Growth curves of LM38 sublines in a three-dimensional assay in liquid medium. Cell suspensions (2 × 105 cells/ml) in complete growth medium were seeded on top of an agar layer. At different time points spheroids were collected and trypsin-treated; cells were then counted. Whereas the LM38-HP cells proliferated at least up to the 10th day in this condition, the LM38-LP subline not only did not proliferate but began to die after the fourth day. The representative data shown correspond to one of two independent experiments. Standard deviations (not shown) were within ± 10% of the mean.