Purpose: It was the aim of our study to establish an extensive panel of non-small cell lung cancer (NSCLC) xenograft models useful for the testing of novel compounds and for the identification of biomarkers. Experimental Design: Starting from 102 surgical NSCLC specimens, which were obtained from primarily diagnosed patients with early-stage tumors (T₂/T₃), 25 transplantable xenografts were established and used for further investigations. Results: Early passages of the NSCLC xenografts revealed a high degree of similarity with the original clinical tumor sample with regard to histology, immunohistochemistry, as well as mutation status. The chemotherapeutic responsiveness of the xenografts resembled the clinical situation in NSCLC with tumor shrinkage obtained with paclitaxel (4 of 25), gemcitabine (3 of 25), and carboplatin (3 of 25) and lower effectiveness of etoposide (1 of 25) and vinorelbine (0 of 11). Twelve of 25 NSCLC xenografts were >50% growth inhibited by the anti-epidermal growth factor receptor (EGFR) antibody cetuximab and 6 of 25 by the EGFR tyrosine kinase inhibitor erlotinib. The response to the anti-EGFR therapies did not correlate with mutations in the EGFR or p53, but there was a correlation of K-ras mutations and erlotinib resistance. Protein analysis revealed a heterogeneous pattern of expression. After treatment with cetuximab, we observed a down-regulation of EGFR in 2 of 6 sensitive xenograft models investigated but never in resistant models. Conclusion: An extensive panel of patient-derived NSCLC xenografts has been established. It provides appropriate models for testing marketed as well as novel drug candidates. Additional expression studies allow the identification of stratification biomarkers for targeted therapies.

In the past, eukaryotic cell-derived complexes of the Myc/Max/Mad network of transcriptional regulators have largely been refractory to DNA binding studies. We have developed electrophoretic mobility shift assay conditions to measure specific DNA binding of Myc/Max/Mad network complexes using a COS7 cell-based overexpression system. With the established protocol, we have measured on- and off-rates of c-Myc/Max, Max/Max, and Mad1/Max complexes and determined relative affinities. All three complexes appeared to bind with comparable affinity to a Myc E-box sequence. Furthermore, our data derived from competition experiments suggested that the Mad3/Max and Mad4/Max complexes also possess comparable DNA binding affinities. The conditions established for COS7 cell-overexpressed proteins were then used to identify c-Myc/Max, Max/Max, and Mnt/Max complexes in HL-60 cells. However, no Mad1/Max could be detected, despite the induction of Mad1 expression during differentiation. Whereas the DNA binding activity of c-Myc/Max complexes was down-regulated, Max/Max binding increased, and Mnt/Max binding remained unchanged. In addition, we have also tested for upstream stimulatory factor (USF) binding and observed that, in agreement with published data, USF comprises a major Myc E-box-binding factor that is more abundant than any of the Myc/Max/Mad network complexes. Similar to the Mnt/Max complex, the binding activity of USF remained constant during HL-60 differentiation. Our findings establish conditions for the analysis of DNA binding of Myc/Max/Mad complexes and indicate posttranslational regulation of the Max/Max complex.