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

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

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

NSC-secreted anti-HER2 antibody is functionally equivalent to trastuzumab.

<p>Flow cytometric analysis (<b>A</b>) of MCF7, MCF7/HER2, and BT474 cells labeled with F3-IgG, trastuzumab, or a human IgG isotype control antibody. Graphs show mean fluorescence intensity (MFI) of labeled cells. Inhibition of cell proliferation (<b>B</b>) of MCF7, MCF7/HER2, or BT474 cells treated for 6 days with F3-IgG, trastuzumab, or isotype control antibody. Graphs show proliferation as a percentage of untreated cells.</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

<i>In vitro</i> migration of NSCs to breast carcinoma conditioned media.

<p>Migration of parental NSCs and anti-HER2-transfected HB1.F3 NSCs to breast tumor-conditioned media in an <i>in vitro</i> chemotaxis assay. In this assay, bovine serum albumin (BSA) was used as a negative control for chemotaxis. Both parental and transfected NSC lines preferentially migrated to MCF7/HER2 compared to negative control (2% BSA) (<i>p</i><0.01).</p

Expression of human IgG in NSCs.

<p>Fluorescence micrograph of parental HB1.F3 NSCs (<b>A</b>), HB1.F3.H2IgG (<b>B</b>), HB1.F3.Adeno-H2IgG NSCs (<b>C</b>), and HB1.F3.Lenti-H2IgG (<b>D</b>). Cells were stained with anti-human IgG (green) and DAPI nuclear stain (blue). Expression was confirmed by intracellular flow cytometry of fixed and permeabilized NSCs (<b>E</b>). Fluorescence of fixed and permeabilized parental NSCs (indicated by gray histogram) was used to set the marker for each graph.</p

Sensitivity of MRI monitoring of FE-Pro-labeled NSCs targeting human glioma.

<p>(A) T2-weighted MR image of mouse brain in Fomblin, showing two distinct signal voids generated by FE-Pro-labeled NSCs that were injected ∼200 µm apart from each other on the left hemisphere and a hypointense signal generated by FE-Pro-labeled NSCs that migrated to the contralateral tumor site (white dotted boxes). Approximately 600 FE-Pro-labeled NSCs constituted a detectable signal void. (B and C) Prussian blue stained section from the region shown in (A). Higher magnification images (B, tumor area denoted by black dotted line) of the regions outlined in (C), showing PB-positive labeled NSCs corresponding to the hypointense signal sites in (A). 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 = 200 µm (B), 500 µm (C).</p

NSCs target breast carcinoma and can deliver anti-HER2 antibody <i>in vivo</i>.

<p>Confocal fluorescence micrographs of tumor sections from MCF7/HER2 xenografts. First three panels in the upper row show the presence of CM-DiI-labeled red NSCs (HB1.F3, HB1.F3.Ad-H2IgG, HB1.F3.Lenti-H2IgG, respectively) in tumors 4 days after intravenous injection. The fourth panel of the upper row shows tumor with no red NSCs in mice treated with trastuzumab alone (sporadic small red dots not associated with cells are visible as autofluorescence background). Middle row shows tumor sections stained with FITC-conjugated anti-human IgG (green). Bar, 50 µm. Insets are 2× magnification (Bar, 20 µm). Bottom row shows confirmation of the presence or absence of HB1.F3 NSCs within tumors by PCR detection of a DNA amplicon (293 bp) of the v<i>-myc</i> transgene, a unique identifier of the HB1.F3 cell line.</p