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

Immunofluorescent labeling of brain tumors reveals microvascular proliferation and perivascular stem-like cell niche formation.

<p><b>(A)</b> Dense populations of stem-like cells (CD44+) around large, healthy neovascular structures (RECA-1+) were visualized within the tumors (white stars represent vessel lumens). <b>(B)</b> The contralateral cerebral hemisphere (control (CTR)) showed low-level CD44 positively and fewer small blood vessels. <b>(C)</b> The neovascular tumor regions also showed perivascular pockets of HIF-1+ cells, a factor linked to tumor angiogenesis and other oncogenic adaptations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174557#pone.0174557.ref045" target="_blank">45</a>] (white star represents vessel lumen). <b>(D)</b> Brain tissue in the contralateral hemisphere showed no evidence of HIF-1+ cells.</p

Histopathology of PDGFA/p53 shRNA-driven brain tumors.

<p><b>(A)</b> Hematoxylin and eosin staining of whole brain slice with colored boxes highlighting areas of particular interest. (B) Red box = representative sample taken from the invasive edge of the tumor. Arrowheads indicate tumor invasion into adjacent brain tissue. <b>(C)</b> Blue box = hypercellular tumor with regions of pseudopallisading necrosis. <b>(D)</b> Yellow box = areas of microvascular proliferation, hypercellularity, and pleomorphism. (Scale bar = 200 μm).</p