Noninvasive contrast-enhanced US quantitative assessment of tumor microcirculation in a murine model: effect of discontinuing anti-VEGF therapy.
OBJECTIVE
To determine, by using contrast material-enhanced ultrasonography (US), how quickly renal tumors grafted in mice begin to revascularize after stopping bevacizumab treatment.


METHODS
All experiments were approved by the regional ethics committee. A human tumor cell line SK-NEP-1 was grafted at day 0 in the left kidney of 50 nude mice. Forty-two mice developed tumors and longitudinal follow-up was performed on 32 surviving mice. From day 13, 14 controls received biweekly saline; 11 mice received biweekly bevacizumab until day 35 (continuous); and seven received biweekly bevacizumab until day 22, then biweekly placebo until day 35 (discontinued). Contrast-enhanced US was performed on days 13, 14, 22, 27, and 35. Once the injected contrast material distribution reached an equilibrium phase, high-acoustic pressure pulses were applied to destroy microbubbles in the capillary bed in the imaged plane. Reperfusion was monitored, and time-signal intensity (SI) curves were obtained from the linear average of SIs in intratumoral and matched-depth renal cortex regions of interest. A kinetic parameter calculated from reperfusion curves reflects local perfusion, normalized with respect to adjacent renal cortex perfusion. Normalized perfusion obtained from each group was compared with that from the other groups and with necrosis percentages and microvascular density assessed histologically at day 35. Comparisons were made by using analyses of variance and Tukey-Kramer tests.


RESULTS
The lowest excised mean tumor weights (+/- standard deviation) corresponded to the longest bevacizumab-treatment duration: 1.4 g +/- 1.1 (continuous-treatment) compared with 2.3 g +/- 2.1 (discontinued) and 3.7 g +/- 1.9 (control) (P = .01). On day 35, the respective control and continuously treated groups had comparable and significantly larger necrotic areas: 37% +/- 14 and 32% +/- 17 larger than the discontinued-treatment group (15% +/- 9; P < .05). Normalized perfusion increased significantly with time (P = .02) in the discontinued-treatment group after therapy ceased (day 22).


CONCLUSIONS
Noninvasively measured contrast-enhanced US parameters demonstrated tumor revascularization after stopping antiangiogenic therapy in this murine tumor model.
Guibal
Aymeric
A
Department of Radiology, GH Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, France.
Taillade
Laurent
L
Mulé
Sébastien
S
Comperat
Eva
E
Badachi
Yasmina
Y
Golmard
Jean Louis
JL
Le Guillou-Buffello
Delphine
D
Rixe
Olivier
O
Bridal
S Lori
SL
Lucidarme
Olivier
O
D000704:Analysis of Variance; D020533:Angiogenesis Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D000068258:Bevacizumab; D045744:Cell Line, Tumor; D003287:Contrast Media; D005260:Female; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008833:Microcirculation; D009374:Neoplasms, Experimental / Q000000981:diagnostic imaging* / Q000188:drug therapy*; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging* / Q000188:drug therapy*; D010743:Phospholipids; D013459:Sulfur Hexafluoride; D014463:Ultrasonography; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors*
D020533:Angiogenesis Inhibitors; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D003287:Contrast Media; D010743:Phospholipids; D042461:Vascular Endothelial Growth Factor A; C420843:contrast agent BR1; D000068258:Bevacizumab; D013459:Sulfur Hexafluoride