Microvascular mechanisms by which the combretastatin A-4 derivative AC7700 (AVE8062) induces tumour blood flow stasis

Abstract

We previously reported that a novel combretastatin A-4 derivative, AC7700, has remarkable antitumour effects because of an irreversible stasis of tumour blood flow (TBF) and subsequent loss of nutrient supply to tumour tissue. Since early 2002, under the new designation AVE8062, AC7700 has undergone clinical trials in Europe and the US. Questions remain, however, concerning how AC7700 blocks TBF and why the TBF stasis does not recover. In this study, using a rat tumour LY80, a variant of Yoshida sarcoma, we examined whether TBF cessation after AC7700 administration is due to a direct action of the agent on tumour blood vessels. We constructed electrodes that can drop a small quantity of the drug solution directly at the site of blood flow measurement and inserted them subcutaneously and into the tumour. We compared the blood flow responses of normal vessels and tumour vessels after administration of 10-microl doses of various concentrations (0.2, 1, 10, and 50 mg ml(-1)) of the AC7700 solution. In addition, we assessed TBF stasis after i.v. and intra-arterial 10 mg x kg(-1) AC7700 administration in an LY80-induced kidney tumour. To determine why the TBF stasis is irreversible, we observed AC7700-induced changes in host arterioles and the tumour vascular network of the Sato lung carcinoma using a vital microscopic rat transparent chamber. Since an increase in tumour interstitial fluid pressure brings about a decrease in TBF, we also measured 10 mg x kg(-1) AC7700-induced changes in this pressure. The sensitivity of the blood flow response after intratumoral application of AC7700 was markedly higher in normal vessels relative to tumour vessels. Intra-arterial administration of AC7700 did not have stronger effects on TBF stasis than did i.v. administration. Intravital microscopy showed that AC7700 induced a powerful and long-lasting constriction of host arterioles, so that complete stasis of blood flow occurred in downstream vessels, which supplied blood to tumours. Owing to this stasis, the lumens of numerous tumour vessels narrowed or completely disappeared, and numerous erythrocytes stagnated in drainage vessels of the tumour vascular network. Haemolysis of these erythrocytes occurred after 2-3 h, resulting in complete thrombosis. There was no indication of reperfusion in vessels showing haemolysis. This haemolysis is thought to be the main cause for the irreversibility of TBF stasis. Since the tumour interstitial fluid pressure decreased after i.v. AC7700 administration, the possibility of stasis of TBF being caused by tumour vascular compression was excluded. All these results strongly suggest that the main target of AC7700 is host arterioles and that the stasis of TBF induced by AC7700 is not triggered by a direct action of the drug on tumour vessels.

Figures

Figure 1

Changes in blood flow induced by topical application of AC7700 or 0.9% NaCl solution to tumours and normal s.c. tissues (A) A sample of 10 μl of AC7700 solution at each concentration (0.2, 1, 10, and 50 mg ml−1) and of 0.9% NaCl solution was dropped on the region of blood flow measurement in tumour and s.c. tissue.The blood flow was measured before and 30 min after application of the solution, and the rate of change in blood flow (%) was calculated. Black bars, normal s.c. tissue; hatched bars, tumour. For each concentration of AC7700 and 0.9% NaCl solution, nine and seven rats were used for measurements in normal s.c. tissues and tumours, respectively. At concentrations of 1 and 10 mg ml−1, the sensitivity of normal s.c. tissue to AC7700 was significantly higher than that of tumour tissue. (B) vital microscopic images of changes in arterioles after topical application of 10 μl of 0.2 mg ml−1 AC7700. Note that an arteriole (arrows) was markedly constricted by AC7700: (a) before droplet application; (b) 19 s after droplet application.

Figure 2

Comparison of changes in TBF after i.a. and i.v. administration of AC7700. AC7700 administration (10 mg kg−1) was completed at 0 min. The difference in TBF reduction between the two routes of AC7700 administration was not significant (P=0.0765): •, i.a. route (n=12); ○, i.v. route (n=11).

Figure 3

Process of irreversible TBF stasis caused by AC7700: (A) before 10 mg kg−1 AC7700 administration; (B) 30 min after the end of drug administration; (C) 2 h later; (D) 2.5 h later. (E) Enlargement of a section of D. (A–D) Photograph with the × 10 the eyepiece and the × 4 objective (bar, 500 μm); (E) photograph with the × 10 the eyepiece and the × 20 the objective (bar, 100 μm). Arterioles (arrows) and a feeding arteriole (*) showed marked contraction induced by AC7700. Tumour blood flow stopped completely 30 min after i.v. AC7700 administration, and many erythrocytes were trapped in drainage vessels located at the tumour periphery. Dramatic haemolysis occurred in those tumour vessels 2–2.5 h after drug administration (C, D). Haemorrhage was not observed (E). Reperfusion of blood into these tumour vessels was never seen.

Figure 4

Disappearance of tumour vascular lumens caused by AC7700-induced TBF stasis. (A) Fluorescence angiography. To confirm functioning tumour vessels, 2% FITC-dextran (m.w. 4400 Da) was administered by a single bolus i.v. injection. Before AC7700 administration, the fluorescent dye did reach the tumour vessels. Many tumour vessels had diameters of 50–100 μm. Bar, 250 μm. (B) histology of the same area 120 min after administration of 10 mg kg−1 AC7700. The tumour tissue within the chamber was locally fixed by formalin before killing. Many tumour vessels greatly constricted or the vascular lumens disappeared, and the vessels showed a fine thread-like appearance. We confirmed this finding by serial section of the tissue. Bar, 50 μm.

Figure 5

Stagnation of blood cells in the drainage vessels caused by AC7700: (A) before administration of 10 mg kg−1 AC7700; (B) 30 min after AC7700 administration. The plasma volume in tumour vessels gradually decreased, and many erythrocytes remained in the vessels. Bar, 100 μm.

Figure 6

Normal microvessels after the administration of AC7700: (A) true capillaries (arrows); (B) arteriole (arrow) and venule (arrowhead); (C) arteriole and precapillary vessel (arrow). Bars, 100 μm. Haematoxylin and eosin staining. A rat with a transparent chamber was killed 2 h after the administration of 10 mg kg−1 AC7700. The tissue was resected, and routine histological studies were performed. Note that the vascular lumens of the normal arterioles, venules, and even true capillaries (with diameters of 10–15 μm) remained open after AC7700 administration.

Figure 7

Simultaneous changes in TIFP, MABP, and TBF caused by AC7700. ○, TIFP; •, MABP; □, TBF. AC7700 administration (10 mg kg−1) was completed at 0 min. Note that TIFP markedly decreased, together with a TBF decrease, immediately after AC7700 administration.

Figure 8

Microvascular mechanisms of AC7700-induced TBF stasis and necrosis formation. Initially, AC7700 causes the powerful and continuing constriction of pre-existing arteriolar vessels, which is why systemic blood pressure is raised after administration of the drug. With the continuing increase in the resistance of arterioles, a fall in perfusion pressure occurs in the tumour-feeding vessels downstream. As a result, blood flow to the tumour vascular bed halts, and that effect, in turn, causes a decrease in the volume of water in the tumour interstitial region, producing a decrease in TIFP. Owing to the complete cessation of TBF, the lumens of tumour vessels with their weak supporting structures become constricted or disappear entirely. The blood flow in the drainage vessels of the tumour vascular bed comes to a halt, leading to the accumulation of erythrocytes, and the erythrocytes undergo haemolysis in 2–3 h. Constriction or disappearance of the tumour vessel lumens and haemolysis cause the exit routes from the tumour to become closed, and recovery of TBF becomes impossible, which makes the stasis irreversible. The halt in TBF causes interstitial convection to stop, and the removal of water from the tumour interstices reduces the efficiency of diffusion. Decreases in both convection and diffusion prevent nutrient supply to the tumour, which is ultimately the cause of necrosis of the solid tumour tissue. Squares, observed or measured events.