Retention of FE-Pro label in HB1.F3.CD NSCs.

<p>Data is displayed as means +/− SD of Prussian blue positive iron-loaded NSCs (% of total cell number). The data were obtained from 5 random fields of each independently labeled triplicate sample at 24, 48 and 96 h post-labeling.</p

Cellular viability of FE-Pro-labeled NSCs.

<p>(A) Cellular biomass normalized to non-labeled NSC cell growth at day 1 as measured by absorbance of protein-bound sulforhodamine B (SRB) at 570 nm. Data are mean±SE of triplicate samples and were analyzed using paired t-test between non-labeled vs. each FE-Pro dosage. P<0.05 was considered statistically significant. (B) Representative FACS plots showing the viable and apoptotic cell populations at 24 hours post-label and before sub-culturing. (C–D) Bar graphs showing the percentage of healthy cells at days 1, 4 and 8 for non-labeled NSCs (C), and FE-Pro-labeled NSCs (D) after sub-culturing passage at each time point. (E): Confocal images of healthy FE-Pro labeled and non-labeled NSCs (left panel) and apoptosis-induced FE-Pro labeled and non-labeled NSCs (right panel) at Day 6 post-labeling. Staining: PI (red), YO-Pro-1 (green). A FE-Pro dosage of 50∶3 µg/ml was used for each labeled sample unless otherwise indicated. Abbreviations: FE-Pro, Ferumoxide-Protamine Sulfate complex; PI, propidium iodide; Magnification: 20×.</p

Host mouse nestin and smooth muscle actin-expressing cells are partners during glioma angiogenesis.

<p>Close contact was observed between host nestin (green) and SMA (red)-positive cells incorporated into blood vessels in a U87 xenograft 28 days post-implantation (<b>A</b>, higher power <b>B;</b> insets, nestin staining; dotted circle, putative ‘peg-socket’ structure). Cell nuclei are stained blue (DAPI). Onset of vascularization involving mouse nestin-(white arrows) and SMA-positive cells (red arrows) surrounding a small tumor satellite in A U87 xenograft 28 days post-implantation (<b>C</b>). Note the localization of mouse nestin- and SMA-positive cells at the periphery of this satellite, which descend in a pair-wise manner into the deeper parts of larger satellites (<b>A</b>, <b>B</b>). High-magnification image of a branching vessel in a D566 xenograft 10 days post-implantation showing SMA immunostained cells at the luminal lining and nestin expression at the sprouting front (<b>D</b>). U87 xenograft (<b>A</b>, <b>B</b>, <b>C</b>), D566 xenograft (<b>D</b>). <i>Bars </i><b>D</b> insert 20 µm, <b>B</b> and <b>B</b> insert 25 µm, <b>A</b> and <b>A</b> insert 50 µm.</p

Host nestin-expressing cells are mobilized by human glioma xenografts in the mouse brain.

<p>Mouse nestin-positive cells (green) at the tumor border and peritumor brain tissue, as well as in deeper regions of the tumor mass (<b>A</b>, <b>D</b>) (boxed area on the left in <b>B</b> is shown enlarged in <b>A</b> and <b>D</b>). A mouse brain implanted with U251 human glioma cells (horizontal section; DAPI, blue) (<b>B</b>). Note the high cell density in the xenograft compared to the normal brain tissue. Higher magnification images of the lateral ventricle (hemisphere contralateral to the tumor) showing mouse nestin-expressing NSPCs (green, arrows) (<b>C</b>, <b>E</b>). Note the scarcity of nestin-positive cells and apparent lack of migration by these cells, suggesting little or no proliferation and migration by NSPCs from this region to the tumor. Recruitment of host nestin-positive cells (green) in the subventricular layer of the lateral ventricle ipsilateral to the tumor (<b>F</b>) (arrowheads indicate the wall of lateral ventricle). In this section, the lateral ventricle stretches from the hippocampal formation (caudal) toward the corpus callosum (rostral). Arrowheads show the apparent migration trajectory and pattern of NSPCs along fiber tracts of the deep cerebral white matter. Double-immunostaining for human (red) and mouse nestin (green) in the tumor, showing interdigitation of human and mouse cells (<b>G</b>). lv, lateral ventricle. <i>Bars </i><b>A</b>, <b>C</b>, <b>D</b>, <b>E</b>, <b>G</b> 100 µm, <b>F</b> 500 µm, <b>B</b> 1 mm.</p

Labeling efficiency of FE-Pro.

<p>(A) Light microscopy images of Prussian blue-stained non-labeled and FE-Pro-labeled NSCs at 24, 48 and 96 hours after labeling. (B) Electron micrographs of Fe-Pro-labeled NSCs. (C) Higher magnification image of outlined area in (B). Red arrows point to internalized FE-Pro complex in membrane-bound organelles. (D–E) T2-weighted MR images of labeled (L), non-labeled (N), and an equal mixture (M) of NSCs grown in soft agar. Each phantom contained three different total numbers of NSCs (1×10⁴, 1×10⁵ and 5×10⁵) each in 500 µl of 20% DMEM and 0.8% agar. Coronal view (D) and axial view at 5×10⁵ (E. left) and 1×10⁵ (E. right) of the phantoms. Decrease in T2-w signal strength correlated with the number of labeled cells in the phantom. (F) Graph of T2-w signal intensity vs. number of labeled NSCs. Data were extracted from 5 random fields of each corresponding phantom using ImageJ and shown as mean±SE. MRI conditions: 7.0 Tesla, Gradient-Echo sequence, voxel size = 0.09 mm³, TR/TE = 5402.5/90 ms. Scale bars = 50 µm (A), 2 µm (B) and 200 nm (C).</p

Expression pattern of SDF-1 and CXCR4 in glioma xenografts.

<p>SDF-1 (brown, A and B; red, C and E) is expressed at the tumor edge (<b>A</b>, <b>B</b>, <b>C</b>) and in the tumor center (<b>E</b>). SDF-1 staining was present in the peritumor blood vessels, where some of the signal appeared to be associated with the luminal surface of such blood vessels, indicating the presence of ‘SDF-1 trap’ in these vessels (<b>B</b>). SDF-1 expression (red) also appeared to extend beyond the tumor edge (<b>C</b>), where reactive astrocytes are known to express high levels of SDF-1. CXCR4 (blue) showed high expression at the tumor edge and in the tumor center (<b>D</b>, <b>F</b>). D566 xenograft (<b>A</b>, <b>B</b>), U251 xenograft (<b>C–F</b>). Dotted lines in <b>C</b>, <b>D</b> indicate tumor edge. <i>Bars </i><b>A</b>, <b>B</b> 100 µm, <b>C–F</b> 200 µm.</p

Mouse nestin expressing cells with different morphologies populate the primary tumor bed and the glioma small satellites.

<p>Immunostaining of U87 human glioma tumor bed and satellites) for human nestin (<b>A</b>) (arrowheads indicate tumor satellites). Host mouse nestin-expressing cells with rich arborizations encapsulate and infiltrate the tumor satellites observed in U87 xenografts (<b>B, D, E</b>). Mouse nestin-expressing cells form blood vessels in the main tumor mass (<b>C</b>; inset, higher magnification). Tumor satellite that was detected in the hemisphere contralateral to the main tumor mass (<b>D</b>). Mouse nestin positive cells displayed various morphologies, including rich arborizations (<b>E</b>) and spindle-shaped morphology (<b>F</b>). <i>Bars </i><b>E</b>, <b>F</b> 50 µm, <b>A</b>, <b>B</b>, <b>C</b> 100 µm, <b>D</b> 150 µm.</p

Functionality of FE-Pro labeled NSCs.

<p>(A) Results from Boyden chamber migration assays, showing inherent NSC migration towards conditioned media from U251 (media collected at 24 and 48 hours), UPN029, U87, and U87ffluc cell lines. P<0.05 was considered statistically significant. (B) Flow cytometry plot, showing expression of Cytosine Deaminase (CD) in non-labeled (red (isotype control) and green (anti-bCD)) and FE-Pro-labeled (brown (isotype control) and blue (anti-b-CD)) HB1.F3.CD cells. Abbreviations: HB1.F3.CD.FE-Pro, FE-Pro-labeled HB1.F3.CD NSCs; Anti-bCD, anti-bacterial CD primary antibody.</p

Localization of GFAP-expressing host cells and rodent nestin-positive cells in glioma xenografts.

<p>In mice, murine nestin-expressing host cells (green) at the U251 tumor border and also infiltrating into the tumor (<b>A</b>). These cells did not stain for GFAP, and were shown to be vascular elements. However, a few cells (yellow) expressed both mouse nestin and GFAP (<b>A</b>, inset, arrows). GFAP-expressing cells with characteristic branched morphology (astrocytes) were more numerous in the host brain tissue further away from the tumor border (<b>A</b>, red, GFAP; <b>B</b>, brown, GFAP). We also observed GFAP-positive cells with astrocyte morphology that extended astrocyte foot-like processes onto blood vessels in the vicinity of D566 tumor implant (<b>B</b> inset). Mouse nestin (brown) was strongly expressed at the immediate tumor border (dotted line) (<b>C</b>). GBM biopsy xenograft lesion showing a less demarcated front toward the brain tissue. Numerous double-immunostained cells were seen at the tumor border. GFAP strongly stained host astrocytes as well as glioma cells (<b>D</b>, red, GFAP; green, rat nestin). GFAP and rat nestin co-stained some host cells that showed diverse morphologies. Double-immunostained cells often had elongated cell bodies, with several terminal processes (<b>E</b>, <b>F</b>, red, GFAP; green, rat nestin). An elongated GFAP and rat nestin positive cell within the tumor center, with branched terminal processes (<b>G</b>). U251 (<b>A</b>), D566 (<b>B</b>, <b>C</b>), biopsy spheroid xenografts (<b>D</b>–<b>G</b>). <i>tu</i> tumor. <i>Bars </i><b>E</b>, <b>F</b> 10 µm, <b>G</b> 15 µm, <b>A</b> insert 25 µm, <b>B</b> insert 75 µm, <b>A</b>, <b>D</b> 100 µm, <b>B</b>, <b>C</b> 500 µm.</p

MRI Visualization of FE-Pro-labeled NSCs targeting human glioma in an orthotopic mouse model.

<p>(A) Consecutive T2-weighted MR images of mouse brain in 30% sucrose and 4% PFA. FE-Pro-labeled NSCs are shown as hypointense (dark) signals (white dotted boxes) in the left hemisphere and in the contralateral right hemisphere, where human U251 glioma cells were implanted. (B) Higher magnification, Prussian blue stained sections from the areas outlined by the boxes in (A) (top, left hemisphere; bottom, right hemisphere, tumor area outlined by black dotted line). (C) Consecutive T2-weighted MRI images of mouse brain in Fomblin that received PBS sham injection on left hemisphere and human glioma U251 on the right hemisphere. No low-intensity signals were detected in this control. (D) Higher magnification, Prussian blue stained sections from the areas outlined by the boxes in (C) (top, left hemisphere; bottom, right hemisphere, tumor area outlined by black dotted line). MRI conditions: 7.0 Tesla, Rapid Acquisition Relaxation Enhancement sequence, 78 µm/pixel, 300 µm/slice, T_R/T_E = 1500/23.1 ms. Scale bars = 100 µm (B and D).</p