3-DIMENSIONAL ENDOTHELIAL-TUMOR EPITHELIAL-CELL INTERACTIONS IN HUMANCERVICAL CANCERS

The put-pose of this study is to understand the multicellular interact ion between tumor epithelial (TEC) and human umbilical vein endothelia l cells (HUVEC). The development of in vitro systems in which to cocul ture these cells as multicellular aggregates is very critical. Cell li nes were established from cervical tumor cells (n = 6) and two from HU VEC (n = 2) and they were cultured as three-dimensional (3-D) multicel lular-cultures using Cytodex-3 microcarrier beads in the rotating wall vessel (RWV). After a 240-h incubation, TEC and HUVEC proliferated ex ponentially to 4.2 x 10(7) and 2.2 x 10(7) cells/ml, respectively, wit hout requiring a feeder layer; in contrast to the two-dimensional (2-D ) cultures that average about 8 x 10(6) cells/ml. Phase contrast micro scopy indicated formation of 3-D aggregates that varied in size from 0 .5 to 5 mm. The size of the aggregates (1-5 mm, 6-14 microcarriers) in creased over time; however, the number of aggregates (0.5-1 mm, 2-5 mi crocarriers) decreased over a long-term incubation (240 h) because the cells merged to form large clumps. Maximum aggregation was observed w ith TEC at 120 h and HUVEC at 96 h. The culture of TEC in the absence of HUVEC produced minimal differentiation in contrast to cocultures. T he TEC and HUVEC as cocultures in RWV proliferated at an accelerated r ate (1.3 x 10(7) cells/ml, 96 h). The TEC-HUVEC coculture presented tu bular structures penetrating the tumor cell masses, forming aggregates larger in size than the monocultures and typically with greater cell mass and number. The cells were viable (trypan blue exclusion) and met abolically active (glucose utilization) until 240 h. These data sugges t that RWV provides a new model that allows us to investigate the regu latory factors that govern turner angiogenesis.