Figure 1.
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 µm. (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 µm. (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 µm.
Table 1.
Alignment of the phage-displayed peptide sequences selected by NSCLC cells.
Figure 2.
Verification of tumor-homing ability of PC5-2 in vivo.
(A) SCID mice bearing human lung cancer xenografts received i.v. injections of PC5-2, and phage was recovered after perfusion. Recovery of PC5-2 from the tumor was markedly higher than from brain, heart or lungs. (B) Targeting activity of PC5-2 to tumor tissues was competitively inhibited by SP5-2 but not by control peptide or mutant peptide. (C) Immunohistochemical localization of PC5-2 after i.v. injection into mice with CL1-5-derived xenografts. The phage was localized on tumor tissues (d and e) and no localization was observed in normal organs such as the brain (a), heart (b), and lungs (c), or in the tissue sections treated with the control phage (f–i). The interaction of PC5-2 with the tumor section was inhibited by synthetic peptide SP5-2 (j). Scale bar: 25 µm.
Figure 3.
Treatment of SCID mice bearing human lung cancer xenografts with SP5-2-LD or SP5-2-LV.
(A) Median tumor volume over time in mice treated with PBS, FD, LD, MP5-2-LD, or SP5-2-LD. Tumor growth was markedly suppressed in the SP5-2-LD treated group. SP5-2-LD has higher therapeutic efficacy than LD and MP5-2-LD (n = 6 in each group; **P<0.01). (B) Mice bearing size-matched CL1-5-derived lung cancer with tumor size of approximately 500 mm3 were treated with SP5-2-LD, LD, or PBS (n = 6 in each group, **P<0.01). (C) Mice bearing H460-derived lung cancer were treated with SP5-2-LD, LD, FD, or PBS (n = 6 in each group, *P<0.05). (D) The effects of different treatments on change in body weight over the treatment period (n = 6 in each group). (E) Mice bearing CL1-5-derived lung cancer were treated with SP5-2-LV, LV, or PBS (n = 6 in each group; **P<0.01). (F) A Kaplan-Meier survival curve showed longer lifespan of mice treated with SP5-2-LV than those treated with LV and PBS (n = 5 in each group).
Figure 4.
Tumor localization of different formulations of liposomal and free doxorubicin in a NSCLC xenograft mouse model.
At selected time points (1, 4, 24 and 48 hours) after injection, doxorubicin concentration in tumor tissues (A) and nuclei of cancer cells (B) was measured. Doxorubicin concentration inside tumor tissues and nuclei in the SP5-2-LD group was higher than in the FD, LD, and MP5-2-LD groups (n = 3 at each time point, *P<0.05, **P<0.01).
Figure 5.
SP5-2 enhanced drug delivery to tumor tissues and increased therapeutic efficacy.
(A) Representative two-color images showing the distribution of doxorubicin (red) in relation to nuclei (blue) in tissue sections. Accumulation of doxorubicin in tumor tissues was shown at 1, 4 and 24 hours post-injection. Bar, 50 µm. (B) Histopathology and fluorescent staining of tumor tissues in each treatment group examined after staining with H&E, TUNEL (green), lectin (red), and DAPI (blue). Bar, 50 µm. Enhancement of drug accumulation in tumor tissues correlated with the increased therapeutic efficacy.