5hmC immunohistochemistry is prognostic of survival in anaplastic astrocytoma and adult glioblastoma.

<p>Patients with anaplastic astroyctoma (A) or adults with glioblastoma (B) were stratified with respect to 5hmC staining level into 5hmC-low (first quartile) and 5hmC-high tumors (quartiles 2–4). The percent survival was graphed using the product limit (Kaplan Meier) method. Log rank test of the curves revealed a significant survival advantage in the 5hmC-high group compared to the 5hmC-low group for anaplastic astrocytoma (p = 0.04) and adult glioblastoma (p = 0.02).</p

IDH1 R132 H mutation status is not associated with a detectable difference in 5hmC content.

<p>Tumors were evaluated for immunoreactivity with antibodies specific for the IDH1 mutant R132H and the corresponding 5hmC levels were correlated with IDH1 immunopositive and immunonegative (IDH1 negative) tumors in (A) grade II astrocytoma, (B) combined low grade gliomas (C) anaplastic astrocytomas, and (D) glioblastoma. No significant difference was identified in 5hmC staining level within any of the specific tumor types.</p

Relationship between 5hmC level and survival in glioma.

<p>Survival data for glioma patients showing low 5hmC compared to other tumors within grade. Survival values are represented in months. P values were generated using the log rank test. P<0.05 was considered significant. N = number of tumors in the designated group.</p

Low expression of TET genes and high expression of APOBEC genes are associated with poor prognosis in malignant glioma.

<p>Gene expression signatures were evaluated for the TET genes, AID/APOBEC genes, or genes of the base excision repair pathway (BER) represented in the Verhaak dataset <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041036#pone.0041036-Verhaak1" target="_blank">[30]</a>. (A) a heatmap of z-normalized expression values separated into the proneural (P), neural (N), classic (C), and mesenchymal (M) subtypes. Seven of eight genes evaluated were upregulated in the mesenchymal subtype of GBM compared the proneural subtype. Significance of p<0.05, p<0.01, or p<0.001 compared to the proneural subtype are designated by (*), (**), or (***), respectively. Survival analysis was performed for mRNA expression of TET genes (B) and selected APOBEC genes (C) from the TCGA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041036#pone.0041036-CancerGenomeAtlasResearch1" target="_blank">[35]</a> or REMBRANDT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041036#pone.0041036-Madhavan1" target="_blank">[37]</a> datasets. For TCGA data low TET gene expression (dashed line) was designated as the first quartile compared to the remaining three quartiles (solid line). For APOBEC3C and APOBEC3G, high expression (dashed line) was defined as the fourth quartile of expression values compared to the remaining quartiles (solid lines). For REMBRANDT data, a value of ≥2 fold overexpression was used as the cutoff for high expression. P-values <0.05 were considered significant.</p

5hmC levels are reduced in the germinal regions of the human fetal forebrain.

<p>Fetal brain sections were stained with hematoxylin and eosin (A-C) and evaluated for 5hmC or TET2 levels using immunohistochemistry. Sections of the forebrain germinal region stained with antibodies for 5hmC (D) showed markedly reduced levels. In contrast, periventriclular regions outside the forebrain germinal matrix (E) and in the fetal cortex (F) showed high levels of 5hmC staining. TET2 immunohistochemistry (G-I) showed a similar distribution to 5hmC staining. Original magnification was 200X for all photomicrographs, except panels A, D, and G which were 100X.</p

MRI of GBM-1-implanted rabbits.

<p>(A) T2-weighted scan showing hyperintense tumor unilaterally near the thalamus (red arrowhead). T1-weighted scan before (B) and after gadolinium injection (C) showing a lack of enhancement within the tumor.</p

Body weight measurements over the course of immunosuppression.

<p>Body weight measurements over the course of immunosuppression.</p

Bioreducible Polymeric Nanoparticles Containing Multiplexed Cancer Stem Cell Regulating miRNAs Inhibit Glioblastoma Growth and Prolong Survival

Despite our growing molecular-level understanding of glioblastoma (GBM), treatment modalities remain limited. Recent developments in the mechanisms of cell fate regulation and nanomedicine provide new avenues by which to treat and manage brain tumors via the delivery of molecular therapeutics. Here, we have developed bioreducible poly­(β-amino ester) nanoparticles that demonstrate high intracellular delivery efficacy, low cytotoxicity, escape from endosomes, and promotion of cytosol-targeted environmentally triggered cargo release for miRNA delivery to tumor-propagating human cancer stem cells. In this report, we combined this nanobiotechnology with newly discovered cancer stem cell inhibiting miRNAs to develop self-assembled miRNA-containing polymeric nanoparticles (nano-miRs) to treat gliomas. We show that these nano-miRs effectively intracellularly deliver single and combination miRNA mimics that inhibit the stem cell phenotype of human GBM cells in vitro. Following direct intratumoral infusion, these nano-miRs were found to distribute through the tumors, inhibit the growth of established orthotopic human GBM xenografts, and cooperatively enhance the response to standard-of-care γ radiation. Co-delivery of two miRNAs, miR-148a and miR-296-5p, within the bioreducible nano-miR particles enabled long-term survival from GBM in mice