Cabozantinib prolongs survival of mice bearing orthotopic E98- xenografts.

<p>Mice were treated with 100 mg/kg cabozantinib from day 12 post tumor inoculation, when tumor was detected via abnormalities in T2 images (see panel A for a representative example). B) Survival curves for placebo (n = 10) and cabozantinib (n = 10) treated animals. Note that, for ethical reasons, mice were sacrificed when excessive weight loss and signs of neurological dysfunction occurred. Median survival was significantly different between the groups (20 and 32 days respectively, log rank test, <i>p</i><0.0001). C) Representative examples of T1-weighted MRI of control (upper row) and treated (lower row) E98 bearing animals before (pre) and 2–3 minutes after (post) Gd-DTPA injection. [Post-pre]/pre represents subtracted images. Note the complete loss of contrast enhancement in treated animals. Panel D shows H&E staining of sections, corresponding to the slices shown in the MR images. Bars: overviews 2 mm, zoom 200 µm.</p

<i>In vivo</i> effects of cabozantinib treatment in E98 xenografts.

<p>Panels A and B show representative examples of IHC for the hypoxia marker MCT4 in control and cabozantinib-treated tumor bearing animals. Hypoxia in compact tumor regions is significantly increased after treatment (Students <i>t</i>-test, p = 0.003, panel C). D and E show examples of Ki67 stainings in compact tumor areas. Proliferation indices were significantly different in these regions (Students <i>t-</i>test p = 0.04), but no difference was detected in diffuse tumor areas (panel F). Panels G and H show representative examples of GLUT-1 vessel staining. Automated quantification revealed no differences between vessel densities of diffuse tumor areas in control vs treated mice (I). Numbers of CD34-positive vessels were lower in cabozantinib treated mice (see panels J and K, arrows point at blood vessels), but these data were not quantified because vessels without CD34 expression were also observed in these mice. L: Western blot analysis of protein extracts (50 µg protein/lane), derived from cabozantinib-treated xenografts reveals a substantial, though not complete, reduction of c-MET phosphorylation. As a loading control, γ-tubulin was included. Immunohistochemistry for phospho-c-MET (Y1234/1235) also shows the presence of phosphorylated c-MET in treated animals, as visualized in panel K. Size bars: A–B 2 mm, D–E 100 µm, G, H, J, K 200 µm,</p

Additional file 1: of Quantification of O-(2-[18F]fluoroethyl)-L-tyrosine kinetics in glioma

Details MR sequences. Details blood sample measurements. Table S1. Patient details. Figure S1. Transaxial views of the tumours on 20–40 min standardised uptake value maps of [18F]FET. Figure S2. Scatter (A) and Bland-Altman plot (B) of volume of distribution, VT, calculated with the 1T2kVb model versus the 2T4kVb model. Shaded areas are 95% confidence intervals. Figure S3. Scatterplot of volume of distribution (VT) versus cerebral blood flow (CBF) (A). The same plot with each patient indicated separately, connecting low, medium and high VOIs with lines (B). CBF data was not available for patient 6. Figure S4. Scatterplot of K1 versus cerebral blood flow (CBF) (A). The same plot with each patient indicated separately, connecting low, medium and high VOIs with lines (B). CBF data was not available for patient 6. Figure S5. Scatterplot of extraction versus cerebral blood flow (CBF). Figure S6. Scatterplots of simplified reference tissue model estimates of binding potential (BPND) (A) and K1-ratio (R1) (B) against the cerebral blood flow ratio (CBF-ratio). Figure S7. Scatter plot of SUV20–40 versus the volume of distribution (VT) calculated with the 2T4kVb model. (PDF 237 kb

c-MET is activated in E98 xenografts.

<p>Panel A shows a Western blot containing protein extracts of different xenografts as indicated (40 µg/lane) and stained with a pan and an Y1234/1235 phosphorylated (P-) c-MET specific antibody. As a loading control, γ-tubulin was included. Immunohistochemical analysis reveals prominent c-MET expression and activation in orthotopic E98 xenografts (C–F). Gross appearances of an E98 tumor are shown in C and E, while D and F show magnifications of the boxed areas in C and E. The H&E section in B illustrates the diffuse nature of these tumors, arrows pointing at white matter tracts and comparison with D shows homogeneous expression of c-MET by tumor cells. Arrow in E points at diffuse infiltrative tumor cells in white matter with activated c-MET, while the arrowhead points at a more compact paraventricular tumor area. The inset in E represents an area with compact leptomeningeal growth partly positive for activated c-MET. The pictures shown are representative for this xenograft model. Size bars: B, D, F 200 µm; C 1 mm and E 500 µm.</p

Additional file 2: of Quantification of O-(2-[18F]fluoroethyl)-L-tyrosine kinetics in glioma

Patient information, TACs, AIFs. (XLSX 443 kb

<i>In vitro</i> effects of Cabozantinib on c-MET and VEGFR2 signaling.

<p>Panel A shows a Western blot of E98NT cell extracts (20 µg per lane) treated for 30 minutes with different concentrations of cabozantinib as indicated. Protein extracts were analyzed for c-MET, phospho-c-MET, AKT, and ERK1/2, using α-tubulin as a loading control. B) MTT assays were done to determine the IC₅₀ concentration of cabozantinib on E98NT cells. Experiments were performed at least in triplicate. C) Effects of cabozantinib on cell migration. Shown are representative examples of DAPI-stained spheroids after 24 hr incubation with indicated concentrations. Number of outgrowing and migrating cells per spheroid are shown in panel D (***: p<0.001). Number of migrating cells were significantly different between groups (one-way ANOVA, p<0.0001). Post-hoc Tukey's Multiple Comparison Test revealed significant differences groups as indicated (***: p<0.001). E) Western blot of cell lysates of E98NT and HUVEC extracts, treated with 10 ng/ml VEGF with or without cabozantinib, and stained for VEGFR2, phospho-VEGFR2 and α-tubulin as an internal control. Note the absence of VEGFR2 in E98NT cells. F) Western blot of treated E98NT cell extracts with the anti-apoptotic antibody U1-70. Control sample consists of Jurkat cells treated with anisomycin.</p

Additional file 4: of Comprehensive protein tyrosine phosphatase mRNA profiling identifies new regulators in the progression of glioma

Immunohistochemical staining for PTPRT on formalin-fixed paraffin-embedded materials. (PDF 303 kb

Western blot for detection of P-gp, MRP1, and BCRP1 in pHGG cultures.

<p>MW represents approximate molecular weight of these proteins, as indicated at the right. The pHGG lanes were loaded with 20 µg of protein, the lanes with positive controls were loaded with 5 µg of protein.</p

a. Cell survival among primary pHGG cultures exposed to classical chemotherapeutic drugs. b. Cell survival among primary pHGG cultures exposed to novel drugs.

<p>a. Cell survival among primary pHGG cultures exposed to classical chemotherapeutic drugs. b. Cell survival among primary pHGG cultures exposed to novel drugs.</p

Tumor and patient characteristics corresponding with the primary cell cultures.

<p>f: female, m: male, AA: anaplastic astrocytoma, GBM; glioblastoma, GC GBM: giant cell glioblastoma multiforme, AO: anaplastic oligodendroglioma, DA2: diffuse fibrillary astrocytoma, GTR: gross total resection, STR: subtotal resection, PR: partial resection. Chemotherapy was administered adjuvant to resection and/or radiotherapy in all treated patients.</p>*<p>hypofractionated radiotherapy 15 fractions of 3 Gy,</p>**<p>survivor, n.a.: does not apply.</p