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 determined by a method utilizing inverted centrifugation. Under optimal conditions, 2 x 10(5) macrophages bound as many as 17,600 +/- 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 less than 0.05) and 64.3% (P less than 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 less than 0.01) and 50.6% (P less than 0.1) increases in
tumor binding and cytolysis, respectively compared with EMEM/5% FCS alone, while
Hepes (25 mM) stimulated 58.3% (P less than 0.01) and 37.5% (P less than 0.1) increases in these activities.
Amphotericin B (2.5 micrograms/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 less than 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.