The BarTeL transgene is specifically expressed in EGL of neonatal mice.

<p>(A) Structure of the BarTeL transgene and RCAS retroviruses. In BarTeL, the mouse <i>Barhl1</i> promoter drives expression of a quail <i>Tva</i> cDNA. An IRES allows co-expression of an eGFP-luciferase fusion protein. The RCAS replication competent retroviral vectors express the inserted oncogenes via a splice acceptor located immediately upstream of the cloning site.(B) Bioluminescence imaging shows expression of the BarTeL transgene in the cerebellar region of three transgenic lines. Pups from lines 1–2, 2–1 and 6–4 were injected with luciferin and examined by optical imaging for bioluminescence. Age at imaging is indicated under the panels. (C) Histology and immunohistochemistry for luciferase transgene protein in brains of BarTeL mouse pups. H&E and successive magnifications (boxed, clockwise) of anti-luciferase staining of sagittal sections of transgenic mouse pup cerebella are shown. (D) eGFP expression in BarTeL cerebellar cells. Dissociated cerebella from BarTeL and nontransgenic mouse pups were analyzed by flow cytometry to demonstrate eGFP expression and the ability to sort eGFP-positive cells, indicative of BarTeL transgene expression.</p

<i>ShhN</i> and <i>Mycn</i>^{<i>T50A</i>,<i>S54A</i>} cooperatively induce bioluminescent medulloblastomas <i>in vivo</i> in BarTeL mice.

<p>(A) BarTeL mice were injected with a mixture of chicken DF-1 cells producing RCAS-<i>ShhN</i> and RCAS-<i>Mycn</i>^{<i>T50A</i>,<i>S54A</i>} viruses and imaged for bioluminescence starting at 14 days post-injection. Shown are images at two week intervals of four mice that developed bioluminescent tumors (left) compared to three mice without tumor (right). (B) Time course of bioluminescent tumor formation in transgenic mice. A cohort of 11 transgenic mice, 10 of which were infected as in (A), were imaged weekly for 12 weeks. At each time point, net ΔLog10 flux values plotted for each mouse were determined by subtracting the mean of the log values of flux of non-tumor-bearing transgenic mice from the log of the flux of each mouse. A graph of unprocessed measurements before conversion to log values is provided separately (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156907#pone.0156907.s002" target="_blank">S2 Fig</a>) to show the loss of bioluminescence by cGNPs during postnatal cerebellar development and the concomitant growth of tumor bioluminescence in those mice that developed medulloblastomas. (C) Medulloblastoma in whole BarTeL brain and immunohistochemistry for SHHN and MYCN. Gross appearance of a medulloblastoma in a formalin-fixed brain (second panel) compared to a normal brain (first panel) from a littermate. Staining with an anti-SHH antibody showed that only a minority of medulloblastoma cells produced SHHN protein (fourth panel). Purkinje cells in control normal (NL) adult cerebellum also stain positively with anti-SHH antibody (third panel). An anti-c-MYC antibody identified the MYC-tagged MYCN^T50A,S54A protein in most cells of the same tumor (fifth panel). The magnified panels above the SHH and MYCN panels show the hypercellularity of these tumors. Bar, 10 μm. (D) Histology and immunohistochemistry of a representative medulloblastoma induced by <i>ShhN</i> and <i>Mycn</i>^{<i>T50A</i>,<i>S54A</i>} viruses. Shown are an H&E-stained tumor section (left panel) and tumor sections stained with antibodies to PGP9.5 (ubiquitin carboxyl-terminal esterase L1), Synaptophysin, Ki67 and GFAP. Bar, 10 μm.</p

<i>Barhl1</i> is expressed in cerebellum of neonatal mice and medulloblastoma cell lines, and an isolated <i>Barhl1</i> promoter is active in medulloblastoma cell lines.

<p>(A) RT-PCR analysis of <i>Barhl1</i> expression in mouse cerebellum and non-cerebellar brain tissue at postnatal days 3, 7, 11 and 24. The PCR cycle number used was limited for <i>Barhl1</i> (32 cycles) and <i>Gapdh</i> (23 cycles) in order to enable distinction of RNA levels in cerebellum samples; high cycle number (40 cycles) showed only a faint <i>Barhl1</i> product in non-cerebellum lanes. The last lane contains 100-bp markers. PCR Primers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156907#pone.0156907.s007" target="_blank">S2 Table</a>. (B) RT-PCR analysis of the expression of <i>BARHL1</i>, <i>NESTIN</i> and <i>ATOH1</i> in human medulloblastoma, glioblastoma and atypical teratoid/rhabdoid tumor cell lines. All PCRs were 40 cycles except <i>GAPDH</i> (23 cycles). PCR Primers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156907#pone.0156907.s007" target="_blank">S2 Table</a>. (C) Expression of <i>BARHL1</i> in the four subgroups of human medulloblastoma. <i>BARHL1</i> gene expression data were obtained from the R2 genomic analysis and visualization platform and plotted according to subgroup. The database used was “Tumor Medulloblastoma (core transcript)–Northcott– 103 –rma_sketch–huex10t”. (D) Testing the <i>Barhl1</i> promoter in a luciferase reporter assay for activity in medulloblastoma cell lines. The Barhl1-luciferase reporter plasmid was co-transfected with a constitutive <i>Renilla</i> luciferase control plasmid into the cell lines shown. Data were corrected for transfection efficiency. The first lane contains 100-bp markers.</p

Neonatal BarTeL cGNP cells infected <i>ex vivo</i> with a mix of RCAS-<i>ShhN</i> and RCAS-<i>Mycn</i>^{<i>T50A</i>,<i>S54A</i>} viruses efficiently develop into bioluminescent medulloblastomas when orthotopically injected into nontransgenic cerebella.

<p>(A) PCR assay to detect the specific infection of transgenic cGNPs in culture. Cerebella from transgenic (Tg) and non-transgenic wild type (WT) mice were dissociated, put into culture, infected with RCAS-<i>ShhN</i> and RCAS-<i>Mycn</i>^{<i>T50A</i>,<i>S54A</i>} viruses and then used for preparation of genomic DNA as described in Materials and Methods. PCRs of these genomic DNAs were performed to test for presence of RCAS-<i>ShhN</i> (upper panels) or RCAS-<i>Mycn</i>^{<i>T50A</i>,<i>S54A</i>} (lower panels) proviral DNAs. Positive control template (+) was DNA from a <i>ShhN</i> and <i>Mycn</i>^{<i>T50A</i>,<i>S54A</i>}-induced mouse medulloblastoma, and negative control template (–) was DNA from an uninfected transgenic cerebellum. Control primers that amplify an intron region of a single-copy gene (<i>Fgfr2</i>) were included in all PCRs. The third lane contains 100-bp markers. Thirty-five PCR cycles were used. PCR Primers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156907#pone.0156907.s007" target="_blank">S2 Table</a>. (B) Bioluminescent tumors induced by transplanting <i>ex vivo</i> infected transgenic cerebellar cells into non-transgenic cerebella. Dissociated cerebellar cells from transgenic (Tg) or non-transgenic (WT) neonatal mice were exposed to RCAS-<i>ShhN</i> and RCAS-<i>Mycn</i>^{<i>T50A</i>,<i>S54A</i>} viruses in culture, harvested, injected into the cerebella of non-transgenic neonatal B6D2F1 mice and imaged at the times shown. (C) Time course of bioluminescent tumor formation in recipient mice orthotopically injected with <i>ex vivo</i>-infected transgenic cerebellar cells. Bioluminescence measurements of the mice shown in (B) were plotted over time. Solid lines (blue) denote mice receiving transgenic cerebellar cells; dotted line (black, empty circles) denotes mouse receiving non-transgenic cells.</p

Bicistronic RCAS virus expressing both <i>Mycn</i>^<i>T58A</i> and <i>ShhN</i> increases tumor incidence and lethality.

<p>(A) Map of the bicistronic RCAS vector. Mycn* denotes <i>Mycn</i>^<i>T58A</i>. Not to scale. (B) Kaplan-Meier curves of time to tumor-related euthanasia of mice infected with the RCASBP(A)ΔF1’-<i>Mycn</i>^<i>T58A</i><i>/ShhN</i> bicistronic virus (maroon) compared to a mixture of the RCASBP(A)ΔF1’-<i>Mycn</i>^<i>T58A</i> and RCASBP(A)ΔF1’-<i>ShhN</i> single-gene viruses (blue). The inset table shows the incidence of medulloblastomas in BarTeL mice infected with the bicistronic virus (ShhN/Mycn), the separate single-gene viruses and a mixture of the two single-gene viruses (ShhN + Mycn) derived from the bicistronic plasmid. Mice were euthanized upon development of tumor-related symptoms. (C) H&E stain of a BarTeL bicistronic medulloblastoma. Typical field from an H&E stain of a medulloblastoma arising in the cerebellum of a BarTeL mouse that was injected with the ShhN/Mycn-containing bicistronic virus. Right panel is a magnified view. Bar, 10 μm. (D) Immunohistochemical staining of a representative medulloblastoma (ShhN/Mycn bicistronic virus) with antibodies to the indicated proteins. Luciferase staining demonstrates the border between normal cerebellum (nl, negative) and the tumor (MB; positive). MYCN expression is detected by staining with antibody to the human MYC epitope tag and shows that almost all cells in the tumor express nuclear <i>Mycn</i>^<i>T58A</i>. Ki67 staining shown is at the border of the tumor, with the upper left area being adjacent normal brain. SHH staining is shown in medulloblastoma (MB) compared to the normal (NL) cerebellum, the latter of which demonstrates SHH expression in three Purkinje cells at the border between the molecular layer (upper left in image) and the IGL. Negative control of medulloblastoma was stained with nonspecific IgG primary antibody. Bar, 10 μm. (E) Leptomeningeal dissemination and spread in brains from bicistronic virus-infected mice. Shown are an H&E stain (upper left; note the overlying meninges) and staining with antibodies to luciferase (lower left) and the MYC epitope tag of MYCN^T58A (lower right) of areas in the same leptomeningeal dissemination over the hemisphere. In a different mouse, the upper right panel demonstrates leptomeningeal spread over normal cerebellum, which is contiguous to the main tumor mass a few millimeters away, outside the right lower area of the panel. Bar, 10 μm.</p

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

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

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

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