Growth Characteristics of Human Melanoma Multicellular Spheroids In Liquid‐Overlay Culture: Comparisons With the Parent Tumour Xenografts

Abstract

The growth characteristics of multicellular spheroids, derived from human melanoma xenografts and cultivated in liquid-overlay culture, were studied and compared with those of the parent tumours. Six of the seven melanomas investigated formed spheroids, which grew exponentially up to a volume of 1-2 x lo7 pm3 (a diameter of 270-340 pm) before the growth rate tapered off. The morphology of the spheroids varied considerably among the melanomas; some spheroids grew as densely packed, spherical structures of cells whereas others were loosely packed and showed an irregular shape. Central necrosis developed when the spheroids attained a diameter of 150-200 ,urn. The histological and cytological appearance of the spheroids was remarkably similar to that of the parent xenograft in five of the six cases. The sixth melanoma contained two subpopulations with distinctly different DNA content, one of which was predominant in the spheroids, the other in the tumours. This gave rise to clear histological and cytological differences. The volume-doubling time of the spheroids during the exponential growth phase ranged from 1.7 k 0.2 to 2.7 0.4 days and the fraction of cells in S from 13 & 1 to 28 & 2%. The volume-doubling time decreased with increasing fraction of cells in S, indicating that the differences in growth rate were due mainly to differences in the growth fraction or to differences in the duration of G,. The spheroid volume-doubling times did not correlate with those of the parent xenografts (T, = 4.2-22.5 days at V = 200 mm3), possibly because the cell loss factors of the xenografts were large and varied among the melanomas. The fractions of cells in G,/G, S and G, + M in the spheroids and the xenografts did not correlate either, but were found to be within the same narrow ranges in the spheroids and the xenografts4.e. 50-80% (G,/G,,), 10-30% (S) and 10-20% (G, + M).Multicellular spheroids are an in v i m tumour model system representing an intermediate level of complexity between monolayer cell cultures and solid tumours (Sutherland el al., 1970;Sutherland & Durand, 1976). Spheroids resemble tumours in that the cells grow in a three-dimensional structure which facilitates intercellular communication (Durand &

Growth and radiosensitivity of malignant melanoma multicellular spheroids initiated directly from surgical specimens of tumours in man

“…Yuhas & Li (1978) have studied the growth in liquid-overlay culture of spheroids initiated from seven murine solid tumours and concluded that the growth fraction was the major determinant of the volumetric growth rate. Previous studies in our laboratory of spheroids initiated from human melanoma xenografts have also shown that differences in volume-doubling time among different spheroid cultures are mainly a consequence of different growth fractions (Rofstad et al, 1986a). One possible explanation of the present observations may therefore be that the culture conditions in vitro through adequate nutrients stimulated cell proliferation, whereby the growth fraction as well as the fraction of clonogenic cells increased.…”

“…One possible explanation of the present observations may therefore be that the culture conditions in vitro through adequate nutrients stimulated cell proliferation, whereby the growth fraction as well as the fraction of clonogenic cells increased. However, there is evidence from studies of human melanoma xenografts that distinctly different stem-cell subpopulations may be predominant in tumours and in the corresponding spheroids (Rofstad et al, 1986a). Consequently, it cannot be excluded that the changes in plating efficiency and volumetric growth rate observed here were due to some stem-cell subpopulations being favoured by the growth conditions in vitro.…”

“…Spheroids initiated from different cell or tumour lines show individual and characteristic growth parameters, e.g. volume-doubling time, cell cycle distribution, cell density and intercellular adhesiveness (Carlsson et al, 1983;Rofstad et al, 1986a). Large spheroids have diffusion gradients for oxygen, glucose and other nutrients, resulting in necrotic areas, radiobiologically hypoxic cells and cells at acid pH (Sutherland & Durand, 1973;Acker 1984).…”

“…Generally, spheroids have been initiated from animal or human cell lines established as monolayer cultures Carlsson et al, 1983) or from disaggregated human tumour xenografts (Jones et al, 1982;Twentyman, 1983;West et al, 1984;Rofstad et al, 1986a). Recently, there has been some interest in growing spheroids directly from human tumour surgical specimens (Darling et al, 1983;Wibe et al, 1984).…”

ras-induced Up-regulation of CTP:Phosphocholine Cytidylyltransferase α Contributes to Malignant Transformation of Intestinal Epithelial Cells

“…Clonogenicity, Soft Agar, and Tumor Growth Assays-A clonogenicity assay was used to measure cell viability as a function of colony formation following anchorage-independent growth on SeaPlaque agarose-coated plates for up to 72 h (44). Briefly, IEC-18, ras3, ras4, and ras7 cells were trypsinized, and 500 cells were seeded onto 60-mm dishes coated with 2 ml of 1% (w/v) SeaPlaque agarose in MEM.…”