We used a phage-displayed random peptide library to isolate phages that were able to bind to NSCLC CL1-5 cells. After five rounds of affinity selection (biopanning) with CL1-5, cells increased the titer of phage by 40-fold (Fig. 1A) . Enriched phages from the third to the fifth biopanning rounds were randomly selected. We then sequenced the phage clones with higher CL1-5binding activities. Using the Genetics Computer Group (GCG) software analysis, we found that these selected phages (PC3-1, PC4-1, PC4-5, PC5-2 and PC5-4) displayed the consensus motif, tryptophan (W)-threonine (T)/tyrosine (Y)-tyrosine (Y) ( Table 1) . Interestingly, the phage PC5-2 appeared in the third (PC3-1), fourth (PC4-1) and fifth (PC5-2) biopanning rounds. During the biopanning rounds, the frequency of PC5-2 increased from 20% (1/5) in the third cycle to 90% (27/30) in the fifth cycle (Table 1) . We chose to focus on the novel peptide displayed by PC5-2, TDSILRSYDWTY, for further study. In vitro phage display screening for peptides that bind to NSCLC. (A) A phage-displayed random peptide library was used to select phages that bind to the NSCLC cell line CL1-5. (B) Visualization of PC5-2 binding to CL1-5 and PC13 lung cancer cells (arrowheads) with immunohistochemical staining. The control phage did not bind to CL1-5 cells. Scale bar: 10 mm. (C) The FITC-labeled peptide SP5-2 bound to five NSCLC cell lines but not to NPC-TW01 cells as detected by immunofluorescent staining. Scale bar: 10 mm. (D) Representative photomicrographs of tumor sections from surgical specimens of human lung cancer were detected using both PC5-2 (a, arrowhead) and biotinylated SP5-2 (c, arrowhead), respectively. In comparison, the control phage or biotinylated control peptide could not bind to these surgical specimens (b and d). PC5-2 was competitively inhibited by the synthetic peptide SP5-2 (e). Mutated peptide, MP5-2, lost this competition ability (f). Scale bar: 25 mm. doi:10.1371/journal.pone.0004171.g001 Identification of phage clones specifically binding to NSCLC cells