Detection of hypoxic cells in a murine tumor with the use of the comet assay.
BACKGROUND
Hypoxic cells within solid tumors are likely to limit tumor curability by radiation therapy and some chemotherapeutic agents.


OBJECTIVE
To quantify a hypoxic fraction in solid tumors, we developed a method which measures radiation-induced DNA single-strand breaks in individual tumor cells and makes use of the fact that three times more strand breaks are produced in aerobic than in hypoxic cells.


METHODS
Immediately after irradiation with doses of 4-20 Gy, SCCVII squamous cell carcinomas growing in C3H mice were removed and cooled, and a single-cell suspension was prepared. These cells were then embedded in agarose, lysed in an alkaline solution, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye. The amount and migration distance of damaged DNA from individual cells were scored by using a fluorescence image processing system, where differentially radio-sensitive aerobic and hypoxic cell populations resulted in bimodal damage distributions. Curve-fitting routines provided quantitative estimates of the fraction of hypoxic cells.


RESULTS
After the mice were exposed to 10-20 Gy, the SCCVII tumors (450-600 mg) were shown to have a hypoxic fraction of 18.5% +/- 10.6% (mean +/- SD for 11 tumors), which compares well with the value of 11.6% observed using the paired survival curve method.


CONCLUSIONS
Our results indicate that this method, which requires only a few thousand cells, is a rapid and sensitive way to detect hypoxic cells in solid animal tumors.


CONCLUSIONS
Estimating hypoxia in accessible human tumors undergoing radiotherapy may be possible if the sensitivity of the method can be improved to allow detection of hypoxic cells after a dose of 2 Gy.
Olive
P L
PL
Medical Biophysics Unit, B.C. Cancer Research Center, Vancouver, Canada.
Durand
R E
RE
D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D004277:DNA, Single-Stranded; D006863:Hydrogen-Ion Concentration; D000860:Hypoxia; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D009374:Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology; D014965:X-Rays
D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded