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Primary and secondary cultures of VX-2 carcinoma produced high levels of collagenase activity in both active and latent forms in serum-free media. These cultures appeared morphologically heterogeneous in phase-contrast microscopy and revealed the presence of mainly three distinct forms; epithelial-like cells (E cells), fibroblast-like cells (F cells), and large rounded-flat cells which may represent a subclass of the F cells. Cell separation techniques such as brief dispase treatment, Percoll gradient centrifugation, thimerosal treatment, and rabbit serum were used to obtain predominantly one form or the other. The E cells never formed a monolayer but rather grew as limited size clusters of intimately associated cells with large nuclei and often appeared multinucleated. These cells were difficult to maintain in culture or serially passed more than a few times. The F cells, rare in early cultures but having the highest growth potential, appeared in various morphological forms ranging from spindle- to stellate-shaped cells. The cells in their third passage were capable of producing palpable tumors, similar in light and electron microscopic studies to the original tumor from which they were derived, when injected intramuscularly into recipient rabbits and produced specific collagenase activity in active and latent forms in serum-free media. Ultrastructural studies suggested that the E cells were of epithelial origin whereas the F cells were similar to stromal fibroblasts. Cytogenetic studies demonstrated that almost all of the E cells showed both numerical and structural chromosomal changes in a modal number of 54 chromosomes. On the other hand, the major cell population of the F cells resembled normal rabbit fibroblasts; both contained a normal diploid (2 n = 44). However, few cells (4–6%) in the F-cell population were hyperdiploid with a modal chromosome number of 54. These cells may represent inadvertent contaminating E cells and account for the apparent limited turmorigenicity observed in early F-cell cultures. The data suggested that the E cells were of tumor origin whereas the majority of the F-cell population appeared to be of host origin. Furthermore, it is suggested that the E cells stimulate tumor-associated stromal cells to produce elevated levels of collagenolytic activity and contribute to collagen degradation during tumor invasion.

Fibroblast-like cells (F-cells) and epithelial-like (E-cells) derived from cultures of rabbit VX-2 carcinoma released collagenase in both active and latent forms in serum-free medium at a level higher than that of normal rabbit fibroblast cultures. The enhanced capacity of the F-cells to release the enzyme, however, continued only for a few passages and then decreased significantly to a low level similar to that of the normal fibroblasts. The enzyme-release by the E-cells continued for a few more passages at a relatively moderate level, higher than that of normal fibroblasts. The release of collagenase in cultures of F-cells was enhanced by the presence of E-cells in mixed cultures as well as by medium conditioned by the E-cells type. Addition of cytochalasin B at 2 micrograms/ml did not significantly effect the enzyme activity released in the cultures. Serum from tumor-bearing rabbits appeared to stimulate the release of enzyme activity in cultures of either cell type.