Separation of clonogenic and differentiated cell phenotypes of ovarian cancer cells (HOC-7) by discontinuous density gradient centrifugation.

We isolated clonogenic cells from differentiated HOC-7 ovarian cancer cells. Both cell subsets were characterised in respect to morphology, growth behaviour, DNA content and expression of tumour-associated antigens and nuclear oncogenes. Ten cell fractions (Fr) were separated by centrifugation in a discontinuous density gradient (Fr 1 less than 1.037 g/ml to Fr 10 greater than 1.069 g/ml, steps 0.004 g/ml). Large adenoid cells containing vacuoles filled with neutral polysaccharides were concentrated in Fr 1-4. These cells were non-clonogenic in soft agar. The growth on solid substrate was highest in Fr 6 and 7, intermediate in Fr 2-5 and Fr 8-10 and lowest in Fr 1. The mean cloning efficiencies of the fractions in soft agar were highest in Fr 6 (8.1%) and lowest in Fr 2 and 3 (0.1%). Diploid and near tetraploid cell subsets were found with similar frequency in all fractions. Immunocytochemistry revealed 4-7% Ki-67 positive cells in Fr 1-6 and 12-20% in Fr 7-10. In Fr 3-10 greater than or equal to 79% of the cells expressed CA 125. Positivity for c-myc, c-myb and c-fos (greater than or equal to 74%) was not correlated with clonogenicity. In conclusion, differentiated cells (Fr 1-4) were separated from cells with higher growth rates (Fr 5-10). Clonogenic cells were enriched in Fr 6. These data indicate that discontinuous density gradient fractionation represents a useful method for separation of cells with different degrees of differentiation, growth potential and clonogenicity.