A New in Vitro Model for Investigation of Tumor Cell-Platelet-Endothelial Cell Interactions and Concomitant Eicosanoid Biosynthesis

We have developed a new in vitro model system to examine tumor cell-platelet-endothelial cell interactions under dynamic conditions. Using the same model, we can determine endogenous eicosanoid metabolism and alterations in the prostacyclin-thromboxane A₂balance associated with interactions among tumor cells, platelets, and endothelial cells. The model consisted of cloned rat aortic endothelial cells grown on gelatin microcarrier beads under dynamic conditions (i.e., spinner culture). Interactions of these endothelial cells with platelets (heparinized rat platelet rich plasma) and/or tumor cells (rat Walker 256 carcinosarcoma) were assessed in an aggregometer. Gelatin beads alone or microcarrier grown endothelial cells did not elicit spontaneous aggregation of platelet rich plasma over a time period of 30 min. Microcarrier grown endothelial cells inhibited tumor cell induced platelet aggregation in a dose dependent fashion (i.e., depending on endothelial cell number). The ability of microcarrier grown endothelial cells to inhibit tumor cell induced platelet aggregation depended on endogenous production of prostacyclin. This conclusion is based on the following results: (a) an increased number of microcarrier grown endothelial cells caused a prolongation of the aggregation lag time; (b) an increased number of microcarrier grown endothelial cells caused a proportionate increase in 6-keto-prostagland in F_1αconcentration; (c) an increased number of microcarrier grown endothelial cells was inversely correlated with thromboxane A₂production by platelets; (d) indomethacin pretreatment of microcarrier grown endothelial cells caused a decrease in prostacyclin production and therefore overcame the associated inhibition of tumor cell induced platelet aggregation; and (e) the inhibition of tumor cell induced platelet aggregation in the presence of endogenous prostacyclin produced by microcarrier grown endothelial cells was the same as that observed in the presence of exogenous prostacyclin. Scanning electron microscopy of aggregometry samples revealed: (a) little or no platelet or tumor cell adhesion to gelatin beads alone, (b) a low basal adhesion of tumor cells to microcarrier grown endothelial cells, and (c) large aggregates of platelets and tumor cells located primarily at gaps in the monolayer of indomethacin treated microcarrier grown endothelial cells. This new in vitro model provides a method for examining the effects of eicosanoid metabolism by endothelial cells on tumor cell-platelet-endothelial cell interactions under dynamic conditions.