine. These monoclonal antibodies (MoAbs) have been used to define and characterize unique antigens on tumor cells. Many MoAbs are known to bind to antigens related to cellular differentiation or to specific cell lineages (4, 5). In bone tissue, expression of proteins such as osteonectin (6), osteocalcin (7), sialoprotein (8), and bone proteoglycans (9) seem to be related to maturational stages of bone development; however, their signiticance as markers of neoplastic bone disorders is unresolved. There have been many attempts to generate MoAbs to sarcoma-associated antigens. Many have cross-reactivity with normal tissues or other types of tumors (lO-14), identify antigens that have not been adequately characterized, and define antigens with no known physiologic role (15-17). Some MoAbs against sarcomas also detect antigens in connective tissue or stages in fibroblast differentiation (18-20). However, none of the reported MoAbs are restricted to primitive mesenchymal cells committed to differentiation along the osteoblastic lineage. This paper reports the use of a recently established osteosarcoma cell line to generate tissue or differentia’ To whom correspondence and reprint requests should be addressed 430

OSTEOSARCOMA

ANTIGENS

431

tion-specific antibodies against cells of mesodermal origin. The antibodies have unique binding distributions which are useful in the characterization of mesenchymal cell differentiation and are helpful in the detection and classification of sarcomas. METHODS

Cell lines. The osteosarcoma cell line 4444T and the skin tibroblast cell line 4444F from the same patient were established in our laboratory (B. P. Croker, manuscript in preparation). URHCL-1, MG63, and U2OS were obtained from the American Type Culture Collection (Rockville, MD). 791T was obtained from Dr. P. Trown (Xoma Corp., Berkeley, CA). The colon carcinoma cell lines H29 and LOVO as well as the melanoma cell line MeWo were provided by Dr. A. Kimura (University of Florida). The carcinoma cell lines RKB and SM and the sarcoma cell lines SynSarc and JR were provided by Dr. Garvin (University of South Carolina, Charleston, SC). JR and SynSarc were maintained as monolayer cultures in serum-free Dulbecco’s minimal essential medium (DMEM), and SM was cultured with 2% supplemental fetal calf serum (FCS). The other cell lines were maintained in DMEM containing 10% bovine calf serum. Immunization. Six-week-old female BALB/c mice were injected intraperitoneally with 4444T cells on Day 0 (3 x lo6 cells), Day 3 (5 x lo6 cells), and then every 7 days with 20 x lo6 cells for 3 weeks. Three days after the last injection the mouse was sacrificed and the spleen was removed aseptically. A single cell suspension was prepared by crushing the spleen between the frosted ends of sterile glass slides. Hybridoma production. Cell fusion was performed as previously described (21). Briefly, spleen cells and an APRT-deficient subclone of NS-1 myeloma cells were fused with 35% polyethylene glycol (PEG, Sigma Chemical Co., St. Louis, MO) adjusted to pH 8.0. The fused cells were seeded in 200-~1 portions into 96-well flat-bottomed wells (Costar, Cambridge, MA). Ten days later culture supernatants were screened for antibody production. Screening of hybridoma supernatants. Initial screening of hybridoma supematants was designed to identify antibodies reactive with surface or intracellular antigens of the 4444T osteosarcoma cell line that were not present in the 4444F fibroblast cell line. Whole cell lysates of the tumor and fibroblast cell lines were prepared by suspending 50 x lo6 cells/ml in a cell lysis buffer containing 0.5% Nonidet-P40 (NP-40), 0.15 M NaCl, 0.05 M Tris, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, pH 8.0. The lysates were then diluted 1:20 for the tumor cell lysate and 1:50 for the fibroblast lysate in binding buffer (0.05 M Tris, 0.15 M NaCl, 0.01% sodium dodecyl sulfate, pH 8.0) prior to application onto nitrocellulose membranes. These lysate dilutions were applied to 0.2~pm nitrocellulose paper (Schleicher & Schuell, Keene, NH) in a minifold dot blot apparatus (Schleicher & Schuell) under vacuum and washed with 150 pi/well of binding buffer. The nitrocellulose paper was removed from the apparatus and, after drying, was treated with 10 ml blocking buffer (Tris-buffered saline (TBS), 10% human serum, 1% bovine serum albumin (BSA) + 0.01% NP-40) for 60 min. The nitrocellulose paper was reclamped in the apparatus including Parafilm to prevent leakage, and

432

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CASSANO,

AND

CROKER

50 ~1 of each culture supernatant (diluted 1: I with TBS) was applied and incubated at 20°C for 30 min. Negative controls were composed of I:500 dilutions of ascites of both IgG and IgM monoclonal antibodies of irrelevant specificity. Positive controls included WCBA23.4, a monoclonal IgM, anti-T-cell antibody with widespread cross-reactivity to normal and neoplastic tissue (W. F. Cassano and B. Aleva. personal communication) and a 150 dilution of mouse serum from the animals immunized to 4444T cells. The nitrocellulose was washed three times in washing buffer (TBS, 1% BSA, 0.01% NP-40) and incubated for 30 min with a 1: 1250 dilution of biotinylated horse anti-mouse antibody (Vector Labs, Burlingame, CA) in washing buffer. After two washes in washing buffer and one wash with TBS alone, the nitrocellulose was incubated for 45 min in ABC-glucose oxidase reagent (Vectastain ABC-glucose oxidase kit, Vector Labs). The paper was washed twice in TBS, once in 0.1 M NaH?PO,, pH 6.9, then 10 ml substrate was added (0.1 M NaH?PO,, pH 6.9, 0.04 M glucose, 2 mg phenazine methosulfate, 10 mg nitroblue tetrazolium, in total of 20 ml), and incubated in the dark for 30 min. A 5-min wash with tap water stopped the reaction. Hybridoma cultures producing antibodies binding to the osteosarcoma cells, but not to the fibroblasts, were expanded in 24-well plates in adenine-thymidine-supplemented medium with a feeder layer of fresh mouse thymocytes. Screening supernatantsfor binding to surfuce antigens. Tumor cells seeded at 2 x lo5 cells/ml and fibroblasts at 1 x IO5 cells/ml in 96-well round-bottom plates (Costar) were cultured overnight. Cells were then washed with phosphatebuffered saline (PBS), cooled to 4”C, and treated for 30 min with blocking buffer (PBS, 1% BSA, 5% human serum). Plates were centrifuged, the supernatant was discarded, and 50 t.~l of a 1:3 dilution in PBS of each hybridoma culture supernatant was added to each well and incubated for 30 min at 4°C. A positive control consisting of a I:40 dilution of the immunized mouse serum and a negative control consisting of a I:200 dilution of ascites of monoclonal. anti-T-cell-specific antibodies were included. Plates were washed three times in surface washing buffer (PBS + 1% BSA) and were incubated with 50 pi/well of a 1:40 dilution of biotinylated horse anti-mouse antibody (Vector) in surface washing buffer for 30 min at 4°C. Cells were then washed three times and incubated with 100 pJwel1 of ABC-PO (ABC-peroxidase kit, Vector Labs) for 45 min at 4°C. After three more washes, 100 kl/well of substrate was added (0.03 M citric acid, 0.06 M Na?HPO,, 0.03% H,O,, 2 mg/ml OPD (o-phenylenediamine dihydrochloride, Sigma Chemical Co.) at pH 5.0 and incubated in the dark at 20°C for 20 min. The reaction was stopped with 100 tJ/well of I .2 N H$O,. Hybridoma cultures producing antibodies binding to the osteosarcoma cells, but not to the fibroblasts, were transferred to 24-well plates in adenine-thymidine-supplemented medium with a fresh feeder layer of mouse thymocytes. Cloning. Hybridomas producing antibodies of the desired specificity were cloned by limiting dilution to 1 cell/well in adenine-thymidine-supplemented medium with a feeder layer of fresh mouse thymocytes in 96-well plates. Tissue specimens. Tumor and normal tissue samples were obtained fresh from surgical specimens or at autopsy. Samples were snap frozen in isopentane, embedded in gelatin or OCT. and stored at -70°C until cryostat sectioning.

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ANTIGENS

Immunoperoxidase histologic staining. Cryostat sections were fixed for 10 min in fresh acetone at - 20°C and staining was performed with the Vectastain Elite kit (Vector Labs) following manufacturer’s instructions. Monoclonal mouse IgG antibodies of irrelevant specificity were used as negative controls. Dilutions of I:30 of the hybridoma culture supematants were used as the primary antibodies. The cell lines were grown on coverslips. fixed in acetone, and stained following the same protocol. Flow cytometry. Blood cells were stained by indirect membrane immunofluorescence by using hybridoma culture supernatants (I:30 dilution) and fluoresceinconjugated goat anti-mouse immunoglobulin (Tago, S. San Francisco, CA). Ten thousand cells were analyzed using a FACStar instrument (Becton-Dickinson, Mountain View, CA) and the data were expressed as a histogram of fluorescence intensity versus number of cells. RESULTS

From approximately 1200 hybridomas formed during the fusion, three stable clones producing an antibody reactive with 4444T and not 4444F were found. One antibody (WCFB25.1) bound to surface antigens while the other two antibodies (WCFB25.2 and WCFB25.3) recognized both surface and intracellular antigens of the cell line. All three hybridomas produced an antibody of the murine IgGl isotype. Analysis of cell lines derived from a variety of human neoplasms demonstrated TABLE REACTIWTY

WITH VARIOUS

I HUMAN

4444F 70% c.O 100% c. 4444T 70% c. 100% c. MG63 79lT uzos URHCL-1 NLF JR SynSarc MEW0 SM HELA RKB HZ9 LOVO

LINES

Antibodies

Cell

line

CELL

WCFB25.1

WCFB25.2

WCFB25.3

Fibroblast

-

-

+

Osteosarcoma

+ + + + -

+ + + + -

+ + + + + -

-

-

-

Origin

Osteosarcoma Osteosarcoma Osteosarcoma MFH Neuroblastoma Rhabdomyosarcoma Synovial sarcoma Melanoma Rhabdoid tumor Cervical carcinoma Transit. cell carcinoma Colon carcinoma Colon carcinoma

a Percentage of confluency in culture.

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that the MoAbs recognize a subset of sarcomas and have no reactivity with carcinomas (Table 1). Three out of four of the osteosarcoma cell lines tested (UZOS, MG63. 444T, 791T) expressed the WCFB25.2 and WCFB25.3 antigens. The 791T cell line was negative for all three MoAbs. WCFB25.1 did not bind to the MG63 line but was reactive with the remaining two osteosarcoma cell lines. In general, the cell lines showed a perinuclear localization of the MoAbs and, in addition, WCFB25.3 exhibited binding to the cell surface membrane (Fig. 1). The malignant fibrohistiocytoma (MFH) cell line URHCL-1 failed to react with all three MoAbs. The synovial sarcoma reacted with WCFB25.1 and WCFB25.3, but not with WCFB25.2. The rhabdomyosarcoma, neuroblastoma, and melanoma cell lines did not react with any of the three MoAbs. The 4444F fibroblast cell line was negative for all three MoAbs except that when the culture reached confluence, WCFB25.3 became positive. A summary of the reactivity of the three MoAbs with normal, human tissues is presented in Table 2. Antibody WCFB25.2 reacted only with the muscularis of arteries and arterioles observed in a variety of organs, while no reactivity was seen with other tissues. Both WCFB25.1 and WCFB25.3 reacted with the connective tissues present in kidney, liver, uterus, skin, and tonsil. WCFB25.1 exhibited a more limited staining pattern than WCFB25.3, but both recognize antigens in the extracellular matrix. Basement membrane staining in the hepatic sinusoids and connective tissue staining in the portal tracts are illustrated in Fig. 2. Blood cells, skeletal muscle cells, and cells from epithelial or endothelial origin were negative in all organs tested. Analysis of surgical specimens of various sarcomas in a frozen section by indirect immunoperoxidase staining is presented in Table 3. MoAbs WCFB25.1 and WCFB25.3 bound to all the osteosarcomas; however. the localization was

FIG. 1. lmmunoperoxidase stains of the osteosarcoma cell line 4444T showing localization of antigens detected by monoclonal antibodies WCFB25.1 (A), WCFB25.2 (B), WCFB25.3 (C), and negative control (D).

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I-Continued

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BLOMMAERT,

CASSANO, TABLE

ANTIGEN

EXPRESSION

AND CROKER

2

ON NORMAL

HUMAN

Trssus.s

Antibodies Tissue Vascular lining Sinusoids Endothelium Smooth muscle Muscularis arteriosus Myometrium Skeletal muscle Cardiac muscle Connective tissue Subbasement membrane Tendon Vascular adventitia Endomysium Mesangium Epithelium Blood cells

Organ

WCFB25.1

WCFB25.2

Liver Skin, kidney, uterus

+ -

-

Skin, liver. uterus, kidney Uterus Muscle Heart

+ + -

Skin, tonsil Extremity Skin, kidney, heart, extremity Heart, extremity Kidney Kidney, skin, liver, uterus Peripheral blood mononuclear cells

+ -

WCFB25.3 i

+ -

+ -

-

+ +

+ +

-

+ f +

-

-

-

-

-

-

primarily to the extracellular matrix of the tumor (Fig. 3). Only in one case was there reactivity with the tumor cells themselves. Antibody WCFB25.2 demonstrated tumor cell-associated binding in 4 of 11 osteosarcomas, 3 of 6 chondrosarcomas, 2 of 2 giant cell tumors (GCT), and in the fibrosarcoma. In all the surgical specimens, antibody WCFB25.2 also bound to the muscularis of the blood vessels as was described above for normal tissues. The GCT displayed cell-associated reactivity for all three MoAbs. In the other tumors studied, WCFB25.1 and WCFB25.3 again localized to the extracellular matrix, although some chondrosarcomas and the rhabdomyosarcoma were entirely negative for WCFB25.1. Fresh colon carcinoma specimens were negative in the five samples studied. DISCUSSION

We have generated three new murine monoclonal antibodies to the osteosarcoma cell line 4444T which are useful in identifying and classifying sarcomas. Cell lines and tumors of endothelial and epithelial origin do not react with the MoAbs. Although the osteosarcoma cell line 791T failed to react with any of the MoAbs, this may be the result of a spontaneous loss of the antigens from this clone of the cell line as has been previously reported in melanoma cell lines (22). All three FIG. 2. lmmunoperoxidase stains of liver sections using monoclonal antibodies WCFB25.1 (A). WCFB25.2 (B), and WCFB25.3 (C).

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BLOMMAERT,

CASSANO, TABLE

ANTIGEN

EXPRESSION

AND CROKER

3

IN SURGICAL.

SEC-rloNs

TUMOR

Antibodies Tumor Osteosarcoma Cell associated Extracellular Giant cell tumor Cell associated Extracellular Chondrosarcoma Cell associated Extracellular Other sarcomas” Cell associated Extracellular

WCFB25.1

WCFB25.2

WCFB25.3

l/11” II/II

4/11 o/11

III I II/II

212 212

212 o/2

212 212

016

316 016

016 516

O/5

II5

415

o/5

o/5 515

216

(1 Number of positive tumors/number examined. b Ewing’s sarcoma, rhabdomyosarcoma, malignant histiocytoma, fibrosarcoma, and an undifferentiated sarcoma.

antibodies identify a subset of sarcoma cell lines. The carcinoma lines were uniformly negative. Cultures of the 4444T cell line at 70% confluence exhibited antigen expression in the cell cytoplasm, but if the cells were grown to full confluence, the localization of WCFB25.1 and WCFB25.3 became mainly extracellular while the cytoplasmic expression became less prominent. This suggests that WCFB25.1 and WCFB25.3 recognize antigens which are secreted from the cell. The WCFB25.2 antigen exhibited a strictly intracellular distribution pattern. Antibody WCFB25.3 was also positive on the 4444F cell line in confluence though negative when subconfluent. This reactivity is far weaker than seen in the tumor cell lines and histologic sections. The antigen distribution patterns of a variety of surgical tumor specimens complemented the results found from studying cell lines. Antibodies WCFB25.1 and WCFB25.3 recognized antigens in the tumors that were predominantly extracellular, although some cell-associated staining was noted. Use of WCFB25.1 resulted in a limited staining of the extracellular matrix while WCFB25.3 resulted in a more extensive staining of the extracellular matrix. One osteosarcoma exhibited some cell-associated binding of WCFB25.1 and WCFB25.3. Antibody WCFB25.2 showed cell-associated staining in a number of osteosarcomas, chondrosarcomas, the giant cell tumor, and a fibrosarcoma. The other tumors failed to react with WCFB25.2. Cells from epithelial and endothelial origin, blood cells, and muscular tissue do not react with these MoAbs. These MoAbs recognize antigens present or secreted from mesenchymal cells. Feit et al. (18) describe MoAbs which react with sarcomas and connective tissue elements. Although some of their MoAbs FIG. 3. Immunoperoxidase stains of tumor sections from a patient with osteosarcoma using monoclonal antibodies WCFB25.1 (A), WCFB25.2 (B), and WCFB25.3 (C).

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AND

CROKER

exhibit distribution patterns similar to our antibodies (23), their MoAbs react with Iibroblasts and ours do not (except for WCFB25.3 with confluent fibroblasts). The distribution of antibodies WCFB25.1 and WCFB25.3 indicate that they may be directed against major components of the extracellular matrix (24, 25). The major collagenous constituents of soft connective tissues are collagen types I and III (26). These collagens are found in most tissues that are also stained by antibodies WCFB25.1 and WCFB25.3. Tendon, which contains both collagen I and III, is positive for WCFB25.3. Endomysium of skeletal muscles which contain collagen types I, IV, and V was also positive for WCFB25.3. The final determination of whether WCFB25.3 recognizes one of the collagens requires further competitive binding studies with purified proteins. Antibody staining patterns of cartilage which contains mainly type II and some type I collagen are different for antibody WCFB25.3. Other major extracellular components found in connective tissue matrix and basement membranes such as fibronectin, laminin, and proteoglycans are also possible targets for our MoAbs (23, 24, 27). Cell migration, cell proliferation, and cell differentiation are influenced by the type of matrix with which cells come in contact (23). Matrix interaction with tumors is reciprocal (28). The extracellular matrix can induce differentiation of tumor cells (29, 30) and tumors can foster the maintenance of stroma or the growth of a fibrous capsule around the tumor. We have produced three different MoAbs which react with sarcomas and are different from previously described MoAbs generated against osteosarcomas (3 l33). Our new antibodies are useful in the detection and classification of neoplasms of mesenchymal origin as well as in the study of extracellular matrix proteins. These antibodies may identify new components of the extracellular matrix and may reveal an interesting role for the extracellular matrix in tumor growth and other pathological disorders (19, 20). Further studies to characterize the antigens detected by antibodies WCFB25.1, WCFB25.2, and WCFB25.3 are in progress. REFERENCES I. Morton, D. L., and Malingren, R. A., Human osteosarcomas: Immunological evidence suggesting an associated infectious agent. Science 162, 1279-1281, 1968. 2. Wood, E. C., and Morton, D. L., Microcytotoxicity test detection in sarcoma patients of Ab cytotoxic to human sarcoma cells. Science 170, 1318-1320, 1970. 3. Kohler, G., and Milstein, C., Continuous cultures of fused cells secreting antibody of predetined specificity. Nature 256, 495-497, 1975. 4. Reinherz, E. L., and Schlossman, S. F., Derivation of human T-cell leukemias. Cancer Res. 41, 47674770, 198 1. 5. Anderson, K. C., Bates, M. P., Slaughenhaupt, B. L.. Pinkus, G. S., Schlossman, S. F., and Nadler, L. M., Expression of human B-cell associated antigens on leukemias and lymphomas; a model of human B-cell differentiation. Blood 36, 1424-1433. 1984. 6. Termine, J. D., Robey, P. G., Fisher, L. W., Shimokawa, H., Drum, M. A., Cann, K. M., Hawkins, G. I., Cruz, J. B., and Thompson, K. G., Osteonectin. bone proteoglycan and phosphophorin defects in a form of osteogenesis imperfecta. Proc. Natl. Acad. Sci. USA 81, 2213-2217, 1984. 7. Hauschka, P. V., Frenkle, J., ReMuth, R., and Gundberg, C. A., Presence of osteocalcin and related higher molecular weight 4-carboxyglutamic acid containing proteins in developing bone. J. Biol. Chem. 258, 176-183, 1983.

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8. Fisher, L. W., Whitson, S. W., Avioli, L. V., and Termine, J. D., Matrix sialoprotein of developing bone. J. Biol. Chem. 258, 6588-6594, 1983. 9. Fisher, L. W., Termine, J. D., Dejter, S. W., Jr., Whitson, S. W., Yanagishita, M., Kimura, J. H., Hascall, V. C., Kleinman, H. K., Hassell, J. R., and Nilsson, B., ProteogIycans of developing bone. J. Biol. Chem. 258, 6588-6594, 1983. 10. Embleton, M. F., Gunn, B., Byers, V. S., and Baldwin, R. W., Antitumor reactions of MoAbs against a human osteogenic sarcoma cell line. Br. J. Cancer 43, 582-587, 1981. 11. Seeger, R. C., Rosenblatt, H. M., Imai, K., and Ferrone, S., Common antigenic determinants on human melanoma, glioma, neuroblastoma and sarcoma cells defined by MoAbs. Cancer Res. 41, 2714-2717, 1981. 12. Brown, J. M., Detection of a human sarcoma associated antigen with MoAbs. Cancer Res. 43, 2113-2120, 1983. 13. Campbell, D. Cl., Price, M. R., and Baldwin, R. W., Analysis of a human osteogenic sarcoma antigen and its expression on various human tumor cell lines. Inr. J. Cancer 34, 31-37, 1984. 14. Heiner. J. P., Miraldi, F., Kallick, S., Naaly, J., Smith-mensah, W. H., and Cheung, N. V., Localization of GD2 specific MoAb 3F8 in human osteosarcoma. Cancer Res. 47, 5377-5381, 1987. 15. Hosoi, S., Nakamura, T., Higashi, S., Yamamuro, T., Toyama, S., Shimomiyo, K., and Mikawa, H.. Detection of human osteosarcoma associated antigen(s) by MoAbs. Cancer Res. 42, 654-659, 1982. 16. Nakamura, T., Gross, M., Yamamuro, T., and Liao, S. K., Identification of a human osteosarcoma associated glycoprotein with MoAbs: Relationship with alkaline phosphatase. Biochem. Cell. Biol. 65, 1091-l 100, 1987. 17. Tanaka, C.. Yamamuro, T., Masuda, T., Tanaka, H., Matsumoto, M., Kataura, Y., Tsuji, T., Toyuchida, J., Miyama-Inaba, M., and Ueda, M., Recognition of serum alkaline phosphatase; murine MoAb against human osteosarcoma cells. Cancer Res. 46, 4853-4857, 1986. 18. Feit, C., Bartal, A. H., Fass, B., Buskin, Y., Cardo, C. C., and Hirshaut, Y., MoAbs to human sarcoma and connective tissue differentiation antigens. Cancer Res. 44, 5752-5756, 1984. 19. Bartal, A. H., Stahl, S., Karev, A., and Lichtig, C., Dupuytren’s contracture studied with MoAbs to connective tissue differentiation antigens. Cfin. Exp. Zmmunol. 68, 45743, 1987. 20. Bartal, A. H., Lichtig, C., Friedman-Bimbaum, R., Anaham, Z., Spivak, N., Fass, B., Feit, C., Robinson, E., and Hirshaut, Y., The interaction of Kaposi’s sarcoma with MoAbs to human sarcoma and connective tissue elements. Cancer 56, 1071-1074, 1985. 21. Cassano, W. F., Murine monoclonal anti-avidin Abs enhance the sensitivity of avidin-biotin immunoassays and immunohistologic staining. J. Zmmunol. Methods 117, 169-174, 1989. 22. Yeh, K. M., Hellstrom, I., and Hellstrom, K. E., Clonal variation in expression of a human melanoma antigen by a MoAb. J. Zmmunol. 126(4), 1312-1317, 1981. 23. Bartal, A. H., Feit, C., Cordon, C., Lichtig, C., Fass. B., Bushkin, Y., Robinson, E.. and Hirshaut, Y., Monoclonal antibodies to human sarcoma and connective tissue differentiation antigens. In “Immunity to Cancer” (A. E. Reif and M. S. Mitchel, Eds.), Academic Press, London, 1985. 24. Vaheri. A., and Alitalo, K., Pericellular matrix glycoproteins in cell differentiation and in mahgnant transformation. In “Cellular Controls in Differentiation” (C. W. Lloyd and D. A. Rees. Eds.), pp. 29-58, Academic Press, London, 1981. 25. Ishimura, E. Sterzel, R. B., Budde, K., and Kashgarian, M., Formation of extracellular matrix by cultured rat mesangial ceils. Am J. Pathol. 134, 843-855, 1989. 26. Furthmayr, H., and von der Mark, K., The use of antibodies to connective tissue proteins in studies on their localization in tissues. In “Immunochemistry of the Extracellular Matrix” (H. Furthmayr, Ed.), Vol. II, pp. 90-117. CRC Press, Boca Raton, FL, 1982. 27. Ruoslahti, E.. Structure and biology of proteoglycans. Annu. Rev. Cell. Biol. 4, 24&244, 1988. 28. Bennett, D. C., The cellular basis of carcinogenesis. In “Biology of Carcinogenesis” (M. J. Waring and B. A. Ponder, Eds.), pp. 65-81, MTP Press Limited, Lancaster, England, 1987. 29. DeCosse, J. J.. Gossens, C. L., Kuzma, J. I., and Unsworth, B. R.. Breast cancer: Induction of differentiation by embryonic tissue. Science 181, 1057-1058. 1973.

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30. Terranova, V. P.. Williams, J. E., Liotta, L. A., and Martin, Ci. R., Modulation of the metastatic activity of melanoma cells by laminin and fibronectin. Science 226, 982-985, 1984. 31. Tsang, K. Y.. Warren, R. O., Bishop, L., Pathak, S.. Koger, B., and LaVia, M. F., MoAbs to human osteosarcoma associated antigen(s). J. Narl. Cancer Inut. 77, 1175-l 180, 1986. 32. Wadw. T., Uede, T., Ishii, S.. Matsayama, K.. Yamawaki, S., and Kikuchi. K., MoAbs that detect different antigenic determinants of the same human osteosarcoma-associated antigen. Guncer Res. 48, 2273-2279, 1988. 33. Bruland, 0. S., Fodstad, 0.. Stenwig, A. E., and Pihl, A.. Expression and characterization of a novel human osteosarcoma-associated cell surface antigen. Cancer Res. 48, 5302-5309. 1988. Received November 30, 1990; accepted with revision May 13, 1991

Mesenchymal specificity of three new monoclonal antibodies generated to the osteosarcoma cell line 4444T.

We have generated three murine monoclonal antibodies to the new human osteosarcoma cell line 4444T. Analysis of their binding patterns to tumor cell l...
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