High Chemotactic Motility and Growth in Hard Agar of a Variant of RSV-Transformed Fibroblasts are Lost in Late Passages

Cloning efficiency in hard agar (0.6%) and high chemotactic migration toward fibroblast conditioned medium have been shown to characterize metastatic tumor cells. We studied growth in 0.6% agar and chemotaxis of two lines of Rous Sarcoma virus-transformed Balb/ c3T3 cells, B77/3T3 (low metastatic) and AA12 (high metastatic), and compared them to their non-transformed counterpart, in order to verify whether these properties were maintained during several subcultivations. Cells were cryopreserved at early passages and thawed for experiments. Both assays were performed on freshly thawed cells (4-6 weeks in culture) and on cells which had been cultured 15-20 weeks after thawing. B77/3T3, which are tumorigenic but low metastatic and which have a very low cloning efficiency in hard agar (0.1-1%), showed a chemotactic response to Balb/c3T3 conditioned medium about two-fold higher than control Balb/c3T3. This response did not change with time in culture. AA12 cells, a genetic unstable variant of B77/3T3 selected for its growth in hard agar (0.6%), had a high cloning efficiency in hard agar and showed a high chemotactic motility (three-fold the controls). High growth in 0.6 % agar and high chemotaxis of AA12 were lost in late passages, where cells behaved as the controls. It seems that besides the already reported variation in anchorage-independent growth, genetically unstable tumor cells can also have important variations in chemotactic motility during subcultivations.