HUMAN TUMOR SPONTANEOUS METASTASIS IN IMMUNOSUPPRESSED NEWBORN RATS .2. MULTIPLE SELECTIONS OF HUMAN-MELANOMA METASTATIC CLONES AND VARIANTS

Using a recently developed model of human tumor spontaneous metastasis in immunosuppressed newborn rats, we selected variants with different metastatic abilities from the human melanoma cell line M4Be. We used 4 in vivo selection approaches, by direct serial tumor transplantations or by techniques involving in vitro reculture of the cells recovered from s.c. tumors or lung and lymph-node metastases. In addition, 3 series of clones were derived in vitro from the M4Be cell line, either by limiting-dilution or by cloning in semi-solid agar and harvesting small and large colonies. A considerable amount of heterogeneity in tumorigenicity and metastatic ability was demonstrated among variants and clones following in vivo selections and in vitro cloning. Four main malignant phenotypes were identified among those expressed by the selected cells: poorly tumorigenic and poorly metastatic; poorly tumorigenic and highly metastatic; highly tumorigenic and poorly metastatic; and highly tumorigenic and highly metastatic. However, while malignant phenotype (i.e., tumorigenicity and metastatic ability) did not appear to be grossly influenced by in vitro cloning procedure, it appeared greatly influenced both by the in vivo selection procedure (direct transplantations or use of in vitro culture between the in vivo passages) and by the origin of the cells under selection (s.c. tumor or metastases). Our study provided us with a large panel of variants and clones with varying metastatic abilities, which represent a model of human melanoma spontaneous metastasis allowing the study of critical determinants in human tumor metastasis.