Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: Intravital microscopy and measurement of vascular permeability

The tumor vascular effects of the tubulin destabilizing agent disodium comb retastatin A-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 tumo r growing in a dorsal skin flap window chamber implanted into BD9 rats. CA- 4-P is in clinical trial as a tumor vascular targeting agent. In animal tum ors, it can cause the shut-down of blood flow, leading to extensive tumor c ell necrosis. However, the mechanisms leading to vascular shut-down are sti ll unknown. Tumor vascular effects were visualized and monitored on-line be fore and after the administration of two doses of CA-4-P (30 and 100 mg/kg) using intravital microscopy. The combined effect of CA-4-P and systemic ni tric oxide synthase (NOS) inhibition using N-omega-nitro-L-arginine (L-NNA) was also assessed, because this combination has been shown previously to h ave a potentiating effect. The early effect of CA-4-P on tumor vascular per meability to albumin was determined to assess whether this could be involve d in the mechanism of action of the drug. Tumor blood flow reduction was ex tremely rapid after CA-4-P treatment, with red cell velocity decreasing thr oughout the observation period and dropping to <5% of the starting value by 1 h. NOS inhibition alone caused a 50% decrease in red cell velocity, and the combined treatment of CA-4-P and NOS inhibition was approximately addit ive. The mechanism of blood flow reduction was very different for NOS inhib ition and CA-4-P. That of NOS inhibition could be explained by a decrease i n vessel diameter, which was most profound on the arteriolar side of the tu mor circulation. In contrast, the effects of CA-4-P resembled an acute infl ammatory reaction resulting in a visible loss of a large proportion of the smallest blood vessels. There was some return of visible vasculature at I h after treatment, but the blood in these vessels was static or nearly so, a nd many of the vessels were distended. The hematocrit within larger drainin g tumor venules tended to increase at early times after CA-4-P, suggesting fluid loss from the blood. The stacking of red cells to form rouleaux was a lso a common feature, coincident with slowing of blood flow; and these two factors would lead to an increase in viscous resistance to blood flow. Tumo r vascular permeability to albumin was increased to <similar to>160% of con trol values at 1 and 10 min after treatment. This could lead to an early de crease in tumor blood flow via an imbalance between intravascular and tissu e pressures and/or an increase in blood viscosity as a result of increased hematocrit. These results suggest a mechanism of action of CA-4-P hi vivo. Combination of CA-4-P with a NOS inhibitor has an additive effect, which it may be possible to exploit therapeutically.