J. Lee et al., DIRECT RELATIONSHIP BETWEEN RADIOBIOLOGICAL HYPOXIA IN TUMORS AND MONOCLONAL-ANTIBODY DETECTION OF EF5 CELLULAR ADDUCTS, International journal of cancer, 67(3), 1996, pp. 372-378
While the potential importance of hypoxia in limiting the sensitivity
of tumor cells to ionizing radiation has long been appreciated, method
s for accurately quantifying the number of radiation-resistant hypoxic
cells within tumors have been lacking. We have used the pentafluorina
ted derivative l-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide] of e
tanidazole (EF5), which binds selectively to hypoxic cells. The adduct
s formed between EF5 and cellular proteins in the hypoxic cells were d
etected using the specific monoclonal antibody (MAb), ELK3-51 conjugat
ed to the flurochrome Cy3, and the number of hypoxic cells was quantif
ied by flow cytometry. To verify the validity of this technique for th
e detection of hypoxic cells, mice bearing KHT sarcomas were treated w
ith various agents to alter tumor oxygenation and hence vary the fract
ion of radiobiologically hypoxic tumor cells. The percentage of EF5 bi
nding cells was then compared directly with the clonogenic survival of
the tumor cells following radiation treatment under the various pretr
eatment conditions. The results showed that allowing the mice to breat
he carbogen (5% CO2/95% O-2) prior to irradiation reduced clonogenic c
ell survival approx. 6-fold and led to an absence of cells binding hig
h levels of EF5. In contrast, pretreating the tumor-bearing animals wi
th either hydralazine, which decreased tumor blood flow, or phenylhydr
azine hydrochloride, which made the mice anemic, increased tumor cell
survival following irradiation 2- to 4-fold, indicative of an increase
in the fraction of hypoxic tumor cells. EF5 measurements made under i
dentical conditions illustrated a shift in the cells in the tumor to h
igh EF5 binding. Our results demonstrate that flow cytometric measurem
ent by fluorescent MAb binding to EF5 adducts may relate directly to r
adiobiological hypoxia in KHT tumors measured by conventional methods.
(C) 1996 Wiley-Liss, Inc.