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Altered Expression in Squamous Carcinoma Cells of an Orientation Restricted Epithelial Antigen Detected by Monoclonal Antibody A9¹

Cancer Research Laboratory, Department of Otolaryngology/Head and Neck Surgery [T. E. C.] and the Department of Microbiology and Immunology [K. A. K., T. E. C.], University of Michigan School of Medicine, Ann Arbor, Michigan 48109

The monoclonal IgG_2a antibody A9 was raised to the UM-SCC-1 human squamous cell carcinoma cell line. Hemadsorption assays using monolayer cultures as target cells revealed a restricted range of A9 reactivity with human cell lines. The A9 antibody was reactive with 29 of 34 squamous cell carcinoma cell lines and with 5 epithelial cancer cell lines of non-squamous origin. In contrast, no antibody binding was detected with twelve malignant melanoma, two fibrosarcoma, two malignant lymphoid, two transitional cell carcinoma, or four adenocarcinoma cell lines. Similarly, normal lymphoid cells, RBC, and fibroblasts from multiple donors were negative. The relative expression of the A9 antigen varied greatly among positive squamous cancer lines such that the 50% endpoint titer of A9 ascites fluid ranged from less than 10¹ for low antigen expressor lines to 10⁶ for strong antigen expressors. Hemadsorption tests with secondary passage cultures of normal squamous cells failed to detect A9 binding to the cell surface. However, in experiments with intact colonies in primary cultures of normal squamous cells, antibody binding was observed at the periphery of individual colonies. Mechanical detachment of such colonies revealed A9 antigen bound to the plastic surface underneath the cells, but the newly exposed surface of the cells, like the upper surface, was negative. In contrast, when cells of similar cultures were detached by trypsinization prior to testing no antigen remained on the plastic, but approximately 30% of the trypsinized cells were positive. Similar experiments with the five surface-negative squamous cell carcinoma cell lines revealed that cryptic A9 antigen could also be detected underneath mechanically detached cells and on the surface of 100% of typsinized cells. Trypsinization of melanomas and fibroblasts did not reveal A9 antigen. Immunoperoxidase assays on frozen sections of normal epidermis localized A9 antigen to the basal cells and the basement membrane region. In frozen sections of squamous cancers, individual tumor cells and particularly the growing edge of the tumors were strongly stained by A9, while in basal cell cancers only very slight staining could be detected. Thus, in normal epithelial cells the A9 antigen appears to be expressed only by the stem cell subset. In squamous cancer cells, the expression of the A9 antigen is not only increased but appears to have escaped the orientation restriction characteristic of normal epithelial cells.

¹ Supported by Biomedical Research Support Grant NIH SO7 RR05383 and by USPHS Grants CA-28564 and CA-35929 from the National Cancer Institute.

² Predoctoral fellow of the Genetics Training Grant T32-GM07544 awarded by the NRSA. Present address: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, NIH, Building 10, Room 11N311, Bethesda, MD 20205.

³ Recipient of USPHS Research Career Development Award CA00621. To whom requests for reprints should be addressed, at Department of Otolaryngology/Head and Neck Surgery, University of Michigan School of Medicine, Box 028, Ann Arbor, MI 48109.

Received 9/30/85. Revised 3/20/86. Accepted 4/ 8/86.

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