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. 2017 Jun 12;12(6):e0177146.
doi: 10.1371/journal.pone.0177146. eCollection 2017.

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

Lars Henning Schmidt  1 , Caroline Brand  1 , Janine Stucke-Ring  1 , Christoph Schliemann  1 , Torsten Kessler  1 , Saliha Harrach  1 , Michael Mohr  1 , Dennis Görlich  2 , Alessandro Marra  3 , Ludger Hillejan  4 , Carsten Müller-Tidow  5 , Georg Lenz  1   6   7 , Eva Wardelmann  8 , Rainer Wiewrodt  1 , Wolfgang E Berdel  1 , Christian Schwöppe  1 , Wolfgang Hartmann  8
Affiliations

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

Lars Henning Schmidt et al. PLoS One. .

Erratum in

Abstract

Background: 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) in vivo in nude mice.

Methods: 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 in vivo effects of the CD13-targeted fusion protein tTF-NGR on tumor growth were tested in CD1 nude mice carrying A549 lung carcinoma xenotransplants.

Results: 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. In vivo, systemic application of CD13-targeted tissue factor tTF-NGR significantly reduced CD13+ A549 tumor growth in nude mice.

Conclusions: 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.

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Conflict of interest statement

Competing Interests: W.E.B. shares a patent application on vascular targeting with TF-constructs. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Expression of CD13 in NSCLC tissues.
Examples for negative immunostaining are shown for squamous cell carcinoma (Fig. 1A) and for adenocarcinoma (Fig. 1B). Positive staining results are given for squamous cell carcinomas (stromal expression of CD13) (Fig. 1C and Fig. 1D) and for adenocarcinomas (tumor cell expression of CD13) (Fig. 1E and Fig. 1F). Co-staining of CD31 (green) and of CD13 (red) demonstrates CD13 expression in endothelial cells and perivascular stroma cells in a case of adenocarcinoma (Fig. 1G) and squamous cell carcinoma (Fig. 1H).
Fig 2
Fig 2. Prognostic impact of CD13 expression in non-small cell lung cancer (NSCLC).
Univariate prognostic models (i.e. microarray-based mRNA expression) are shown for CD13 of all patients (Fig. 2A) and for stage I tumor patients (Fig. 2B) according to “The Kaplan-Meier plotter” database (www.kmplot.com [25]). 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 (Fig. 2C), for squamous cell carinoma patients (Fig. 2D), for stage III NSCLC tumor patients (Fig. 2E), and for patients with pN2 lymph node status (Fig. 2F).
Fig 3
Fig 3. In vivo therapeutic activity of systemic tTF-NGR against CD13+ A549 tumor xenografts.
To investigate CD13 expression flow cytometry was performed with a monoclonal PE-labeled anti-CD13 antibody. CD13 expression was found in 47% of the A549 lung cancer cells (green, control; purple, CD13) (Fig. 3A). Following treatment with tTF-NGR (1 mg tTF-NGR/kg x4 (arrows); i.v.; n = 4 CD-1 nude mice) tumor growth of subcutaneous A549 xenotransplants was reduced as compared to the saline control group (n = 6) CD-1 nude mice (Fig. 3B). The CD13 expression in subcutaneous A549 xenotransplant is demonstrated by immunofluorescence; since the antibodies used for CD13 and CD31 staining are species-specific for human CD13 and CD31, vascular and perivascular staining were not assayed in the xenografts (Fig. 3C).
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Grants and funding

This study was supported by grants from the Else Kröner-Fresenius-Stiftung (2013_A284), by grants from the Deutsche Krebshilfe e.V. (110886). The laboratories of W.E. B. and G. L. are supported by Deutsche Forschungsgemeinschaft (DFG EXC1003, Cells in Motion).

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