Immunohistochemistry of PTK787 and vehicle treated tumor showing expression of VEGFR2, VEGFR3 and EGFR.

<p>Immunohistochemistry confirmed the findings of SPECT studies, where PTK787 treated tumors showed increased expression of VEGFR2 and VEFGR3 at the peripheral parts of the tumors, especially around the vessels (arrows) compared to that of vehicle treated tumors (right column). Both PTK787 (lower panel, left column) and vehicle treated (lower panel, right column) showed expression of EGFR in the tumors. Lower panel, middle column show no brown cells in negative control slide.</p

SPECT analysis of <i>in vivo</i> accumulation of Tc-99m-HYNIC-VEGF-c.

<p>VEGF-c (which targets both VEGFR2 and VEGFR3) was tagged with HYNIC chelators and then labeled with Tc-99m-pertechnetate (Tc-99m) and injected intravenously in PTK787 and vehicle treated rats. One hour after injection, SPECT images were obtained using dedicated animal scanner. All PTK787 treated rats showed increased accumulation of Tc-99m-HYNIC-VEGF-c in the tumors (lower panel, arrows) compared to that of vehicle treated tumors (upper panel, arrows).</p

Western blot images and densitometry analysis of western blots.

<p>(<b>A</b>) Expression of different angiogenic factors (left panel) in the vehicle- and PTK787 treated tumors from representative cases at the peripheral (P), central part of the tumors (C) and contralateral brains (B). Note the increased expression of VEGF, SDF-1 and HIF-1α at the peripheral part of PTK787 treated tumors. Right panel shows the densitometry analysis of the blot (normalized to β-actin and contralateral brain). The analysis also confirmed the finding of the blot. Note the patterns of VEGF, SDF-1 and HIF-1α in PTK787 treated tumors which are different from that of vehicle treated tumors. (<b>B</b>) Densitometry analysis of VEGFR2, VEGFR3 and EGFR blot (normalized to β-actin and contralateral brain). Expression of VEGFR2, VEGFR3 and EGFR showed higher normalized values at the peripheral part of the PTK787 treated tumors compared to that of central part and the expression patterns are different in vehicle treated tumors. Please note that PTK787 treated tumors showed lower normalized values of VEGFR2 and EGFR both at the periphery and central parts of the tumors compared to that of corresponding contralateral brain; whereas vehicle treated tumors did not show any changes in the normalized values of VEGFR2 and EGFR compared to that of corresponding contralateral brain. Data are expressed as mean ± SEM, n = 3.</p

Immunohistochemistry of PTK787 and vehicle treated tumor showing expression of VEGF, HIF-1α, SDF-1 and vessel morphology.

<p>Expression of vascular endothelial growth factor (VEGF) (dark brown colored) at different parts of the PTK787 treated and vehicle treated tumors. There were no differences observed in the expression of VEGF on immunohistochemistry at different parts of the tumors treated with either PTK787 or vehicle. However, delineation of vessels using FITC tagged tomato lectin indicated higher number of dilated vessels at the tumor periphery in rats that received PTK787 treatment. These dilated vessels may be indicative of increased permeability, fPV and signal intensity changes in PTK787 treated tumors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008727#pone-0008727-g001" target="_blank"><b>Figures 1</b></a><b> and </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008727#pone-0008727-g002" target="_blank"><b>2</b></a>). Increased permeability may also be due to increased VEGF expression. HIF-1α expression was mostly seen in the central part of the vehicle treated tumors (arrows), however, SDF-1 expressions were observed both in PTK787 and vehicle treated tumors (arrows).</p

MRI detection of FePro labeled long-term cultured frozen and fresh CB AC133+ EPCs in glioma.

<p>At days 10–15 (A, B) and 25–30 (E, F) of the primary culture, cells were labeled with FePro and cryopreserved for few weeks. On the day of IV administration, the cells were thawed, incubated for 1–2 hours in stem cell media, washed and IV injected. A control group of rats received freshly prepared FePro labeled cells at 10–15 (C, D) and 25–30 (G, H) days of cultures. Seven days after cell administration multi-echo gradient-echo MRI were obtained using a 7 Tesla small animal MRI system. All animals receiving either frozen or fresh FePro labeled cells exhibited low signal intensity areas around tumors (arrows). Corresponding DAB enhanced Prussian blue stained sections showed iron positive cells at the tumor margins. Both frozen and fresh FePro labeled cells migrated and accumulated in tumor sites.</p

Effect of cryopreservation on <i>in vivo</i> angiogenic properties of CB AC133+ EPCs-immunohistology.

<p>At days 10–15 and 25–30 of the primary culture cells were labeled with FePro and cryopreserved for few weeks. Seven days after IV administration of thawed FePro labeled cells to glioma bearing rats, tissues were harvested and analyzed by DAB enhanced Prussian blue staining, FITC conjugated tomato lectin (green) and Rho conjugated antibodies that recognized vWF and CD31 expression. Panels A–C depict tissue sections of animals receiving frozen labeled cells that were cultured for 10–15 days. Control animals received 10–15 days cultured non-cryopreserved FePro labeled cells (D–F). Same experiments were done with cells expanded for 25–30 days. Tissue sections from the animals receiving frozen AC133+ are shown in panels G–I, while the section from control group receiving fresh cells are shown in J–L. Scale bar = 100 µm.</p

CB AC133+ EPCs-expression of cell surface markers during long term <i>in vitro</i> culture.

<p>The data depicts CD133 and CD34 protein expression levels in cells cultured for 5, 11 and 25 days and the levels of CD117 in cells cultured for 25 days (<b>A</b>). Flow cytometric histograms from one representative experiment are shown (n = 3). At least 10,000 live gated cells were analyzed for FITC, PE or PE-Cy5 expression. Isotype controls are shown as black histograms. Panel B shows cells induced to differentiate at day 25–30 of primary culture.</p

MRI relaxation parameters in tumors.

<p>Analyses of R2* values normalized to contralateral normal hemisphere (an indirect indicator of iron positive cell accumulation ) showed significantly higher (p<0.05) accumulation of iron positive cells in animals that received previously cryopreserved, FePro labeled CB AC133+EPCs that were <i>in vitro</i> expanded for 10–15 and 25–30 days. Bars: means ± SD. * p<0.05 frozen day 10–15 versus fresh day 25–30; <b>^§</b> p<0.05 frozed day 25–30 versus fresh day 10–15 and fresh day 25–30.</p

CB AC133+ EPCs expression of CD31, vWF and KDR and DiI-Ac-LDL uptake in differentiated progenitors – effect of FePro labeling and cryopreservation.

<p>Expression of CD31, VEGFR2 and vWF in differentiated cells that were prior to differentiating (at days 25–30 of the primary culture) labeled with FePro and cryopreserved for few weeks (D, E, F and G). Control cells were induced to differentiate at days 25–30 of the primary culture without previous FePro labeling and cryopreservation (A, B and C). Positive signals for CD31, VEGFR2 and vWF were visualized with a FITC conjugated secondary antibody (green). Nuclei were visualized with DAPI (blue). VEGFR2 positive (middle panels B and E) cells also exhibited the uptake of DiI-Ac-LDL (red). Representative photomicrographs (40×) of differentiated cells. Scale bar = 100 µm.</p