PMC

Percent reduction of total blood vessels after treatment with 2-FG. The total blood vessel percent reduction for all blood vessel calibers associated with 2-FG treatment was statistically significant regardless of the vessel-caliber analyzed (P = 0.05 for small-caliber [28%], P = 0.008 for medium-caliber [37%], and P = 0.05 for large-caliber [49%]).

Reduction of tumor burden after treatment with 2-FG. Tumor burden is significantly different between the treatment schedules used (*P < 0.001). Eyes treated with 2-FG for 3 weeks showed a significant decrease in tumor burden (P = 0.009); whereas those treated for 1 d and 1 wk did not show a decrease in hypoxia (P = 0.203 and P = 0.836, respectively).

Reduction of tumor hypoxia after treatment with six injections of 2-FG for 3 weeks. Hypoxia is significantly different between the one injection treatment schedules used (1 d and 1 wk) and the six injections treatment schedule (1 wk; P < 0.001). Eyes treated with 2-FG for 3 weeks showed a significant decrease in hypoxic density (*P = 0.001).

Tumor burden reduction after treatment with 2-FG. Representational H&E pictures of sections (40 × HPF) containing LH_BETAT_AG retinal tumors that were treated for 1 week (A, B) and 3 weeks (C, D). Only those eyes that were treated with 2-FG for 3 weeks showed a significant decrease in tumor burden (P = 0.009).

Intratumoral hypoxia after treatment with one injection of 2-FG. Hypoxia is significantly different between the treatment schedules used (P < 0.001), even though the difference between one injection (inj) treatment and control is nonsignificant for both 1 day (1 d) and 1 week (1 wk) postinjection (P = 0.373 and P = 0.782, respectively). However, hypoxia is decreased substantially at 1 d post treatment after one injection.

Hypoxia reduction after treatment with 6 injections of 2-FG. There were highly significant interactions between the vehicle control (A, B) and the 2-FG treated group (C, D; P < 0.001). Hypoxia is statistical significantly reduced after treatment with 2-FG for 3 weeks (C, D; P = 0.001). Blue, DAPI stain for all the cell nuclei; green, pimonidazole stain for hypoxic regions. Pictures were obtained at magnification ×100 high power field.

Hypoxia density after two treatment schedules with one injection of 2-FG. There were highly significant interactions between the control groups (A, B, and E, F) and the 2-FG treated groups (C, D, and G, H; P < 0.001). Hypoxia is decreased after treatment with 2-FG for 1 d, although not significantly (C, D; P = 0.001). Blue, DAPI stain for all the cell nuclei; green, pimonidazole stain for hypoxic regions. Pictures were obtained at magnification ×100 high power field.

Spatial distribution of hypoxia after treatment with 2-FG. The spatial distribution of hypoxia in the different areas of the tumor (i.e., apex, center, lateral, base) was evaluated by counting the amount of small, medium, large, and very large areas of hypoxia in each of the intratumoral regions (i.e., apex, center, lateral, and base; 100 × HPF). Grading scale measuring the size of the hypoxic areas and the amount of these areas per intratumoral region: Small size hypoxic areas were represented by ≤20 hypoxic cells and were assigned a value of 1; medium size hypoxic areas were represented by 21 to 40 hypoxic cells and were assigned a value of 2; large size hypoxic areas were represented by 41 to 80 hypoxic cells and were assigned a value of 4; and very large size hypoxic areas were represented by ≥81 hypoxic cells and were assigned a value of 8. The difference between treated and untreated was highly significant (*P < 0.001) and there is a statistically significant interaction in the differences between treatments by location (P < 0.008). 2-FG has a differential effect on hypoxia depending on the intratumoral area: apex (88%), center (81%), lateral (79%), and base (95%).