Summary of non-synonymous mutations for EPHB6 (NM_004445 and NP_004436) found in tumors.

<p>Note: The table contains data from the databases of <a href="http://www.sanger.ac.uk/genetics/CGP/cosmic/" target="_blank">http://www.sanger.ac.uk/genetics/CGP/cosmic/</a>, <a href="http://strubiol.icr.ac.uk/extra/mokca" target="_blank">http://strubiol.icr.ac.uk/extra/mokca</a>, and the references were listed in the column of “Pubmed Id”. The NSCLC mutations identified in this study were marked as “not reported”. Two sequence homology-based tools were used to predict the potential impact of the identified non-synonymous substitutions on protein function: Sort Intolerant from Tolerant (SIFT; <a href="http://sift.bii.a-star.edu.sg/" target="_blank">http://sift.bii.a-star.edu.sg/</a>) and Polymorphism Phenotype (PolyPhen-2; <a href="http://genetics.bwh.harvard.edu/pph2/" target="_blank">http://genetics.bwh.harvard.edu/pph2/</a>). If the SIFT prediction tolerance index score was less than 0.05, the variation was considered possibly damaging. Predictions made by PolyPhen-2 were assigned as “probably damaging,” “possibly damaging” or “benign.” Deletion mutations cannot be tested by either SIFT or PolyPhen-2.</p

Proliferative activity and cell size of EPHB6 wildtype and mutant cells.

<p>A) Proliferative activity of empty vector control, EPHB6 wildtype and mutant cells were analyzed using a colorimetric MTT assay after 72 hours. Data are shown as means +/− standard deviation of three independent experiments. Differences were statistically not significant (ANOVA). B) Cell size of individual cells (n = 20) growing on plastic dishes was analyzed by live video microscopy and recorded. EPHB6 mutant cells showed a significantly reduced cell size in comparison to EPHB6 wild type and to control cells (p<0.05, t-test).</p

Migration analysis of EPHB6 expressing NSCLC cells.

<p>A) Protein expression of stably transfected A549 cell lines expressing wild type EPHB6 or the EPHB6 deletion mutant. Cells were co-transfected using an EGFP -pcDNA3.1⁺ vector for identification of selected clones. Multiple clones were pooled and further selected as bulk cultures. B) Transwell migration assays were performed with empty vector control cells, EPHB6 mutant and EPHB6 wildtype cells. Five different experiments in triplicates were analyzed. *: significant (p<0.05) differences by (EITHER ANOVA OR t-test) The provided p-value between the three different cell lines was statistically analyzed from all migrated cells by using the OneWay ANOVA-test. The analysis of the pair-wise t-test results in a significant p-value for the control cells vs. EPHB6-wt cells (p<0.015) and between the EPHB6-wt cells and the EPHB6-mut cells (p<0.005). C) <i>In vitro</i> wound healing scratch assay. Cells were scratched by a 10 µl pipette tip. The scratch areas were recorded over a periode of 17 hours. Shown are means of three different experiments, calculated as percentage from one initial point for all three cell lines. The ANOVA-test (p<0.002) indicated statistically significant differences between the three cell lines. D) Representative images of the scratch assays at the beginning and the end of the experiments.</p

Development of metastasis <i>in vivo</i>.

<p>A) Number of pulmonary metastases in evaluable NOD/SCID mice four weeks after transplantation, each with 3×10⁵ stably transfected A549 cells expressing EPHB6-wt (n = 9), EPHB6-del915-917 (n = 9) or empty vector control cells (n = 2). Dots represent individual mice and horizontal lines the median value of metastases. B) Images from representative whole lungs of NOD/SCID mice, transplanted with A549 cells expressing EPHB6-wt, EPHB6-del915-917, or empty vector control. Lung metastases are marked by black arrows. C) Images from lung sections of NOD/SCID mice, stained with hematoxylin. Metastases are marked by black arrows. Three representative examples are shown each for mice transplanted with A549 cells expressing EPHB6-wt or EPHB6-del915-917.</p

Potential therapeutic impact of CD13 expression in non-small cell lung cancer

<div><p>Background</p><p>Aminopeptidase N (CD13) is a zinc-binding protease that has functional effects on both cancerogenesis and tumor angiogenesis. Since CD13 is an antigen suitable for molecular targeted therapies (e.g. tTF-NGR induced tumor vascular infarction), we evaluated its impact in NSCLC patients, and tested the effects of the CD13-targeted fusion protein tTF-NGR (truncated tissue factor (tTF) containing the NGR motif: asparagine-glycine-arginine) <i>in vivo</i> in nude mice.</p><p>Methods</p><p>Expression of both CD13 and CD31 was studied in 270 NSCLC patients by immunohistochemistry. Clinical correlations and prognostic effects of the expression profiles were analyzed using univariate and multivariate analyses. In addition, a microarray-based analysis on the basis of the KM plotter database was performed. The <i>in vivo</i> effects of the CD13-targeted fusion protein tTF-NGR on tumor growth were tested in CD1 nude mice carrying A549 lung carcinoma xenotransplants.</p><p>Results</p><p>CD13 expression in tumor endothelial and vessel associated stromal cells was found in 15% of the investigated samples, while expression in tumor cells was observed in 7%. Although no significant prognostic impact was observed in the full NSCLC study cohort, both univariate and multivariate models identified vascular CD13 protein expression to correlate with poor overall survival in stage III and pN2+ NSCLC patients. Microarray-based mRNA analysis for either adenocarcinomas or squamous cell carcinomas did not reveal any significant effect. However, the analysis of CD13 mRNA expression for all lung cancer histologies demonstrated a positive prognostic effect. <i>In vivo</i>, systemic application of CD13-targeted tissue factor tTF-NGR significantly reduced CD13+ A549 tumor growth in nude mice.</p><p>Conclusions</p><p>Our results contribute a data basis for prioritizing clinical testing of tTF-NGR and other antitumor molecules targeted by NGR-peptides in NSCLC. Because CD13 expression in NSCLC tissues was found only in a specific subset of NSCLC patients, rigorous pre-therapeutic testing will help to select patients for these studies.</p></div

Baseline characteristics of the NSCLC study population.

<p>Baseline characteristics of the NSCLC study population.</p

Prostate specific membrane antigen (PSMA) expression in non-small cell lung cancer

<div><p>Objectives</p><p>PSMA (prostate-specific membrane antigen) is overexpressed in prostate cancer cells and is reported to be a promising target for antibody-based radioligand therapy in patients with metastasized prostate cancer. Since PSMA expression is not restricted to prostate cancer, the underlying study investigates PSMA expression in non-small cell lung cancer (NSCLC).</p><p>Material and methods</p><p>Immunohistochemistry was used to identify PSMA expression in n = 275 samples of NSCLC tissue specimens. By means of CD34 co-expression, the level of PSMA expression in tumor associated neovasculature was investigated. The impact of PSMA expression on clinicopathologic parameters and prognosis was evaluated.</p><p>Results</p><p>PSMA tumor cell expression in NSCLC is as low as 6% and was predominantly found in squamous cell carcinoma (p = 0.002). Neovascular PSMA expression was found in 49% of NSCLC. High neovascular PSMA expression was associated with higher tumor grading (G3/G4) (p < 0.001). Neither for PSMA tumor cell expression, nor for PSMA neovascular cell expression prognostic effects were found for the investigated NSCLC cases.</p><p>Conclusion</p><p>Here, we report on the expression of PSMA in NSCLC tissue samples. Against the background of a potential treatment with radiolabeled PSMA ligands, our data might serve for the future identification of patients who could benefit from this therapeutic option.</p></div

Prognostic impact of CD13 expression in non-small cell lung cancer (NSCLC).

<p>Univariate prognostic models (i.e. microarray-based mRNA expression) are shown for CD13 of all patients (<b>Fig. 2A</b>) and for stage I tumor patients (<b>Fig. 2B</b>) according to “The Kaplan-Meier plotter” database (<a href="http://www.kmplot.com" target="_blank">www.kmplot.com</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177146#pone.0177146.ref025" target="_blank">25</a>]). With regard to the immuno-histochemical protein expression of CD13 in endothelial cells and vessel-associated stroma cells of the tumors prognostic analyses were performed for our complete NSCLC study collective (<b>Fig. 2C</b>), for squamous cell carinoma patients (<b>Fig. 2D</b>), for stage III NSCLC tumor patients (<b>Fig. 2E</b>), and for patients with pN2 lymph node status (<b>Fig. 2F</b>).</p

Univariate log rank test results for the association of CD13 with overall survival for the study collective of n = 1926 lung cancer patients as provided by “The Kaplan-Meier plotter” (KM plotter) database (transcriptomic data).

<p>Univariate log rank test results for the association of CD13 with overall survival for the study collective of n = 1926 lung cancer patients as provided by “The Kaplan-Meier plotter” (KM plotter) database (transcriptomic data).</p

PSMA expression in non-small cell lung cancer (NSCLC).

<p>A (100x, anti-PSMA) displays strong PSMA expression in tumor cells. In B (100x, anti-PSMA), neovascular PSMA expression is shown. Neovasculature was identified by CD34 coexpression (insert, anti-CD34).</p