Effects of assay medium composition on macrophage-mediated tumor cell binding and lysis

Abstract

Activated macrophages express selective, high avidity tumor binding and cytotoxicity for tumor targets. The reported macrophage-mediated tumor binding and killing activities vary considerably among studies. Whereas, some of these studies utilized identical tumor target cells and similar sources of macrophages, the composition of the assay media used by different groups varied with respect to: type of medium (Eagle's minimal essential medium (EMEM), RPMI-1640, Dulbecco's modified Eagle's medium with 1.0 g/l glucose (DMEMLG), or with 4.5 g/l, glucose (DMEMHG)), concentration of serum (0–20%), and the addition of certain reagents (amphotericin B, pyruvate, and Hepes). The purpose of this study was to evaluate the effects of varying these parameters of medium composition on macrophage-mediated high (HA) and low avidity tumor binding and cytolysis. Tumor cytolysis was measured with an 18 h 51Cr release assay using peritoneal macrophages from C3H/HeN mice primed in vivo with pyran copolymer and further stimulated in vitro with lipopolysaccharide (10 ng/ml). P815 tumor cells were used as targets at a 10:1 effector: target ratio. Binding of targets to macrophages was termined by a method utilizing inverted centrifugation. Under optimal conditions, 2 × 10 5 macrophages bound as many as 17600 ± 3565 tumor cells and caused up to 50.4 ± 3.6% cytolysis. Assays performed in DMEMHG compared with the other three media tested resulted in HA tumor binding and cytolysis which were decreased by up to 42.5% ( P < 0.05) and 64.3% ( P < 0.01), respectively, compared with the three other types of medium. The addition of pyruvate (1 mM) to EMEM with 5% fetal calf serum (FCS) stimulated 98.8% ( P < 0.01) and 50.6% ( P < 0.1) increases in tumor binding and cytolysis, respectively compared with EMEM/5% FCS alone, while Hepes (25 mM) stimulated 58.3% ( P < 0.01) and 37.5% ( P < 0.1) increases in these activities. Amphotericin B (2.5 μg/ml) completely abrogated tumor cytolysis, but it caused no change in tumor binding. Serum produced variable effects on macrophage-mediated tumor killing. Five of six lots of FCS inhibited tumor lysis, by 16 to 98% (32.6 ± 28.6%; mean ± SD). However, the same lot of FCS which inhibited cytolysis by 98% enhanced HA binding by 152% ( P < 0.05). Finally, several commercially available serumless medium preparations supported macrophage-mediated tumor binding; however, none of the serumless media tested supported macrophage-mediated tumor cytolysis. We conclude that common differences in assay medium composition can markedly alter macrophage-mediated tumor cell binding and cytolysis.