Fig 1.
Inhibition of proliferation of pediatric solid tumor cell lines in vitro by regorafenib.
33 ITCC pediatric rhabdomyosarcoma (A), Ewing sarcoma (B), neuroblastoma (C), medulloblastoma (D) and osteosarcoma (E) cell lines were exposed to regorafenib at the indicated concentrations and cell viability was measured after 72 hours by MTS assay. The adult RET mutated TT thyroid cancer cell line is included as reference. Dose-response to regorafenib of all cell lines is plotted as the percentage of relative cell viability compared to untreated controls.
Table 1.
GI50s (μmol/L) of regorafenib in ITCC cell lines after 72h exposure determined by MTS Assay.
Fig 2.
Antitumor activity of regorafenib in vivo against various subcutaneous pediatric tumor xenografts.
Animals bearing subcutaneous RMS-1 rhabdomyosarcoma, STA-ET-1 and EW7 Ewing sarcoma, SJ-N-B8 and SK-N-AS neuroblastoma xenografts were treated orally with regorafenib at 10 mg/kg/d (light blue; R-10) and/or 30 mg/kg/d (dark blue, R-30), or with vehicle (grey, C) for a minimum of 21 days; treatment periods are indicated by bars above the graphs. (A) Graphs show arithmetic means ± standard error of mean (SEM) of tumor volumes. (B) Times to reach 5 times the initial volume (Vi) of 3–14 tumors per group is displayed as box plot, + represents means and error bars minimum to maximum values. Statistical significance was estimated by Mann-Whitney or Kruskal-Wallis tests, ****p<0.0001 ***<0.001, **<0.01., *<0.05.
Table 2.
Anti-tumor activity of regorafenib alone and in combination against pediatric xenografts
Fig 3.
Regorafenib inhibits orthotopic IGR-N91-Luc neuroblastoma through antiangiogenic effects and induction of apoptosis.
Animals bearing orthotopic adrenal IGR-N91-Luc tumors were treated orally with regorafenib at 30 mg/kg/d (blue lines) or vehicle (grey line) for 28 days. (A) Graph shows growth curves of individual regorafenib treated mice and arithmetic means (± SEM) of tumor volumes of 7 untreated and 6 treated tumors. Tumor volumes were determined by ultrasonography and representative images of adrenal tumors and kidneys at Day 22 post treatment initiation are displayed (E). Paraffin-embedded entire sections of adrenal IGR-N91-Luc xenografts from Day 29 post treatment initiation were stained immunohistochemically with antibodies against the endothelial cell marker protein CD34 and the apoptosis marker cleaved caspase 3. (B) Microvessel area, quantified as number of CD34-positive vessel using Calopix software, is presented as means ± SEM of 6 controls and 4 treated tumors (p = 0.0095; Mann-Whitney). (C) Apoptosis index is determined as number of caspase 3 cleavage positive cells in 6 controls and 6 treated tumors (p = 0.0043). Examples of histological stainings are shown at 100x magnification. Positive staining appears as brown color (E). (D) Total lysates of three individual tumors from Day 29 post treatment initiation were subjected to Western blot analyses using antibodies against phosphorylated (p-) and non-phosphorylated PDGFR, AKT, ERK1/2 and against cleaved PARP-1. β-actin was used as reference. C: control, R: regorafenib.
Fig 4.
Antitumor activity of regorafenib alone and in combination against xenografts of PDGFRA amplified (IGRG93 and IGRM57), and non-amplified (NEM14) brain tumors.
Animals bearing subcutaneous (A) IGRG93 and (C) NEM14 gliomas, and (B) IGRM57 medulloblastoma, were treated orally with regorafenib at 10 mg/kg/d (R-10) and/or 30 mg/kg/d (R-30) for a minimum of 21 days, total body irradiation of 1 Gy (RT) for 5 consecutive days (IGRG93, NEM14), or irinotecan at 27 mg/kg intravenously (Iri) for 5 consecutive days (IGRM57), respective combinations thereof as indicated, or vehicle (C). Graphs show arithmetic means ± SEM of tumor volumes of 6–14 tumors per group. Bars above graphs indicate treatment periods. Fluorescence in situ hybridization for PDGFRA gene amplification is shown in the right upper corner for each model (PDGFRA gene in red, centromere of Chromosome 4 in green); picture shows macroscopic aspect of IGRG93 tumors after 3 days of treatment.
Fig 5.
Regorafenib activity alone and in combination against patient-derived tumor models is mediated by anti-angiogenic effects and induction of cell death.
Paraffin-embedded sections of IGRG93 (left column), IGRM57 (central column) and NEM14 (right column) tumors, harvested at Day 3 post-treatment initiation, were stained immunohistochemically with anti-CD34 and anti-cleaved caspase 3 antibodies and histologically with Hematoxylin-Eosin-Saffron (HES). (A) Microvessel area and (B) apoptosis index are presented as means ± SEM of 3 controls and regorafenib 30 mg/kg/d treated tumors each (*p<0.05; Mann-Whitney). Due to extensive necrosis only one sample could be evaluated in the combination group of the IGRG93 study. For IGRM57, the assessment of viable tumor by excluding necrotic areas was estimated on HES stained sections. (C) Total lysates from individual tumors were subjected to Western blotting for expression analyses of phosphorylated (p-) and non-phosphorylated PDGFRA, PDGFRB, AKT and ERK1/2. β-actin was used as reference. C: control, R: regorafenib; X: omitted sample due to limited availability of tumor 1; RT: irradiation, Iri: irinotecan.