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

Abstract

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 < 1.037 g/ml to Fr 10 > 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 ≥ 79% of the cells expressed CA 125. Positivity for c-myc, c-myb and c-fos (≥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.