Tumor-infiltrating macrophages influence the glycosphingolipid composition of murine brain tumors.

A procedure was developed to analyze glycosphingolipids (GSLs) in tumor-infiltrating macrophages (TIMs). The procedure entailed dissociating tumors into single cell suspensions with a concurrent metabolic labeling of GSLs using [14C]galactose. TIMs were then separated from tumor cells and other host cells by magnetic activated cell sorting and CD11b (Mac-1) microbeads. Gangliosides and neutral glycosphingolipids were analyzed in the TIM-enriched and TIM-depleted fractions in two different murine brain tumors (EPEN and CT-2A). The TIM gangliosides consisted of over 30 structures as assessed by two-dimensional high performance thin-layer chromatography. GSLs enriched in TIMs, relative to the tumors, included Gg4Cer (asialo GM1), GM1b, and GD1alpha. TIM GSLs were similar in EPEN and CT-2A despite their differences in growth and morphology. TIM GSLs were similar whether TIMs were isolated from tumors grown intracranially or subcutaneously. TIM GSLs were also similar to activated peritoneal macrophage GSLs, although differences in the ceramide structure were observed. Knowledge of TIM GSLs will be important in determining the function of these molecules in macrophage-tumor interactions. In addition, these methods will be helpful in determining the cellular origin of human brain tumor GSLs and in identifying tumor-associated GSLs for immunotherapy.