Relationships between cycling hypoxia, HIF-1, angiogenesis and oxidative stress

Abstract

This Failla Lecture focused on the inter-relationships between tumor angiogenesis, HIF-1 expression and radiotherapy responses. A common thread that bonds all of these factors together is microenvironmental stress caused by reactive oxygen and nitrogen species formed during tumor growth and angiogenesis or in response to cytotoxic treatment. In this review we focus on one aspect of the crossroad between oxidative stress and angiogenesis, namely cycling hypoxia. Understanding of the relative importance of this feature of the tumor microenvironment has recently expanded; it influences tumor biology in ways that are separate from chronic hypoxia. Cycling hypoxia can influence angiogenesis, treatment responses and metastatic behavior. It represents an important and relatively less well understood feature of tumor biology that requires additional research.

FIG. 1

Relationship between red cell flux variation and interstitial pO₂ in a skin fold window chamber tumor. Panel A: Tracing of vascular field, taken from a video monitor, indicating direction of flow for segments surrounding an interstitial location for pO₂ measurement. Panel B: Time tracings of red cell flux and interstitial pO₂ for an 80-min observation period. Note that the interstitial pO₂ drops toward the end of the period, commensurate with a reduction in red cell flux. Panel C: Summary figure from several experiments showing the median and magnitude of fluctuations in pO₂ as a function of distance from the microvessel in each preparation with the highest red cell flux. These data strongly suggest that cycling hypoxia can exist near the diffusion limit of oxygen. Oxygen tension measurements were made using recessed tip oxygen microelectrodes with tip diameters <10 μm. Figure adapted from Lanzen et al. with permission from the author and publisher (34).

FIG. 2

Analogy between tides and waves and cycling hypoxia. Oxygen transport in tumors is somewhat analogous to the effect of high and low tides on how far waves crash up on to the beach of an island. If the tide is low, then the waves do not travel very far up the beach, but when tide is high, the waves can travel much farther. In an analogous fashion, some networks of tumor vessels can have relatively little oxygen available (low tide), whereas in the same tumor, other networks may have an overabundance of oxygen (high tide). In both cases, though, the amount of oxygen delivered is unstable over time (analogous to the waves), and the tumor cells (analogous to the beach) experience the same instability. The upper panel shows a map of hemoglobin saturation (Hb_sat) in a window chamber tumor. The central portion (blue) shows blood vessels with low Hb_sat, indicating that they are not carrying very much oxygen (they were perfused, however, based on visual inspection). The peripheral portion of the tumor exhibits microvessels with much higher Hb_sat. The lower panel depicts the analogy with the island, showing the net effect of the tides and waves on water coverage over the beach. In the case of cycling hypoxia in tumors, however, the kinetics of oxygen instability is quite complex (not like regular waves), with high (<1 cycle/h), intermediate (<24 h) and slow (>24-h cycle times). In addition, there are circumstances where pO₂ drops to a very low level for long periods (severe chronic hypoxia), which would be insufficient to support cell survival.

FIG. 3

Changes in perfusion within an A-07 melanoma xenograft, as assessed by two DCE-MRI studies done 1 h apart. The parameter E*F is derived from the Kety equation (78) and is an estimate of perfusion. ΔE*F is the difference between the two studies. Panel A: The color code indicates a change in perfusion of more than 0.03 ml/(g*min), which was considered significant. Note that in the same tumor, there are regions that increase (yellow) and decrease (blue) between the two studies. Panel B: ΔE*F is plotted as a function of pixel number (arbitrary assignment). Corroborating panel A, there are significant increases and decreases in ΔE*F within the same tumor. The connection between contiguous pixels, with respect to direction of change, suggests that changes in vascular network perfusion patterns are responsible for this effect. DCE-MRI refers to dynamic contrast enhanced MRI. This is a method to assess the dynamic changes in MRI contrast as a function of time after injection in a tissue of interest. Figure adapted from Brurberg et al. with permission from the author and publisher (50).

FIG. 4

Spatial relatedness of cycling hypoxia obtained using phosphorescence lifetime imaging of a skin fold window chamber containing the R3230Ac mammary carcinoma. Watershed segmentation results are shown for 2.5-min intervals. Time increased from left to right, top to bottom, in 2.5-min increments. Watershed segmentation creates boundaries at sharp gradients in pO₂; segmented regions can be thought of as pO₂ isobars. Segments are color-coded by their deviations from the median pO₂ of the image. Red, high deviations from the median; blue, no deviation from the median. Figure adapted from Cardenas Navia et al. with permission from the author and publisher (51).

FIG. 5

Changes in stress granule prominence in response to reoxygenation after radiation therapy (RT) in the 4T1 mouse mammary tumor. Hoechst 33342, a perfusion marker dye, was administered to mice intravenously a few minutes prior to tumor removal. Perfused vessels will show perivascular Hoechst 33342 staining of tumor cell nuclei (blue). HIF-1-GFP is a reporter gene for HIF-1 expression (green). Stress granules were identified using an antibody to one of the constituent proteins that form the granules (red). The upper row shows a sham-irradiated tumor. Hoechst 33342 shows the heaviest uptake in regions of low HIF-1 GFP expression. Stress granules are in high density in regions of HIF-1 GFP expression (yellow shows overlap). The lower row shows a tumor treated with 3 × 5 Gy and removed 24 h after last radiation dose. GFP expression is higher than the control, but there is a reduction in the density of stress granules and overlap with HIF-1 GFP. In data not shown, the tumors exhibited strong reoxygenation during this period, even though HIF-1 GFP expression was elevated. Thus a reduction in stress granule density was associated with a period of reoxygenation after radiotherapy. It is likely that stress granule formation and disaggregation occurs during cycling hypoxia. Bar = 50 μm. Figure adapted from Moeller et al. with permission of the author and publisher (75).

FIG. 6

Differences in ¹⁸F misonidazole uptake as assessed by PET scans taken 3 days apart in patients with head and neck cancer. In these scans, the ¹⁸F FDG (fluorodeoxyglucose) avid area is outlined in white and the areas positive for ¹⁸F misonidazole are outlined in red (time 0) and yellow (time 3 days). In patient 1, the region of hypoxia remains in the same location but shrinks between day 0 and day 3. In patient 2, the regions of hypoxia change in size and distribution between the 2 days. This is the first evidence for cycling hypoxia in human subjects. Data courtesy of Clif Ling, John Humm and Nancy Lee. Related data can be found in Lin et al. (77).