[CANCER RESEARCH 38. 199-203. January 1978] Anti-Squamous Tumor Antibodies in Patients with Squamous Cell Carcinoma1 Howard Sofen and Carol O'Toole2 Department of Microbiology and Immunology-lmmunobiology Group ¡H.S.¡and Department ol Surgery-Urology [C. O'T.j, UCLASchool of Medicine, Los Angeles, California 90024 ABSTRACT Patients with advanced squamous cell carcinomas were shown to have serum antibodies directed towards cul tured squamous tumor cells as shown by quantitative membrane immunofluorescence. The sera of these same patients did not react with a variety of other cultured tumor cells. Serum obtained from normals or from patients with other forms of cancer (transitional cell carcinoma, adenocarcinoma, and melanoma) did not give positive reactions. When the sera of squamous carcinoma patients were chromatographed on Sephadex G-150, tumor-reac tive antibodies were recovered solely in the 19 S fraction, suggesting ¡mmunoglobulin M as the ¡mmunoglobulin isotype involved. Identification of the squamous tumor cell-reactive immunoglobulin as ¡mmunoglobulin M was confirmed by quantitative immunofluorescence with the use of class monospecific antisera to human immuno- globulins. INTRODUCTION The presence of antibodies against human tumors has been established for a variety of histological types of neo plasms (6, 9, 13). However, the role of these antibodies remains controversial. Patients with squamous cell carci nomas of the head and neck have been reported to possess serum antibodies to autologous tumor cells (17). Antibodies reacting with nonvirion antigens of Herpeswrus-infected cells have also been detected in the sera of patients with squamous cell carcinomas of various anatomical sites (8). Some insight into possible roles of the putative antitumor antibodies may be gained from the isotypes (classes) of antibody involved, inasmuch as the different immunoglobu lin isotypes have been shown to have different in vivo functions. With the use of a quantitative immunofluorescent technique for the measurement of membrane immunofluo rescence. we demonstrated that sera from patients with squamous cell carcinomas derived from different anatomi cal locations contain antibodies that react with cultured autologous and allogeneic tumor cells derived from squa mous cell carcinomas. Furthermore, we have characterized these antibodies as pentameric 19 S IgM. ' This work was supported by Grant CA16880 from the National Bladder Cancer Project and by the University of California Cancer Research Coordi nating Committee Grant 76LA34. 2 Present address: Department of Urology. University of Tennessee. Mem phis, Tenn. 38163. Received March 23. 1977; accepted October 17, 1977. MATERIALS AND METHODS Patients and Controls. Nine patients with diagnoses of squamous cell carcinoma were studied. Tumors were clas sified where possible according to the criteria for cancer staging of the American Joint Committee. Sixteen control donors were tested. Eight had neoplasms unrelated to squamous cell carcinomas, and 8 were healthy or had nonmalignant disease (urethritis or cystitis) (Table 1). Indicator Cells. Three established cell lines were used as reference material: COLO-16, derived from a squamous cell carcinoma of skin (11); SCaBER, derived from a squa mous cell carcinoma of the urinary bladder (16); and T24, originated from a transitional cell carcinoma of urinary bladder (5). SCaBER and COLO-16 cells were used between passages 10 and 20. Other cells tested included: MEL-1. originating from a metastasis of cutaneous melanoma (20); HCV-29, derived from nonmalignant bladder epithelium (J. Fogh, unpub lished observations, J82 and TCCSuP, derived from transi tional cell carcinoma (14); and primary cultures of normal skin fibroblasts and kidney epithelial cells. All cells were grown in monolayer tissue culture with Medium 199 with Hanks' salts containing 10% heat-inactivated FCS.' penicil lin, 100 ID/ml; streptomycin, 100 /¿g/ml; and glutamine, 0.3 mg/ml. All cultures were free of Mycoplasma; routine monitoring was performed as described by O'Toole et al. (16). Quantitative Membrane Immunofluorescence. Indicator cells were harvested by treatment with 0.02% EDTA-0.05% trypsin. The cells were washed twice in Medium 199 con taining 10% FCS and incubated for 3 hr at 37° in humidified air + 5% CO2. Cells were then aliquoted into Fisher centri fuge tubes (2.5 x 10"' cells/tube), and 25 /J of test serum or phosphate-buffered saline, pH 7.2, were added. After a 30- min incubation on ice, the cells were washed twice in PBS + A. Fluorescein-conjugated polyvalent goat anti-human immunoglobulin antiserum (Meloy No. 520) diluted 1:20 was reacted with the cells for 30 min at 4°.For removal of aggregates, goat antisera had been ultracentrifuged at 45,000 x g for 90 min, stored at -70°, and centrifuged at 10,000 x g for 30 min just prior to use. After 2 more washes in PBS + A, the cells were fixed in 4% formal dehyde for 20 min (4) and subsequently were washed twice more in PBS + A before microscopic examination. Fluorescence was quantitated as described by Neri ef al. (15) with the use of a Leitz Orthopan microscope with a vertical illuminator. Microfluorometric measurements were • The abbreviations used are: FCS. fetal calf serum; PBS + A, phosphate- buffered saline containing 0.1% sodium azide. JANUARY 1978 199 Research. on December 9, 2021. © 1978 American Association for Cancer cancerres.aacrjournals.org Downloaded from