Quantifying transient hypoxia in human tumor xenografts by flow cytometry

Transient hypoxia is a poorly understood and potentially important factor that may limit tumor response to various forms of therapy. We assessed transient hypoxia on a global scale in two different human tumor xenografts by sequentially administering two hypoxia markers followed by quantification of hypoxic cells using flow cytometry. High levels of the first hypoxia marker (pimonidazole) were maintained in the circulation over an 8-hour period by multiple hourly injections, providing a "time-integrated" hypoxia measure showing an asymptotic increase in the total number of hypoxic cells. Subsequent administration of a second hypoxia marker (CCI-103F) showed that substantial numbers of the previously pimonidazole-labeled cells were no longer hypoxic during the circulation lifetime of the second marker. The overall fraction of tumor cells that demonstrated changes in hypoxic status with time increased with different kinetics and by different magnitudes in the two xenograft systems. Specifically, up to 20% of the cells in SiHa (human cervical squamous cell carcinoma) tumors and up to 8% of the cells in WiDr (human colon adenocarcinoma) tumors were intermittently hypoxic over an 8-hour period. Also, the tumor cells that demonstrated transient hypoxia were typically not adjacent to functional tumor blood vessels. Similar approaches could be used in the clinic to provide information on the duration of intermittent hypoxia episodes and the fraction of transiently hypoxic tumor cells, which would, in turn, have important implications for the strategic improvement of cancer therapy.