title: 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: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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 of specifically bound microbubbles under therapy, consistent with a reduced expression of VEGFR2.
authors.affiliation: Laboratory for Experimental Radiology, Institute for Clinical Radiology, Ludwig-Maximilians-University Hospital, Munich, Germany.
authors.forename: Ralf Stefan
authors.identifier: http://orcid.org/0000-0002-9489-0707
authors.initials: RS
authors.lastname: Eschbach
authors.affiliation: Laboratory for Experimental Radiology, Institute for Clinical Radiology, Ludwig-Maximilians-University Hospital, Munich, Germany.
authors.forename: Dirk-Andre
authors.initials: DA
authors.lastname: Clevert
authors.affiliation: Laboratory for Experimental Radiology, Institute for Clinical Radiology, Ludwig-Maximilians-University Hospital, Munich, Germany.
authors.forename: Heidrun
authors.initials: H
authors.lastname: Hirner-Eppeneder
authors.affiliation: Josef Lissner Laboratory for Biomedical Imaging, Institute for Clinical Radiology, Ludwig-Maximilians-University Hospital, Munich, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Ingrisch
authors.affiliation: Laboratory for Experimental Radiology, Institute for Clinical Radiology, Ludwig-Maximilians-University Hospital, Munich, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Moser
authors.affiliation: Laboratory for Experimental Radiology, Institute for Clinical Radiology, Ludwig-Maximilians-University Hospital, Munich, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Schuster
authors.affiliation: Laboratory for Experimental Radiology, Institute for Clinical Radiology, Ludwig-Maximilians-University Hospital, Munich, Germany.
authors.forename: Dina
authors.initials: D
authors.lastname: Tadros
authors.affiliation: Josef Lissner Laboratory for Biomedical Imaging, Institute for Clinical Radiology, Ludwig-Maximilians-University Hospital, Munich, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Schneider
authors.affiliation: Laboratory for Experimental Radiology, Institute for Clinical Radiology, Ludwig-Maximilians-University Hospital, Munich, Germany.
authors.forename: Philipp Maximilian
authors.initials: PM
authors.lastname: Kazmierczak
authors.affiliation: Laboratory for Experimental Radiology, Institute for Clinical Radiology, Ludwig-Maximilians-University Hospital, Munich, Germany.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Reiser
authors.affiliation: Laboratory for Experimental Radiology, Institute for Clinical Radiology, Ludwig-Maximilians-University Hospital, Munich, Germany.
authors.forename: Clemens C
authors.initials: CC
authors.lastname: Cyran
mesh_terms: D000818:Animals; D003110:Colonic Neoplasms / Q000188:drug therapy*; D003287:Contrast Media; D005260:Female; D019073:HT29 Cells; D006801:Humans; D007150:Immunohistochemistry; D008279:Magnetic Resonance Imaging; D045423:Microbubbles; D010671:Phenylurea Compounds / Q000627:therapeutic use*; D011725:Pyridines / Q000627:therapeutic use*; D051381:Rats; D011923:Rats, Nude; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
chemical_list: D003287:Contrast Media; D010671:Phenylurea Compounds; D011725:Pyridines; C559147:regorafenib; D040301:Vascular Endothelial Growth Factor Receptor-2