Morphology and serum dependence of cloned cell lines undergoing spontaneous malignant transformation in culture.

A number of morphological changes were found to correlate with the occurrence of spontaneous neoplastic transformation in sublines of five rigidly isolated clones of mouse embryo fibroblasts. These morphological changes included increased cytoplasmic basophilia, reduced spreading of cells on the substrate, increased nuclear:cytoplasmic ratio, greater heterogeneity in the size and shape of cells and nuclei, and more random orientation of cells. Because these changes were reproducible, occurring in some sublines of all five clones, they have been described and illustrated to serve as a guide for identifying spontaneous transformants among rodent fibroblasts in culture. Neoplastic transformation was determined by the growth of the cells as malignant neoplasms in syngeneic hosts. The spontaneous transformants, as compared with nonneoplastic fibroblasts derived from the same cell, showed similar saturation densities and serum dependence. Some clones had a higher transformation frequency than the parental line, which remained nonneoplastic for years. Thus, the capacity for continuous growth in vitro can be independent of malignant potential. The use of horse serum as supplement to the medium did not accelerate or increase the frequency of neoplastic transformation.

Colony morphology and growth in agarose as tests for spontaneous neoplastic transformation in vitro.

Adherent fibroblast-like cells from paired lines, one non-neoplastic and the other "spontaneously" transformed neoplastic, were compared in simultaneous in vivo and in vitro assays. The in vivo assay was the i.m. implantation of 10(6) or 10(7) cells in irradiated syngeneic animals, and the two in vitro assays were the evaluation of colony morphology on plastic and the enumeration of colony growth in semisolid agarose. The percentage of colonies diagnosed from their morphology as neoplastic correlated with tumorigenicity as follows: 100% always indicated a tumorigenic cell population with tumor latent periods from 6 to 230 days and tumor incidence from 40 to 100%; 0% always indicated a nontumorigenic cell population; 1 to 32% indicated either a tumorigenic cell line with long tumor latent period (218 days) with 70% tumor incidence or a nontumorigenic cell line. Growth in agarose, as measured by colony number and size, correlated with tumorigenicity as follows: nontumorigenic cell lines produced no colonies; tumorigenic cell lines produced colonies, but not always larger than 0.1 mm in diameter. The number of size or colonies did not correlate with the tumor latent period or tumor incidence. Therefore, both in vitro tests were reliable qualitative assays of spontaneous neoplastic transformation, but they did not correlate directly with the tumor incidence or mean tumor latent period. The relative success of the agarose assay emphasizes the importance of decreased anchorage dependence for progressive growth of injected cells as a malignant neoplasm in vivo.

The cytology of spontaneous neoplastic transformation in culture.

Thirteen cell lines derived from embryos of the mouse, rat and hamster were sampled at closely spaced intervals for cytology and spontaneous neoplastic transformation. Transformation, determined by growth of the cells as malignant neoplasms in compatible hosts, occurred in eight lines and appeared to be unrelated to time in culture, passage number, proliferative activity of the cells or type of serum used to supplement the culture medium. In an effort to establish cytologic criteria for malignancy of rodent fibroblasts, cultures were examined for 19 cytologic abnormalities. The percentage of cells or colonies showing each of the abnormal properties was recorded. Five of the 19 were found to correlate with neoplastic transformation in all cell lines. These were cytoplasmic basophilia, reduced cytoplasmic spreading on substrate, cording, high nuclear: cytoplasmic ratio and clumping. Each of these properties is amenable to quantitation or computerized image analysis. On the basis of the five cytologic criteria, the percentage of colonies diagnosed neoplastic, borderline and non-neoplastic was determined for each line. Although these percentages varied from one transplant generation to another, the percentage of neoplastic colonies tended to increase with time in all lines which became malignant, and the borderline colonies tended to decrease, suggesting a progression from borderline to neoplastic state.

Serum-induced chromosome damage and neoplastic transformation of mouse cells in vitro.

In previous studies, mouse cells grown in medium supplemented with horse serum (HS) developed more chromosomal aberrations and underwent malignant transformation earlier than cells from the same pool grown with fetal bovine serum (FBS) supplement. In the present study cells derived from C3Hf/HeN mouse embryos were grown in medium NCTC-135 supplemented with various combinations of large- and small-molecule fractions of HS and FBS in an effort to determine the effective components. The results indicate that the large-molecule fraction of HS (mare or stallion) produces alterations in chromosome number and structure. HS is also shown to cause chromatid breaks and exchanges at or near the centromere in contrast to fluorescent-light-induced breaks and exchange at or near the centromere in contrast to fluorescent-light-induced breaks which occur randomly along the chromatid. However, efforts to control completely chromosome stability and malignant transformation through the use of large- and small-molecule fractions of HS and FBS or combinations thereof were unsuccessful. In connection with this study, diagnosis of malignant transformation in vitro was made by a direct sampling method based on cytologic criteria previously described and documented. With one exception, the diagnoses of 11 different cell lines were consistent with results of in vivo assays.