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

Abstract

ObjectivesTo 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.Materials and MethodsHuman 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.ResultsCEUS 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.ConclusionsCEUS 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 of specifically bound microbubbles under therapy, consistent with a reduced expression of VEGFR2.

Evolution of regorafenib from bench to bedside in colorectal cancer: Is it an attractive option or merely a &ldquo;me too&rdquo; drug?

“…Eschbach et al studied the role of contrast-enhanced ultrasound (CEUS) with VEGFR-2-targeted microbubbles to monitor the functional and molecular therapy effect of regorafenib in rat HT-29 CRC xenografts. 51 Tumor-bearing rats received daily treatment with regorafenib or placebo (10 mg/kg) for 1 week. In CEUS, tumor perfusion was assessed during an early vascular phase (wash-in area under the curve) and VEGFR-2-specific binding during a late molecular phase.…”

Dynamic contrast-enhanced ultrasonography (D-CEUS) for the early prediction of bevacizumab efficacy in patients with metastatic colorectal cancer

“…CI confidence interval [20]. In our study, the optimal cut-off value corresponded to a 10 % decrease in the AUC baseline value.…”

“…Semiquantitative perfusion and tumour vascular density such as relative blood volume (rBV) and relative blood flow (rBF) can be determined from the time-concentration curve constructed by perfusion software which represents the kinetics of microbubble contrast agent flow through the tumour [16,17]. Although the accuracy of D-CEUS in microvascular perfusion assessment and in the evaluation of changes in tumour perfusion after therapy has been documented in both experimental models [18][19][20] and patients with a wide range of cancers [6,21,22], its impact on clinical practice remains modest.…”

EGFR-targeted nonviral NIS gene transfer for bioimaging and therapy of disseminated colon cancer metastases

“…This pattern of increased vascularization of the tissue surrounding metastases along with low vascularization inside metastases could be confirmed by immunofluorescence analysis of vascularization (CD31-staining). Moreover, the extensive spread of the LS174T derived metastases in control mice lead to highly necrotic areas in the center of metastases, further diminishing vascularization inside metastases [ 44 , 45 ].…”