[CANCER RESEARCH 43, 669-678, February 1983] 0008-5472/83/0043-0000502.00 Immunohistochemical Localization of the Mouse Stage-specific Embryonic Antigen 1 in Human Tissues and Tumors I Niles Fox, 2 Ivan Damjanov, Barbara B. Knowles, and Davor Solter Department of Pathology and Laboratory Medicine, Hahnemann Medical College [I. D.] and The Wistar Institute lB. B. K., N. F., D. S.], Philadelphia, Pennsylvania 19104 ABSTRACT Normal human tissues and various human tumors were sur- veyed by immunohistochemical techniques for expression of the stage-specific embryonic antigen 1 (SSEA-1). The antibody reacted with many normal and neoplastic human tissues. In most instances, equivalent human and mouse tissues ex- pressed SSEA-1 ; however, different tissue localization patterns were sometimes seen between these two species. Most SSEA- 1-positive tumors originate from tissues that normally ex- pressed this antigen; however, some breast and ovarian tumors are SSEA-1 positive, and these organs are SSEA-1 negative. SSEA-l-positive tumors were composed of both immunoreac- tive and nonreactive tumor cells. These data show that SSEA- 1, initially defined as a mouse embryonic antigen, represents a heterogenetic antigen present in many normal human tissues. It is retained on many but not all neoplastic cells originating in these normal tissues and also appears on the surface of some tumor cells developing in SSEA-l-negative tissues. INTRODUCTION The SSEA-13 is a carbohydrate antigenic determinant de- fined by a monoclonal antibody produced by immunizing mice with murine embryonal carcinoma F9 cells (8, 9, 1 3). Although all mouse embryonal carcinoma cell lines react with this mon- oclonal antibody, we could not demonstrate SSEA-1 on any other mouse tumor cell lines. Although SSEA-1 is selectively expressed on mouse embryonic cells in a stage-specific man- ner, certain fetal and adult tissues also express this determinant (5-7). Nevertheless, despite widespread distribution of SSEA- 1 in normal murine tissues, the monoclonal antibody to SSEA- 1 injected i.v. into tumor-bearing mice can be localized to embryonal carcinoma (1). In a previous study (2), we have shown that the monoclonal antibody to SSEA-1 reacts with human yolk sac carcinoma as well as germ cells in human fetal (but not adult) testis. Because of the potential diagnostic or therapeutic value of the anti- SSEA-1 antibody in clinical medicine, it was essential to deter- mine whether this antibody reacts with other human tumors and normal human tissues. We report that antibody to SSEA-1 reacts with many human tissues and that tumors originating from immunoreactive tissues also express this antigenic deter- minant. Most tumors, however, show considerable heteroge- neity and are composed of both SSEA-1 -positive and -negative 1This investigation was supported by Grants CA-23097, GM-29040, CA- 18470, CA-10815, CA-27932, and HD-12487 from the NIH and Grant PCM- 8118801 from the National Science Foundation. 2 To whom requests for reprints should be addressed. 3 Abbreviations used are: SSEA-1, stage-specific embryonic antigen 1; PBS, phosphate-buffered saline; PMN, polymorphonuclear leukocytes. Received July 29, 1982; accepted November 2, 1982. cells. We also show that, in the breast and ovary, SSEA-1- positive tumors develop from the epithelial tissues that are normally unreactive with this monoclonal antibody. MATERIALS AND METHODS Tissue Samples and Tumors. The normal tissues listed in Table 1 were removed within five hr of death from 2 male and 2 female autopsy cases at the Hahnemann Medical College, Philadelphia, Pa. All speci- mens were immediately frozen in 2-methylbutane, precooled in liquid nitrogen, and sectioned immediately on a cryostat or stored at -70 ~ Fresh tumor samples were obtained from specimens surgically re- moved at Hahnemann and similarly processed. In addition, paraffin- embedded tumors from previous surgical cases were sectioned and tested for SSEA-1. Antiserum to SSEA-I. The anti-SSEA-1 monoclonal antibody was produced previously (13) by fusing the spleen cells of a mouse immu- nized with the nullipotent mouse embryonal carcinoma cell line F9 with cells of the murine myeloma cell line P3X63AgS. From this fusion, a cloned hybrid cell line was obtained which secreted monoclonal anti- bodies of the IgM isotype. This monoclonal antibody defined the SSEA- 1 antigenic determinant. Ascites fluid from mice injected i.p. with this antibody-secreting hybrid cell clone was used throughout this investi- gation. It was diluted 1:10 in PBS containing bovine serum albumin (1 mg/ml) and sodium azide (0.04 mg/ml). Ascites fluid (diluted 1:10 as above) from a mouse injected i.p. with the P3X63Ag8 myeloma line was used as the primary antibody on control sections. Immunohistology. Indirect immunofluorescence or immunoperoxi- dase tests were performed on cryostat sections which were briefly fixed in cold acetone (4 ~ for 10 min. Paraffin sections were first deparaffinized in xylene, cleared and rehydrated in graded alcohols, treated with trypsin (1:250) (M. A. Bioproducts, Walkersville, Md.) for 30 rain at 37 ~ and rinsed 3 times in PBS. The slides were incubated with either anti-SSEA-1 or control ascites for 1.5 hr at room tempera- ture in a humidified chamber. After rinsing in PBS (4~ either horse- radish peroxidase-conjugated or fluorescein-conjugated goat (IgG frac- tion) anti-mouse IgM (heavy chain specific) (Cappel Laboratories, Cochranville, Pa.) were similarly incubated on the slides for 1 hr. After a rinsing in PBS, the slides were either reacted with diaminobenzidine (5) for light microscopy or mounted with glycerine for fluorescent microscopy. RESULTS SSEA-1 in Normal Tissues The normal tissues tested for SSEA-1 are listed in Table 1. In each tissue where SSEA-1 was detected, reactivity was always limited to the epithelial components. Withthe exception of the central nervous system (see below), no reactivity was detected on the stromal or connective tissue elements in ~y tissue. Hemopoietic-Lymphoid Tissues. In peripheral blood smears FEBRUARY1983 669 Research. on July 21, 2021. © 1983 American Association for Cancer cancerres.aacrjournals.org Downloaded from