Journal of Leukocyte Biology 45:322-328 (1989) © 1989 Alan R. Liss, Inc. Formalin-Fixed Macrophages Bind Tumor Targets Similarly to Viable Macrophages Stephen Keith Chapes Division of Biology, Kansas State University, Manhattan Macrophages (MPs) fixed with 1% formalin in PBS bound targets similarly to viable MP5. Like binding between viable MP5 and tumor cells, the process was temperature and calcium dependent. Fixed MP5 discriminated targets similarly to viable MP5. Targets not bound by viable MPs were not bound by fixed MPs. The lectin Bandeiraea simpllclfolla (ASI-B4) was able to enhance MP binding to tumor cells regardless of whether MP5 were fixed or viable. However, it did not appear that ASI-B4-llke molecules were involved in the direct recognition of F5b tumor cells by MP5. Target cells could not be fixed in 1% formalin for binding to occur. These data suggest that the receptor on the MP for tumor cell binding is functional in the absence of active physiological processes. In contrast, tumor cell processes that are dependent upon target cell viability are required for binding. Key words: macrophage-tumor cell binding, tumor cell recognition, formalin fixation INTRODUCTION The ability of activated macrophages (MPs) to kill tumor cells but not normal cells is often cited as evidence of their role in neoplastic surveillance [13,20]. In some instances killing by MPs is mediated by a soluble product, tumor necrosis factor-alpha (TNF) [8,22,26,28]. How- ever, many tumor cells are resistant to TNF yet they are killed by activated MPs. This process appears to be de- pendent upon recognition and binding of MPs to tumor cells [1 ,6]. The molecular recognition of tumor cells by MPs is poorly understood. Our laboratory is attempting to define the molecular interactions that occur between cells and activated MPs during contact-dependent killing. We previously described an SV4O-transformed tumor cell line, F5b, that is susceptible to killing by activated MPs [4-6]. Killing is not mediated by soluble products, such as TNF, and appears to be dependent on contact [6]. During the course of these studies, several experimental protocols were attempted to determine how tumor cell binding is affected by various treatments of the MP membrane. One was to determine whether fixing MPs with 1% formalin would affect MP binding to tumor cells. This report presents data to demonstrate that MPs fixed with 1% formalin are able to bind tumor cells comparably to viable MPs. MATERIALS AND METHODS Animals C3H.OL mice were bred and maintained in our animal colony in the Division of Biology at Kansas State University. They were used as the source of MPs. Cell Cultures SV4O-transformed cells-E8b10, F5b, and F5m-and their susceptibilities to MP-mediated cytotoxicity have been described previously [4-6]. VERO cells were obtained from the American Type Culture Collection. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 2% fetal bovine serum (FBS; Hazelton Research Products, Lenexa, KS), 10% L-glutamine, and 10% Opti-MEM (GIBCO, Grand Island, NY). No antibiotics or antimycoties were used in the continuous culture of these cells. Macrophage Cytotoxicity Assay Macrophage tumoricidal activity was measured by a 16-18-h 51Cr release assay. We have described this technique previously [5]. Macrophages were activated in vivo with 700 jig Propionibacrerium acnes (Corynebac- terium parvum, CN6 134; Wellcome Biotechnology Ltd., Research Triangle Park, NC). Macrophage-Tumor Cell Binding Assays Assessment of tumor cell binding was done using a modification of the inverted mierotiter plate- centrifugation procedure described by Somerset al. [25], which we have described elsewhere [6]. Briefly, P. acnes-aetivated MPs were seeded into 96-well polyvinyl Received July 15, 1988; accepted October 24, 1988. Reprint requests: Stephen Keith Chapes. Division of Biology, Kansas State University. Manhattan, KS 66506.