Five continuous cell lines have been established from 29 ocular melanomas and maintained for periods ranging from 3 to 9 years in medium identical to that in which 3 concomitantly studied lines of cutaneous melanoma cells were cultured as controls. The long-term problems to be overcome in establishing uveal cell lines are related to cell-doubling times which ranged from 72 to 432 hr, and plating efficiency, which ranged from 0.5%-6.5%. Tumors and cell lines were found to contain melanosomes. The morphology of uveal cells during the early subcultures exhibited multiple changes. Two different established cell lines were obtained from one ciliary-body tumor. Biochemical studies revealed markers of melanogenesis and neuroendocrine compounds. Cytogenetic studies revealed chromosomal abnormalities that differed between uveal and conjunctival melanomas.

The effects of variations in the concentrations of L-cystine (Cys), L-methionine (Met), and L-glutamine (Glu) on the establishment of melanocyte cell lines obtained from a primary tumor and its metastasis in the same patient were studied. The special role of Glu was also studied in 4 lymph node metastases from other patients. Differentiation in vitro was dependent on the culture conditions, as assessed by morphologic and biochemical studies. Karyologic expression, doubling time, cloning efficiency, and tumorigenicity in nude BALB/c mice varied widely among the cell lines. Cys was an indispensable amino acid and Glu was not. Met and Glu were implicated in melanogenesis. From these observations arose the question of the accuracy of comparative results, concerning differentiation and tumorigenicity, that had been collected for cell lines obtained under different culture conditions.

Variants of the B16 melanoma exhibiting markedly different tumorigenic and metastatic potential in Ay/a and a/a C57BL/6J syngeneic mice were investigated and compared, with respect to their relative growth and metastatic potential after culture under different conditions. Over 2 to 6 months of growth, in vitro cells demonstrated a rapid and significant decrease in their ability to form spontaneous lung colonies. Such a decrease depended on the conditions of culture. We suggest that the in vitro environment influences the phenotype of cells with respect to their capacity for spontaneous metastasis. This possibility must not be ignored, when conclusions obtained from studies of established cell lines are extended to cancers.

Dielectrophoretic experiments on pearl-chain formation and collection of human melanocytes were performed as a function of the frequency of the applied nonuniform electric field. Evidence is reported showing that the behavior of malignant melanocytes is markedly dependent upon the type of established cell line (pigmented or achromic), age, and drug treatment (e.g., chlorpromazine).

The purpose of this study was to examine the differentiation of variant tumors of the B16 metastatic melanoma when tumors were grown serially under different culture conditions and transplanted into C57BL/6J black mice, lethal yellow Ay/a, albino c/c, and C+/c mutant mice. Morphological and biochemical markers of melanogenesis were examined in cells in culture and in the corresponding tumors. Cellular pigmentation was assessed in terms of the levels of DOPA and 5-S-CD and in terms of tyrosinase activity in the various cell lines and tumors. The observed change from high to low metastatic capacity, which was dependent on culture conditions, appeared to be unrelated to melanogenesis even though changes were observed in the biochemical melanotic phenotype. Overall, tumor cells from spontaneous pulmonary metastases appear to differentiate in ways that are unrelated to the instability of experimental metastatic capacity. The melanotic phenotype in albino c/c and C+/c mice was dependent on the phenotype of the parental tumors. A marked difference was observed between two pigmentation compartments, one of which was stable in the B16 control, while the other was unstable in YB16 and MB16 variant cells and in the tumors derived from them. It appears, therefore, that the metastatic capacity of B16 metastatic variants is changeable and is independent of the unstable melanogenic behavior. The production of metastases and the differentiation of tumors in the present experiments appeared to be related to the genetic background of the mice and the epigenetic metabolic environment of tumors and cells.

No abstract

A system of tumor transplantation has been developed to select metastatic variants of B16 in mutants of the C57BL/6J black strain of mice. The effects of transplantation into nonagouti a/a and mutant recipients on the production of melanin and on the metastatic potential of tumors were investigated. Transplantation of the pigmented B16 melanoma from a nonagouti black a/a host to a yellow mutant Ay/a recipient resulted in an achromic and metastatic variant melanoma, designated YB16. The amelanotic phenotype occurred consistently after more than ten passages through yellow mice and simultaneously with an increase in the incidence of pulmonary metastases. When YB16 was transplanted back to the nonagouti black a/a host, a second variant, MB16, characterized by its variable pigmentation, was obtained. Pigmented and/or entirely achromic tumors were observed. MB16 was dramatically more metastatic than B16 and YB16 when injected s.c. or i.v. Metastases in the lungs were pigmented and/or achromic. The properties of tumor cells derived from artificially induced metastases were investigated after s.c. and i.v. injections. Whereas the metastatic cells expressed a potent ability to generate metastases when injected s.c., no differences in the incidence of metastases, as compared to the metastatic potential of cells of parental origin, were observed after i.v. injection. In the MB16 variant, there appeared to be an inverse relationship between differentiation (production of melanins) and malignancy. Our results demonstrate that differentiation and metastatic behaviour are dependent on specific mutations in the host environment which generate a pool of tumor cells from which highly metastatic variants can be selected.

The concept of cell adhesiveness was analyzed by looking for correlations between the adhesive behavior and measurable biological properties of different cell populations. Ten established lines of melanoma cells were assayed for passive deformability (by micropipet aspiration), active spreading (by measuring the height/diameter ratio after incubation on different surfaces), density and mobility of concanavalin A binding sites (by quantitative analysis of fluorescence microscopic images), spontaneous and concanavalin A-mediated agglutination (by measuring the number of cell conjugates resisting calibrated shearing forces), and binding to glass capillary tubes (with a quantitative assay of binding strength). Forty-four different parameters were thus measured, and each set of determinations was repeated 2 or 3 t at different days on each cell line. Analysis of variance was performed to assess the capacity of each parameter to discriminate between different lines. Correlations between different parameters were studied in order to understand a possible influence of cell intrinsic properties on the behavior of individual cells. The following conclusions were suggested by experimental data 1. Cell spreading ability, resistance to slow deformation within a micropipette and ability to form shear-resistant bonds, are independent properties. It is therefore suggested that different mechanisms rule the cell deformations on time scales of several minutes, tens of seconds, and fractions of a second. 2. Cell spreading ability may effectively influence binding strength only when adhesive stimuli are low, since in this case, cell stiffness is likely to impair the formation of extensive contact areas. 3. Individual cells may display marked heterogeneity within a given population, that emphasizes the danger of using averaged parameters to predict rare events (such as metastasis formation). 4. The most useful parameters to discriminate between different cell lines were, spreading ability and shear-resistant lectin agglutination, and substrate adhesion. It is concluded that cell adhesion is influenced by several measurable cellular properties that may display independent variations. The importance of a given parameter depends on the conditions of bond formation and rupture.