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Combretastatin-A4 disodium phosphate (CA4DP) is a vascular-disruptive agent that causes an abrupt decrease in tumor blood flow. The direct actions of CA4DP include increases in vascular permeability and destabilization of the endothelial cytoskeleton, which are thought to contribute to occlusion of the tumor vasculature. It has been proposed that increased permeability causes a transient increase in interstitial fluid pressure (IFP), which in turn could collapse intratumoral blood vessels. We examined the immediate effects of CA4DP on tumor IFP in C3H mammary carcinoma. Mice were treated with 100 mg/kg CA4DP by intraperitoneal injection. Tumor perfusion was recorded by laser Doppler flowmetry at separate time points, and IFP was recorded continuously by the wick-in-needle method. In this study, we found that CA4DP treatment resulted in a rapid reduction in tumor perfusion, followed by a decrease in IFP; no increases in IFP were observed. This suggests that CA4DP-induced reductions in tumor perfusion are not dependent on increases in IFP.

Angiopoietins (Ang) are involved in the remodeling, maturation, and stabilization of the vascular network. Ang-4 was discovered more recently; thus, its effect on angiogenesis and its interplay with other angiogenic factors have not been equivocally established. The role of Ang-4 in angiogenesis was tested in Matrigel chambers implanted into the subcutaneous space of nude mice. Ang-4 inhibited the angiogenic response of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and GLC19 tumor cells. In Matrigel chambers with Ang-4-transfected cells, the mean response was significantly lower than that of mock cells. Subcutaneous tumor interstitial fluid pressure (IFP) was significantly lower in Ang-4-transfected GLC19 tumors than in mock-transfected tumors. IFP reduction in Ang-4-transfected tumors was comparable to the reduction seen after bevacizumab treatment. In vitro, we examined the effect of recombinant Ang-4 on endothelial cell migration in Boyden chambers. Human umbilical vein endothelial cell (HUVEC) migration induced by bFGF and VEGF was inhibited by Ang-4 to control levels. In conclusion, we show that rhAng-4, as well as transfection with Ang-4, inhibits angiogenesis induced by GLC19 tumor cells and that Ang-4 expression reduces elevated tumor IFP. In addition, we demonstrate that rhAng-4 inhibits HUVEC migration and growth factor-induced angiogenesis.

The effect of combining SU5416 with fractionated radiotherapy or with low molecular weight (LMW) heparin (dalteparin) was studied in U87 human glioblastoma xenografts in nude mice. SU5416 is antiangiogenic by a specific inhibition of the vascular endothelial growth factor receptor 2 (VEGFR-2), and heparins are assumed to bind VEGF. Both SU5416 (100 mg/kg every second day in 5 days) and 3 Gyx5 produced moderate, yet significant, growth inhibition. Tumors treated with concomitant irradiation and short-term SU5416 maintained a lower growth rate during regrowth than the other treatment groups (P=.007). Dalteparin (1000 IE/kg subcutaneously once a day) had no growth-inhibitory effect on its own, but when this LMW heparin was added to the SU5416 schedule, a significantly enhanced growth inhibition was obtained. VEGF protein content in tumors was not significantly altered by SU5416, but a significant decrease in VEGF levels was found in tumors treated with concomitant dalteparin and SU5416 compared with controls (P=.03). We conclude that: 1) an additive growth-inhibitory effect is obtained by combining SU5416 and fractionated radiotherapy; and 2) LMW heparin (dalteparin), in combination with SU5416, decreases the level of VEGF in tumors and increases the growth-inhibitory effect of SU5416.

The potential of noninvasive laser Doppler flowmetry (LDF) and near infrared spectroscopy (NIRS) to detect acute effects of different vascular-modifying agents on perfusion and blood volume in tumors was evaluated. C3H mouse mammary carcinomas (approximately 200 mm(3)) in the rear foot of CDF1 mice were treated with flavone acetic acid (FAA, 150 mg/kg), 5,6-dimethylxanthenone-4-acetic acid (DMXAA, 20 mg/kg), combretastatin A-4 disodium phosphate (CA4DP, 250 mg/kg), hydralazine (HDZ, 5 mg/kg), or nicotinamide (NTA, 500 mg/kg). Tumor perfusion before and after treatment was evaluated by noninvasive LDF, using a 41 degrees C heated custom-built LDF probe with four integrated laser/receiver units, and tumor blood volume was estimated by NIRS, using light guide coupled reflectance measurements at 800+/-10 nm. FAA, DMXAA, CA4DP, and HDZ significantly decreased tumor perfusion by 50%, 47%, 73%, and 78%, respectively. In addition, FAA, DMXAA, and HDZ significantly reduced the blood volume within the tumor, indicating that these compounds to some degree shunted blood from the tumor to adjacent tissue, HDZ being most potent. CA4DP caused no change in the tumor blood volume, indicating that the mechanism of action of CA4DP was vascular shut down with the blood pool trapped in the tumor. NTA caused no change in either tumor perfusion or tumor blood volume. We conclude that noninvasive LDF and NIRS can determine acute effects of vascular modifying agents on tumor perfusion and blood volume.