PDGFRA represents the most functionally consequential miR -34a target.

<p><b>A</b>) TS543 cells were transfected with either miR-34a or control oligonucleotides for 24 hours, and NOTCH1 protein levels were measured by immunoblotting. <b>B</b>) 48 hours post-transfection, TS543 cells transfected with either miR-34a, siRNAs against PDGFRA, siRNAs against NOTCH1, or control oligonucleotides were pulsed with BrdU and assayed for cell proliferation by S-phase BrdU incorporation. siRNAs against PDGFRA and NOTCH1 are effective at reducing PDGFRA and NOTCH1 protein levels, respectively, as measured by immunoblotting 24 hours post-transfection. Error bars represent standard deviation within a single experimental set containing multiple replicates. Similar results were obtained in at least 3 independent experiments. *, P<0.05. **, P<0.005.</p

miR-34a induces cell cycle arrest and inhibits tumorigenesis in proneural GBM cells.

<p><b>A</b>) 48 hours post-transfection, proneural TS543 were pulsed with BrdU and assayed for cell cycle distribution and <b>B</b>) apoptosis by Annexin V flow cytometry. Staurosporine was added as positive control. <b>C</b>) <i>Ex vivo</i>-transfected TS453 cells stably expressing a luciferase reporter plasmid were xenografted into the brains of ICR scid mice and tumor formation and growth was followed over 11 days. Error bars represent standard deviation within a single experimental set containing multiple replicates. Similar results were obtained in at least 3 independent experiments. *, P<0.05. **, P<0.005.</p

miR-34a is selectively downregulated in proneural glioma, likely in response to activated PDGF signaling.

<p><b>A</b>) RNA was extracted from KP cells treated with DMSO control, KP cells treated with imatinib for 72 hours, and parental NIH 3T3 cells treated with DMSO control. miRNA expression profiling identified miR-34a as being responsive to PDGF signaling. <b>B</b>) Data from TCGA demonstrate a strong negative correlation of miR-34a with proneural subclass. <b>C</b>) Expression of miR-34a in proneural gliomas and in gliomas of other subtypes. *, P<0.05. **, P<0.005. ***, P = 1e-12.</p

miR-34a specifically inhibits proliferation of proneural glioma cells.

<p><b>A</b>) aCGH analyses of cell lines used. Proneural TS543 and TS667 cells both harbor PDGFRA amplification on chromosome 4, and mesenchymal TS600 cells harbor EGFR amplification on chromosome 7. <b>B</b>) Cells were transiently transfected with miR-34a mimics or control oligonucleotides and for assayed cell numbers over 3–8 days by MTT assay.</p

Immunohistochemical analysis of <i>Ntv-a</i> rat glioma.

<p>Immunohistochemical labeling of PDGFA/p53 shRNA-driven brain tumors reveals features in common with human GBM. Immunohistochemical (IHC) labeling (brown) for <b>(A)</b> Ki-67, demonstrating the high degree of proliferation in the tumor compared to the adjacent brain, and nests of proliferative cells in the invasive rim. <b>(B)</b> GFAP staining revealed variable staining with the greatest degree at the invasive tumor rim and regions of the tumor core. Notably, a significant portion of the tumor had minimal GFAP signal, reinforcing the proneural features of the tumor. <b>(C)</b> SMA (blood vessel marker) labeling detailed the extensive neo-vascularization within the tumor. <b>(D)</b> Olig2, considered a marker of glioma stemness and glial lineage, was strongly positive throughout the tumor. <b>(E)</b> Nestin labeling revealed persistent activation of neural stem-like progenitor cells throughout the tumor but not within the rest of the brain. <b>(F)</b> CD44 was broadly expressed through the brain pockets of strong positivity at the invasive areas near the tumor margins and associated white matter tracts. (Scale bar = 200 μm).</p

Schematic depiction of the RCAS-<i>Ntv-a</i> rat glioma model.

<p><b>(A)</b> Transgenic Sprague-Dawley rats were created using site-specific transposase technology [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174557#pone.0174557.ref034" target="_blank">34</a>] with the <i>tv-a</i> gene under control of the <i>nestin</i> promoter. <b>(B)</b> Chicken fibroblast DF1 cells are transfected with RCAS constructs (e.g. PDGFA and p53 shRNA) that propagate and release RCAS virions into the culture media <b>(C)</b> DF1 cells are injected into the transgenic rats and the RCAS virions bind to the <i>tv-a</i> receptor expressed on neural precursor cells in which the nestin gene promoter is active. Once inside the cell, the DNA insert is integrated to produce stable gene expression changes leading to tumor formation. RCAS virions do not enter differentiated brain cells where the nestin promoter is inactive. (adapted from Ahronian <i>et al</i>., 2014 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174557#pone.0174557.ref035" target="_blank">35</a>]).</p

Glioma-bearing <i>Ntv-a</i> rat natural history study.

<p>Kaplan-Meir survival curve for PDGFA/p53 shRNA brain tumor-bearing animals. Median survival for PDGF-A/p53 shRNA tumor bearing animals from injection date was 92 [Interquartile Range [70–115]] days. Median survival from the first appearance of a tumor on the MRI was 62 days. All injected animals developed large malignant brain tumors.</p

Immunofluorescent labeling of brain tumors shows infiltration of lymphocytes of varying immunophenotypes.

<p><b>(A)</b> Sparse lymphocyte infiltration within the tumors is demonstrated by CD3 and CD8 co-labeling revealing <b>(B)</b> CD3+/CD8+ cells suggesting cytotoxic T-cells, <b>(C)</b> CD3+/CD8- cells reflecting helper and/or regulatory T-cells, and <b>(D)</b> CD3-/CD8+ cells, possibly NK or dendritic cells.</p

MRI and MRS analyses of brain tumor progression.

<p><b>(A)</b> Early and late MRI time points using T2- and T1-weighted imaging. The early MRI findings are consistent with low-grade glioma, including T2 hyperintensity with discrete, homogeneous features. T1 imaging with (T1C) and without (T1) contrast enhancing dye shows little to no enhancement in the early timepoint image. The later imaging reveals a marked transformation in the same animal to a large, heterogeneous tumor with significant mass effect and midline shift, as well as early signs of obstructive hydrocephalus. This is confirmed by T1C imaging, which reveals marked and heterogeneous enhancement within the tumor. <b>(B)</b> The MRS comparing early and later brain tumor regions versus spectra from the contralateral cerebral hemispheres provides additional detail regarding malignant progression with evidence of increased cellular proliferation (red box: higher Cho/Cr) and expansion of non-neuronal tumor elements (blue box: decreased NAA).</p

Immunofluorescent labeling of brain tumors reveals dense and diverse monocytic tumor infiltration.

<p><b>(A)</b> Labeling of tumor-associated macrophages and microglia (Iba-1+) showed dense infiltration within the tumor and a high degree of cellular activation (ED-1+). <b>(B)</b> Further delineation of microglia (P2Y12+)- and <b>(C)</b> bone marrow-derived monocytes (CD49d+) revealed separate contributions from each of these unique cell populations and lineages to the tumor ecosystem. Of note, co-labeling with Iba-1 and each P2Y12 and CD49d showed some overlap (white arrows) but also some distinct, Iba-1 only positive cells.</p