Representative H&E, SMA and CD31 stains of PRS-050-PEG40, Avastin or PBS treated tumors at day 2 after onset of therapy.

<p>The representative Anticalin treated tumor shows extensive necrosis (black star) and necroptosis (white star), the Avastin treated tumors shows extensive necrosis and a mitosis (block dot). In contrary the PBS treated control tumor shows no necrosis or apoptosis. The black arrows indicate vessels, black arrowheads indicate musculature and black dash indicates apoptosis.</p

Representative DWI (transverse slices; b = 20, b = 200, ADC map), T1 map (T1 values are shown in color scale (ms)) of DCE-MRI from a single time point (transverse slices; pre and post i.v. injection of Gadolinium) used for T1 quantification and FDG-PET (from left to right: transverse, coronal and sagittal reconstruction) images of the A673 rhabdomyosarcoma xenografts (arrows) subcutaneously implanted in the right lateral flank.

<p>Representative DWI (transverse slices; b = 20, b = 200, ADC map), T1 map (T1 values are shown in color scale (ms)) of DCE-MRI from a single time point (transverse slices; pre and post i.v. injection of Gadolinium) used for T1 quantification and FDG-PET (from left to right: transverse, coronal and sagittal reconstruction) images of the A673 rhabdomyosarcoma xenografts (arrows) subcutaneously implanted in the right lateral flank.</p

Representative transverse slices of T2w-MRI, DWI and FDG-PET of the subcutaneously implanted A673 rhabdomyosarcoma xenografts (arrows) pre and 2 days after therapy onset (p.i.) with injection of PRS-050-PEG40, Avastin or PBS as control group.

<p>On T2-weighted MR images the treated tumors showed discreet central hypointense changes indicating apoptosis while the control group did not show significant signal alterations of the tumor tissue. DWI (b = 200) of the PRS-050-PEG40 and Avastin treated tumors shows a small increase in signal intensity while the control group shows a discreet decrease in concordance with the ADC value. FDG-PET demonstrates a decrease in FDG uptake (radioactivity is shown in color scale (Bq/mL)) in the PRS-050-PEG40 and Avastin treated tumor tissue, while the control tumor shows an increase in FDG uptake. Of note, the PET images reveal physiologic cardiac FDG uptake (arrowheads).</p

Percentage change of the tumor size.

<p>On day 3 (day 2 after therapy onset) tumor volume between the different groups did not reach a statistical significance (PRS-050-PEG40/Avastin/PBS, P = 0.09). On day 8 (day 7 after therapy onset) tumors in the control group were significantly larger compared to the treatment groups (PRS-050-PEG40/Avastin in comparison to PBS, each P = 0.001). There was no significant difference in the tumor growth between the therapy groups PRS-050-PEG40 and Avastin neither on day 2 nor day 7 after therapy onset (P = 0.13/0.30). Data are mean values.</p

Percentage change (1.0 = 100%) of all subjects (mean and standard deviation depicted as horizontal and vertical line) of the tumor size, DWI, DCE-MRI and FDG-PET on day 2 after onset of therapy (PRS-050-PEG40 or Avastin) or injection of PBS as control group.

<p>Percentage change (1.0 = 100%) of all subjects (mean and standard deviation depicted as horizontal and vertical line) of the tumor size, DWI, DCE-MRI and FDG-PET on day 2 after onset of therapy (PRS-050-PEG40 or Avastin) or injection of PBS as control group.</p

Scatter plots illustrating the degree of correlation between the percentage change of tumor size (pre vs. day 7 after onset of therapy) plotted along the horizontal axis versus DWI (hollow circle, r = −0.58, P = 0.001), DCE-MRI (grey circle, r = 0.71, P = 0.001) and FDG-PET (black circle, r = 0.67, P<0.001) (pre vs. day 2 after onset of therapy) plotted along the vertical axis.

<p>Scatter plots illustrating the degree of correlation between the percentage change of tumor size (pre vs. day 7 after onset of therapy) plotted along the horizontal axis versus DWI (hollow circle, r = −0.58, P = 0.001), DCE-MRI (grey circle, r = 0.71, P = 0.001) and FDG-PET (black circle, r = 0.67, P<0.001) (pre vs. day 2 after onset of therapy) plotted along the vertical axis.</p

Semiquantitative analysis of the microvessel density (MVD) as assessed by CD31 and SMA staining.

<p>Quantification (%) of the necrotic areas in the tumor tissue before and 2 or 7 days post therapy (p.t.).</p

Small-animal [¹¹C]PiB PET/MRI overview.

<p>[¹¹C]PiB PET co-registered to in vivo 1.5T cranial MRI of the same mouse. Overview of cranial tracer uptake shows images of four representative animals from the major study groups in radiological orthogonal perspective (20–30 min frame). (<b>A</b>) 23 month old female hemizygous APP/PS1 mouse (weight: 20.8 g, injected dose: 14.7 MBq, color scale 37–144 kBq/cc), (<b>B</b>) 9 month old female homozygous APP/PS1 mouse (weight: 22.2 g, injected dose: 15.2 MBq, color scale 60–350 kBq/cc) (<b>C</b>) 21 month old female homozygous APP/PS1 mouse (weight: 24.5 g, injected dose: 24.2 MBq, color scale 73–280 kBq/cc), (<b>D</b>) 23 month old female C57BL/6J control mouse (weight: 29.9 g, injected dose: 15.1 MBq, color scale: 66–300 kBq/cc). <i>Columns</i> from left to right show horizontal (<b>1</b>), coronal (<b>2</b>) and sagittal (<b>3</b>) views. The <i>right column</i> (<b>4</b>) shows corresponding neocortical (<i>yellow</i>) and cerebellar (<i>magenta</i>) time-activity curves (TACs). <i>Inset</i> (<b>5</b>) shows initial tracer dynamics on a smaller time scale (1 to 3 min) to delineate the peak of uptake required for quantification of PET data. Difference between transgenic and control animals is significant for each study group visibly and analytically. For the young homozygous animal, it is seen in the lower color scale range. Cortex in B₂ shows uptake towards <i>blue</i> and <i>cyan</i>. Same structures show lowest uptake in D₂ (<i>magenta</i>, corresponding to cerebellum). TACs confirm visual perception: neocortex TAC in B₄ intersects cerebellum TAC and stays above it (neocortex-to-cerebellum ratio >1) while neocortex TAC in D₄ remains below the cerebellum TAC (ratio <1). PET color look-up-table is <i>UCLA</i> (Pmod) with lower thresholds set to still visualize the cerebellum. Arrowheads indicate slice positions. Slice coordinates (corresponding to Paxinos atlas) are: horizontal Bregma −1.90 mm, coronal Bregma −0.10 mm and sagittal 0.65 mm lateral. Image scale is double size of reality. Further results for these animals are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031310#pone-0031310-g007" target="_blank">Figure 7</a>.</p

Histological Aβ plaque burden and plaque size distribution in neocortex.

<p>Aβ plaque burden and size of individual plaques were analyzed on histological sections stained with Thioflavin S and double immunofluorescence against Aβ40 and Aβ42 by applying a semi-automatic imaging algorithm. All animals were analyzed in PET, before. Shown here, are the results for neocortex of the transgenic study groups: tg-old (<i>orange</i>), tgtg-young (<i>yellow</i>) and tgtg-old (<i>red</i>). (<b>A</b>) Aβ plaque burden of each transgenic group as measured by Thioflavin S, compound anti-Aβ40/42, anti-Aβ42 and anti-Aβ40. Compound anti-Aβ40/42 result shows co-localization of both Aβ species. (<b>B</b>) Plaque size distribution in each transgenic study group. Here, the anti-Aβ42 signal was used for its highest signal-to-noise. Its strong association with the Thioflavin S signal is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031310#pone.0031310.s007" target="_blank">Figure S7</a>.</p

Extracerebral tracer retention.

<p>High [¹¹C]PiB uptake in regions frontal to the brain were accurately validated to be extracerebral. (<b>A</b>) Cranial [³H]PiB ex vivo autoradiography. 15 µm thick section of a complete mouse head showing exact anatomical locations of unspecific tracer retention (male tgtg, 16 month old). Exposition time needed to be shortened to achieve good resolution of extracerebral tissues. For this reason, only few plaques can be seen in the brain. Color table: <i>Red Hot</i> (ImageJ) (<b>B</b>) CNS removal during [¹¹C]PiB PET. 9 month old male homozygous APP/PS1 mouse was scanned in vivo for 30 min before the complete brain was extracted and scanned for further 30 min together with the skull. The skull of the ex vivo [¹¹C]PiB PET scan is co-registered to a cranial CT for better orientation and shown on six horizontal slices which are 1 mm apart (top left horizontal level at about −1.9 mm Bregma in correspondence to all other figures). Both parotid glands can be seen on bottom section. Color table is <i>UCLA</i> (Pmod) (<b>C</b>) Ex vivo biodistribution of [¹¹C]PiB relative to cerebellar uptake in (extracerebral) glandular tissues and eyebulbs in both homozygous and both control study groups. Cerebral biodistribution data from the same animals as presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031310#pone-0031310-t002" target="_blank">Table 2</a> is included graphically as reference. Data show that olfactory bulb does not contribute to high surrounding uptake in harderian glands and eyebulbs. Column heights represent means, error bars represent standard deviation.</p