Characterization of firefly luciferase—eGFP infected B16-F1 melanoma, LLC1 lung carcinoma and TRAMP-C2 prostate carcinoma cells.

<p>(A) Cells were lentivirally transduced to express firefly luciferase, enhanced GFP and puromycin. Representative phase contrast and green fluorescence image of B16-luc, LLC1-luc and TRAMP-C2-luc cells (bar represents 100 μm). (B) Analysis of luciferase activity by spectrometer demonstrates relative light units (RLU) emission by lysed cells. Bars represent mean ± SD, Student’s two tailed t-test (B16-luc vs. B16: p = < 0.0001, n = 7, LLC1-luc vs. LLC1: p = < 0.0001, n = 6, TRAMP-C2-luc vs. TRAMP-C2: p = < 0.0001, n = 6). (C) Viability was measured by absorbance of MTT. The absorbance was significantly higher in B16-luc cells seeded at 10’000 cells per well compared to control B16 cells (p < 0.0001). There was no viability difference in LLC1-luc and TRAMP-C2-luc cells compared to LLC1 and TRMAP-C2 cells respectively. Mean ± SD, n = 5 for all experiments shown, two way ANOVA with Bonferroni post hoc analysis. (D) Wound healing 2D migration analysis shows no difference in B16-luc, LLC1-luc andTRAMP-C2-luc cell migration 24 hours post scratch. Mean ± SD, n = 3 for all experiments shown, two way ANOVA with Bonferroni post hoc analysis.</p

Comparison of B16-luc, LLC1-luc and TRAMP-C2-luc tumor cell dissemination versus microsphere dissemination (7 and 16 μm diameter) in osseous organs.

<p>(A) Only ~5% of tumor cells and microbeads disseminated to the spine. No significant difference was found between the groups. (B) Long bones revealed dissemination fractions between ~10–25%. TRAMP-C2-luc disseminated significantly higher to long bones compared to LLC1-luc cells (LLC1-luc vs. TRAMP-C2-luc: p = 0.0406). No other significant dissemination differences were found. (C) Low tumor cell and microbeads dissemination was found in the thorax (~5–10%). No significant difference was found between the groups. (D) Dissemination to the cranium was ~2–6%. No significant dissemination differences were found. Mean ± SD, (B16-luc n = 4, LLC1-luc n = 5, TRAMP-C2-luc n = 5, 7 μm beads n = 5, 16 μm beads n = 5) for all experiments shown, one way ANOVA with Bonferroni post hoc analysis.</p

B16-luc, LLC1-luc, TRAMP-C2-luc and microsphere dissemination (7 and 16 μm diameter) in soft tissues.

<p>(A) Individual lung dissemination of tumor cells varied between ~30–40%. In contrast, very few microbeads (independent of their size) were found in the lung. All cell types disseminated significantly higher to the lung compared to microbeads (B16-luc vs. 7 μm beads: p < 0.0001, B16-luc vs. 16 μm beads: p < 0.0001, LLC1-luc vs. 7 μm beads: p = 0.0025, LLC1-luc vs. 16 μm beads: p = 0.0039, TRAMP-C2-luc vs. 7 μm beads: p = 0.0024, TRAMP-C2-luc vs. 16 μm beads: p = 0.0037). (B) Tumor cell and microbeads dissemination to the brain was relatively low (~5%). LLC1-luc cells disseminated significantly higher to the brain compared to B16-luc. Cell sized (16 μm) microbeads also disseminated significantly higher to the brain compared to B16-luc and even TRAMP-C2-luc (B16-luc vs. LLC1-luc: p = 0.0288, B16-luc vs. 7 μm beads: p = 0.0386, B16-luc vs. 16 μm beads: p = 0.0012, TRAMP-C2-luc vs. 16 μm beads: p = 0.0019). (C) The kidneys’ dissemination fraction ranged from ~10 to 25%. Small (7 μm) microbeads disseminated significantly higher to the kidneys compared to B16-luc and LLC1-luc tumor cells (LLC1-luc vs. 7μm beads: p = 0.0327). (D) Microbeads dissemination was high in the liver (~20%), whereas cell dissemination varied by cell type. LLC1-luc cells disseminated significantly stronger to the liver compared to the other cells. Comparably high amounts of microbeads were found in the liver, 7 μm beads disseminated significantly higher to the liver compared to B16-luc and TRAMP-C2, 16 μm beads showed significantly higher dissemination compared to TRAMP-C2 (B16-luc vs. LLC1-luc: p = 0.0029, B16-luc vs. 7μm beads: p = 0.0116, B16-luc vs. 16μm beads: p = 0.0338, LLC1-luc vs. TRAMP-C2-luc: p < 0.0001, TRAMP-C2-luc vs. 7μm beads: p = 0.0003, TRAMP-C2-luc vs. 16μm beads: p = 0.0003). (E) The skin showed high dissemination of microbeads and TRAMP-C2-luc cells, with a significant effect compared to B16-luc cells (B16-luc vs. TRAMP-C2-luc: p = 0.0366, B16-luc vs. 7μm beads: p = 0.0082, B16-luc vs. 16μm beads: p = 0.0084). Mean ± SD, (B16-luc n = 4, LLC1-luc n = 5, TRAMP-C2-luc n = 5, 7 μm beads n = 5, 16 μm beads n = 5) for all experiments shown, one way ANOVA with Bonferroni post hoc analysis.</p

Expression of ZEB1 in human glial tumors and normal brain.

<p>(A) Biopsy samples of human glial tumors show abundant nuclear expression of ZEB1. Sections from non-neoplastic normal brain show weak cytoplasmic staining of neurons and nuclear expression in astrocytes. (B) Quantification of ZEB1 in full histological sections of gross total resections of human glial tumors. ZEB1 positive cells were quantified using automated image analysis. Dots represent the mean of multiple regions of interest (ROI) per tumor. Box plots show the distribution of the ZEB1 labelling in tumor with indicated integrated diagnosis according the 2016 WHO classification of CNS tumors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185376#pone.0185376.ref029" target="_blank">29</a>]. (C) <i>ZEB1</i> mRNA expression in public GBM cDNA microarray datasets. Boxplots show distribution in normal brain vs. GBM. Data were retrieved via the GlioVis portal [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185376#pone.0185376.ref030" target="_blank">30</a>].</p

Intertumoral heterogeneity of ZEB1 expression.

<p>(A) Box plots of ZEB1 labelling index (percent) with respect to EGFR amplification and IDH1 mutation. (B,C) Correlation of ZEB1 expression and Ki67 labelling index or cellularity, respectively, in N = 193 glioblastoma TMA samples. Trend lines indicate linear regression estimates. Note log scale for Ki67 index. (D) Kaplan-Meier estimates of overall survival time (months) with respect to ZEB1 expression. The observed data range of ZEB1⁺ percentages was split into two equally sized bins at the threshold value of 49%.</p

ZEB1 expression in the tumor microenvironment.

<p>(A) Cell type specific expression of ZEB1 in reactive brain tissue. Human brain biopsy samples from cases of seizure-induced reactive gliosis or subacute infarction were co-stained for ZEB1 and Iba1 for microglia, CD45 for leucocytes, CD68 for macrophages and GFAP for astrocytes, respectively. Scale bars 50 μm. (B) Co-labeling of ZEB1 and CD68 or HLA-DR, respectively in human GBM. (C,D) Correlation of microarray-based gene expression levels of <i>ZEB1</i> and myeloid cell markers <i>CD68</i> (C) and <i>AIF1</i> (D), respectively. Processed log₂-transformed intensities from 157 GBM cases studied by Gravendeel et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185376#pone.0185376.ref034" target="_blank">34</a>] were obtained via the GlioVis portal [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185376#pone.0185376.ref030" target="_blank">30</a>].</p

Intratumoral heterogeneity of ZEB1 expression.

<p>(A) Co-labeling of IDH1 R132H and ZEB1 in a case of IDH1 mutant glioblastoma. The image shown has been taken from one out of ten fields of view that were subjected to separate and blinded manual scoring of mutant IDH1 and ZEB1 expression. (B) ZEB1 gradient along the tumor edge of a mesenchymal GBM (BLN-7 parental tumor). ZEB1 IHC, overall cellularity (<i>blue</i>), the relative frequency of ZEB1+ cells (<i>red</i>) and the mean nuclear intensity of ZEB1+ cells (<i>green</i>) at the tumor edge are shown. (C,D) Expression of ZEB1 (<i>pink</i>) and CD68 (<i>brown</i>) in perinecrotic regions with (C) or without (D) pseudopalisades.</p