title: Reducing tumor growth and angiogenesis using a triple therapy measured with Contrast-enhanced ultrasound (CEUS).
abstract: BACKGROUND
  To evaluate the in vivo response by detecting the anti-angiogenic and invasion-inhibiting effects of a triple-combination-therapy in an experimental-small-animal-squamous-cell-carcinoma-model using the "flash-replenishment" (FR) method to assess tissue hemodynamics via contrast-enhanced-ultrasound (CEUS).
  
  
  METHODS
  Human hypopharynx-carcinoma-cells were subcutaneously injected into the left flank of 22-female-athymic-nude-rats. After seven days of subcutaneous tumor growth, FR-measurements were performed on each rat. Treatment-group and control-group were treated every day for a period of one week, with the treatment-group receiving solvents containing a triple therapy of Upamostat®, Celecoxib® and Ilomastat® and the control-group solvents only. On day seven, follow-up measurements were performed using the same measurement protocol to assess the effects of the triple therapy. VueBox® was used to quantify the kinetic parameters and additional immunohistochemistry analyses were performed for comparison with and validation of the CEUS results against established methods (Proliferation/Ki-67, vascularization/CD31, apoptosis/caspase3).
  
  
  RESULTS
  Compared to the control-group, the treatment-group that received the triple-therapy resulted in a reduction of tumor growth by 48.6% in size. Likewise, the immunohistochemistry results showed significant decreases in tumor proliferation and vascularization in the treatment-group in comparison to the control-group of 26%(p ≤ 0.05) and 32.2%(p ≤ 0.05) respectively. Correspondingly, between the baseline and follow-up measurements, the therapy-group was associated with a significant(p ≤ 0.01) decrease in the relative-Blood-Volume(rBV) in both the whole tumor(wt) and hypervascular tumor(ht) areas (p ≤ 0.01), while the control-group was associated with a significant (p ≤ 0.01) increase of the rBV in the wt area and a non-significant increase (p ≤ 0.16) in the ht area. The mean-transit-time (mTT) of the wt and the ht areas showed a significant increase (p ≤ 0.01) in the follow-up measurements in the therapy group.
  
  
  CONCLUSIONS
  The triple-therapy is feasible and effective in reducing both tumor growth and vascularization. In particular, compared with the placebo-group, the triple-therapy-group resulted in a reduction in tumor growth of 48.6% in size when assessed by CEUS and a significant reduction in the number of vessels in the tumor of 32% as assessed by immunohistochemistry. As the immunohistochemistry supports the CEUS findings, CEUS using the "flash replenishment"(FR) method appears to provide a useful assessment of the anti-angiogenic and invasion-inhibiting effects of a triple combination therapy.
authors.affiliation: Institute for Clinical Radiology, Ludwig Maximilian University Hospital, Munich, Germany. philipp.paprottka@med.uni-muenchen.de.
authors.forename: Philipp Marius
authors.initials: PM
authors.lastname: Paprottka
authors.affiliation: Institute for Ear, Nose and Throat Medicine, Ludwig Maximilian University Hospital, Munich, Germany. svenja-rosspunt@web.de.
authors.forename: Svenja
authors.initials: S
authors.lastname: Roßpunt
authors.affiliation: Institute for Clinical Radiology, Ludwig Maximilian University Hospital, Munich, Germany. michael.ingrisch@med.lmu.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Ingrisch
authors.affiliation: Institute for Clinical Radiology, Ludwig Maximilian University Hospital, Munich, Germany. clemens.cyran@med.lmu.de.
authors.forename: Clemens C
authors.initials: CC
authors.lastname: Cyran
authors.affiliation: Institute for Clinical Radiology, Ludwig Maximilian University Hospital, Munich, Germany. konstantin.nikolaou@uni-tuebingen.de.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Nikolaou
authors.affiliation: Institute for Clinical Radiology, Ludwig Maximilian University Hospital, Munich, Germany. maximilian.reiser@med.lmu.de.
authors.forename: Maximilian F
authors.initials: MF
authors.lastname: Reiser
authors.affiliation: Institute for Ear, Nose and Throat Medicine, Ludwig Maximilian University Hospital, Munich, Germany. brigitte.mack@med.lmu.de.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Mack
authors.affiliation: Institute for Ear, Nose and Throat Medicine, Ludwig Maximilian University Hospital, Munich, Germany. olivier.gires@med.lmu.de.
authors.forename: Olivier
authors.initials: O
authors.lastname: Gires
authors.affiliation: Institute for Clinical Radiology, Ludwig Maximilian University Hospital, Munich, Germany. dirk.clevert@med.lmu.de.
authors.forename: Dirk A
authors.initials: DA
authors.lastname: Clevert
authors.affiliation: Institute for Ear, Nose and Throat Medicine, Ludwig Maximilian University Hospital, Munich, Germany. pamela.zengel@med.lmu.de.
authors.forename: Pamela
authors.initials: P
authors.lastname: Zengel
mesh_terms: D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage*; D000068579:Celecoxib / Q000008:administration & dosage* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D006801:Humans; D006877:Hydroxamic Acids; D007012:Hypopharyngeal Neoplasms / Q000000981:diagnostic imaging* / Q000188:drug therapy* / Q000473:pathology; D007211:Indoles / Q000008:administration & dosage* / Q000627:therapeutic use; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging / Q000188:drug therapy / Q000473:pathology; D010091:Oximes; D010879:Piperazines / Q000008:administration & dosage* / Q000627:therapeutic use; D051381:Rats; D013449:Sulfonamides / Q000008:administration & dosage* / Q000627:therapeutic use; D016896:Treatment Outcome; D014463:Ultrasonography; D023041:Xenograft Model Antitumor Assays
chemical_list: D006877:Hydroxamic Acids; D007211:Indoles; D010091:Oximes; D010879:Piperazines; D013449:Sulfonamides; C119203:ilomastat; D000068579:Celecoxib; C580590:upamostat