Contrast-Enhanced Ultrasound with VEGFR2-Targeted Microbubbles for Monitoring Regorafenib Therapy Effects in Experimental Colorectal Adenocarcinomas in Rats with DCE-MRI and Immunohistochemical Validation

• Published in: PloS one 2017-01, Vol.12 (1), p.e0169323-e0169323
• Main Authors: Eschbach, Ralf Stefan, Clevert, Dirk-Andre, Hirner-Eppeneder, Heidrun, Ingrisch, Michael, Moser, Matthias, Schuster, Jessica, Tadros, Dina, Schneider, Moritz, Kazmierczak, Philipp Maximilian, Reiser, Maximilian, Cyran, Clemens C
• Format: Article
• Language: English
• Subjects: Adenocarcinoma; Angiogenesis; Animals; Apoptosis; Biology and Life Sciences; Biomarkers; Cancer therapies; Colon; Colon cancer; Colonic Neoplasms - drug therapy; Colorectal cancer; Complications and side effects; Contrast Media; Correlation; Cytotoxicity; Diagnosis; Dosage and administration; Drug therapy; Female; Functional magnetic resonance imaging; Genetic aspects; Growth factor receptors; HT29 Cells; Humans; Immunohistochemistry; Inhibitor drugs; Laboratory animals; Magnetic Resonance Imaging; Medicine and Health Sciences; Microbubbles; Monitoring; Morphology; Perfusion; Phenylurea Compounds - therapeutic use; Physiological aspects; Pyridines - therapeutic use; Rats; Rats, Nude; Regorafenib; Research and Analysis Methods; Studies; Targeted cancer therapy; Therapy; Tumors; Ultrasonic imaging; Ultrasound; Vascular endothelial growth factor; Vascular Endothelial Growth Factor Receptor-2 - metabolism; Xenografts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0169323

To investigate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring therapy effects of regorafenib on experimental colon carcinomas in rats with correlation to dynamic contrast-enhanced MRI (DCE-MRI) and immunohistochemistry. Human colorectal adenocarcinoma xenografts (HT-29) were implanted subcutaneously in n = 21 (n = 11 therapy group; n = 10 control group) female athymic nude rats (Hsd: RH-Foxn1rnu). Animals were imaged at baseline and after a one-week daily treatment with regorafenib or a placebo (10 mg/kg bodyweight), using CEUS with VEGFR2-targeted microbubbles and DCE-MRI. In CEUS tumor perfusion was assessed during an early vascular phase (wash-in area under the curve = WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity after 8 (SI8min) and 10 minutes (SI10min)), using a conventional 15L8 linear transducer (transmit frequency 7 MHz, dynamic range 80 dB, depth 25 mm). In DCE-MRI functional parameters plasma flow (PF) and plasma volume (PV) were quantified. For validation purposes, CEUS parameters were correlated with DCE-MRI parameters and immunohistochemical VEGFR2, CD31, Ki-67 and TUNEL stainings. CEUS perfusion parameter WiAUC decreased significantly (116,989 ± 77,048 a.u. to 30,076 ± 27,095a.u.; p = 0.005) under therapy with no significant changes (133,932 ± 65,960 a.u. to 84,316 ± 74,144 a.u.; p = 0.093) in the control group. In the therapy group, the amount of bound microbubbles in the late phase was significantly lower in the therapy than in the control group on day 7 (SI8min: 283 ± 191 vs. 802 ± 460 a.u.; p = 0.006); SI10min: 226 ± 149 vs. 645 ± 461 a.u.; p = 0.009). PF and PV decreased significantly (PF: 147 ± 58 mL/100 mL/min to 71 ± 15 mL/100 mL/min; p = 0.003; PV: 13 ± 3% to 9 ± 4%; p = 0.040) in the therapy group. Immunohistochemistry revealed significantly fewer VEGFR2 (7.2 ± 1.8 vs. 17.8 ± 4.6; p < 0.001), CD31 (8.1 ± 3.0 vs. 20.8 ± 5.7; p < 0.001) and Ki-67 (318.7 ± 94.0 vs. 468.0 ± 133.8; p = 0.004) and significantly more TUNEL (672.7 ± 194.0 vs. 357.6 ± 192.0; p = 0.003) positive cells in the therapy group. CEUS parameters showed significant (p < 0.05) correlations to DCE-MRI parameters and immunohistochemistry. CEUS with VEGFR2-targeted microbubbles allowed for monitoring regorafenib functional and molecular therapy effects on experimental colorectal adenocarcinomas with a significant decline of CEUS and DCE-MRI perfusion parameters as well as a significant reduction