The pattern of in vitro anchorage-independent growth of tumor cells from the murine UV-2237 fibrosarcoma correlated with their ability to produce experimental metastasis in vivo. When seeded into 0.3% Noble agar semisolid medium, cells of metastatic clones developed into larger tumor colonies at a faster rate than did cells of clones with low metastatic potential. Furthermore, when tumor cells were plated into 0.6% Noble agar, colony development by cells of low metastatic potential clones was almost completely restricted. Tumor cells from the heterogeneous parent UV-2237 fibrosarcoma were plated into dishes containing 0.6% agar semisolid medium. In separate experiments, 16 colonies were isolated 2 weeks thereafter and were established as individual cell lines in monolayer cultures. All of these cell lines produced experimental metastases as determined by in vivo lung colony assay. The data suggest that anchorage-independent growth of UV-2237 tumor cells in 0.6% Noble agar semisolid medium is selective and permits the isolation of metastatic subpopulations.

The metastatic stability of clones, which were derived from the murine UV-2237 fibrosarcoma and which exhibit low or high metastatic potential, was examined after 60-72 days of continuous growth in vitro and in vivo. Subclones of the high metastatic clone exhibited a 140-fold variation in the production ofexperimental pulmonary metastases after intravenous injection into syngeneic C3H-mice. In contrast, subelones from the low metastatic clone varied only slightly (8-fold). Using cloned cells from three mouse tumors with differing metastatic potential, we determined the spontaneous mutation rates of cells with low or high metastatic capacities with respect to the selective genetic markers, 6-thiopurine resistance or ouabain resistance, or both. In all cases, cells with high metastatic potential had a 3-to 7-fold increase in the rate of mutation (per cell generation) at both genetic loci, as compared with their low metastatic but tumorigenic cell controls. These results support the hypothesis that the evolution of tumors from the benign to the malignant state could be the consequence of acquired genetic instability in the neoplastic cells.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (IWGT), comprised of experts from Japan, Europe, and the United States, met on August 29, 2003, in Aberdeen, Scotland, United Kingdom. This meeting of the MLA Workgroup was devoted to reaching a consensus on the appropriate approach to data evaluation and on acceptance criteria for both the positive and negative/vehicle controls. The Workgroup reached consensus on the acceptance criteria for both the agar and microwell versions of the MLA. Recommendations include acceptable ranges for mutant frequency, cloning efficiency, and suspension growth of the negative/vehicle controls and on criteria to define an acceptable positive control response. The recommendation for the determination of a positive/negative test chemical response includes both the requirement that the response exceeds a defined value [the global evaluation factor (GEF)] and that there also be a positive dose-response (evaluated by an appropriate statistical method).

The activities of a colony-stimulating factor (CSF), which stimulates granulocyte-macrophage colony formation by mouse hemopoietic cells, and macrophage growth factor (MGF), which stimulates proliferation of activated peritoneal macrophages, have been demonstrated by various criteria to reside in the same molecular species. These criteria include occurrence in various sources and copurification of the activities in mouse L-cell-conditioned medium as well as the biological, physicochemical, and antigenic properties of the activities of L-cell-conditioned medium. CSF and MGF activities of L-cell-conditioned medium are ascribable to a glycoprotein of mol wt approximately 60,000 which migrates electrophoretically with alpha-globulin. Human urinary CSF, which also possesses MGF activity, has similar properties and can be neutralized by antiserum to highly purified L-cell medium CSF. A procedure is described for the partial purification of material from L-cell medium that has activity at 1 ng/ml in both MGF and CSF assays.