Apoptosis on SKOV-3 cells after treatment with panobinostat, carboplatin or a combination of these drugs for 72 hours.

<p><b>The combination of panobinostat and carboplatin resulted in synergism. A:</b> Nuclear morphology was evaluated by Hoechst 33342 staining. Abnormal nuclei with condensed chromatin were consistent with cell apoptosis/death (highlighted in white). <b>B:</b> The percentages of apoptotic nuclei are shown as mean ± SD (<i>n</i> = 3). <b>C:</b> Cell viability for SKOV-3 is measured with the use of the WST-1 assay. <b>D:</b> The effect of combining panobinostat and carboplatin on SKOV-3 was calculated from the response to each of the drugs alone, compared to the expected response by combining similar concentrations (calculated by Bliss independence analysis). A positive difference between actual response and expected was then ascribed to synergy. <b>E,F:</b> Cell viability for CaOv3, and Bliss independence analysis of expected and actual responses for combinational therapy of panobinostat and carboplatin for CaOv3.</p

Surgical in vivo cohorts.

<p><b>A:</b> Illustration of weekly bioluminescent image analysis of representative group of xenografted mice: a) control, b) surgery, c) surgical and panobinostat/carboplatin-treated and d) combination of panobinostat and carboplatin. <b>B:</b> Relative tumour growth measured by BLI. <b>C:</b> Kaplan-Meyer cumulative survival curves of control, panobinostat-, carboplatin- and a combination of panobinostat- and carboplatin-treated mice. <b>D:</b> Median survival time and increase in survival time (%) for the variously treated groups.</p

Panobinostat inhibits colony formation and induces cell death of SKOV-3 cells assessed by flow cytometry after 72 hours of treatment.

<p><b>A:</b> Representative images of the colony formation after exposure to various drug concentrations. The blue dots represent vital cell clusters. <b>B:</b> Percentages of viable colonies relative to control (untreated) cells. <b>C:</b> Apoptosis for SKOV-3 measured by annexin V/PI staining. Viable cells (lower left quadrant) were negative for both annexin V and PI, apoptotic cells were positive for annexin V staining and negative for PI, and the dead/late apoptosis cells were positive for both annexin V and PI (upper right quadrant). <b>D:</b> Percentages of the different cell death stages for SKOV-3. All results were presented as mean ± SD (<i>n</i> = 3), * <i>P</i> < 0.05, ** <i>P</i> < 0.005 and *** <i>P</i> < 0.001 compared to controls, calculated by unpaired t-test. <b>E, F</b>: Apoptosis by annexin V/PI staining and percentage of the different cell death stages for CaOv3.</p

Exposure of SKOV-3 cells to panobinostat for 24 hours results in cell cycle arrest and upregulation of proteins regulating cell cycle arrest, histone acetylation and cell death.

<p><b>A, B:</b> Cell cycle arrest revealed by cell cycle analysis. <b>C:</b> Upregulation of p21 and H2B. PARP1 and caspase-3 cleavage and activation of cdc2 detected by immunoblotting techniques. <b>D, E:</b> Increased phosphorylation of H2AX examined by phosphoflow cytometry.</p

Morphological evaluation of human vs xenografted mice ovarian serous adenocarcinomas.

<p>The left column shows a high-grade serous adenocarcinoma from human ovary. The right column shows a mouse with a representative ovarian xenograft derived from human SKOV-3^luc+ cells. (<b>A</b>), Formalin fixed paraffin embedded H+E stained sections of human (left) vs xenografted (right) mice (10× magnification), (<b>B–F</b>), Detection of various cancer protein biomarkers (Ber-EP4, cytokeratin, TAG72, vimentin, and WT1) by immunohistochemistry in human (left) vs xenografted (right) mice (10× magnification).</p

Surgical procedures and monitoring of tumor growth in xenografted mice by bioluminescence image analysis.

<p>(<b>A</b>), Preoperative bioluminescence imaging of a representative xenografted (SKOV-3^luc+ cells) mouse (dorsal aspect) with colour bar illustrating photon counts per raster scan point (1 mm²). (<b>B–D</b>), Illustrations of various routine surgical procedures with exposure of right tube (B), ovary (C) and after closure of the incision in the mouse abdominal wall (D). (<b>E</b>), Routine surgical resection specimen illustrating uterus (U), ovaries (O) and the ovarian tumor (T). (<b>F</b>), Immediate postoperative (dorsal view) bioluminescent negative view indicating apparent complete surgical removal of xenografted SKOV-3^luc+ cells.</p

Tumor engraftment and tumor progression.

<p>Xenograft disease characteristics. Data is derived from 10 xenografted mice.</p

Characterization of the experimental <i>in vivo</i> mouse model.

<p>(<b>A</b>), DNA fingerprinting illustrating unique shared microsatellite DNAs between native SKOV-3, mutant SKOV-3^luc+, and <i>in vivo</i> xenografted SKOV-3^luc+ cells. (<b>B</b>), Illustration of <i>in vivo</i> bioluminescence imaging of orthotopic SKOV-3^luc+ cells from one representative untreated control mouse. (<b>C</b>), Relative mean tumor growth vs time as determined by bioluminescence image analysis. (<b>D</b>), Kaplan-Meier survival curve for untreated xenografted mice (n = 10). E, Illustrations of tumor manifestations in various organs in surgical specimens (photographs), by bioluminescence imaging (BLI), and morphology (H+E). Tumors are denominated “T.” Tumor delineation against normal tissue is indicated by dashed line on H-E staining.</p

Multimodal imaging of the same mouse by MRI, ¹⁸F-FDG PET, ¹⁸F-FLT PET and BLI.

<p>MRI three weeks presacrificed (A) depicting large uterine tumour tissue in the left uterine horn (thin arrows) with intrauterine fluid cranial of the tumour (filled large arrow) and small amounts of free intraperitoneal fluid cranial to the right kidney (K) (small arrows). The tumour tissue is moderately enhancing on T1-weighted series after contrast and the tumour exhibits restricted diffusion with hyperintensity on high b-value DWI with corresponding low apparent diffusion coefficient (ADC) value (1.11 x 10⁻³ mm²/s) on the ADC map (A). BLI 4 to 1 weeks presacrificed (B) shows increasing BLI signal corresponding to the tumour of the left uterine horn; the corresponding tumour tissue was evident macroscopically and confirmed microscopically at necropsy (B). ¹⁸F-FDG PET-CT two weeks presacrificed (C) depicts a large ¹⁸F-FDG-avid tumour in the left uterine horn (arrows) with estimated metabolic tumour volume of 33 ml. ¹⁸F-FLT PET-CT one week presacrificed (D) depicts large ¹⁸F-FLT-avid tumour in the left uterine horn (arrows) with estimated metabolic tumour volume of 44 ml. ¹⁸F-FDG/¹⁸F-FLT-avidity in a VOI in the nuchal muscular tissue (C and D; small arrows) was used as reference tissue to define a threshold for likely tumour tissue (activity of x2 and of x6 for ¹⁸F-FLT and ¹⁸F-FDG, respectively) to be included in the estimated metabolic tumour volume. B: bladder; H: heart.</p

Orthotopic injection of Ishikawa^Luc cells results in weight loss and reduced survival.

<p>Mice injected with Ishikawa^Luc cells were monitored weekly for signs of disease development. Weight loss (A) was detected as an early sign of disease. Mice developing symptoms of severe disease were sacrificed and the overall survival is visualized in a Kaplan-Meier survival plot (B).</p