Detection of hypoxic cells in murine tumors using the comet assay: Comparison with a conventional radiobiological assay

The comet (single-cell electrophoresis) assay has been developed as a metho d for measuring DNA damage in single cells after irradiation. We have devel oped our own methods and image analysis system for the comet assay to ident ify hypoxic fractions. In vitro, we tested our system using a cultured tumo r cell line (SCCVII), In vivo, we compared the hypoxic fractions detected b y this assay with those determined by the in vivo-in vitro clonogenic assay using two rodent tumors (SCCVII/C3H, EMT6/KU/balb/c), which exhibit differ ent types of hypoxia: acute and chronic. In vitro, our method could differe ntiate hypoxic cells from oxic cells, using the parameter of tail moment. I n vivo, there were good correlations between the hypoxic fractions determin ed by the comet assay and by the clonogenic assay, in SCCVII/C3H (r=0.85) a nd in EMT6/KU/balc/c (r=0.75) tumors. By comparison of the two methods in c hronically hypoxic and acutely hypoxic tumors, we further confirmed that th e comet assay is clinically useful for estimating hypoxic fractions of soli d tumors.