Korean J Gastroenterol.  2019 Jul;74(1):30-41. 10.4166/kjg.2019.74.1.30.

Development of a Pancreatic Cancer Specific Binding Peptide Using Phage Display

• Lee DW
• Park JM
• Yang SM
• Kwak MH
• Roh YJ
• Lee IS
• Choi MG

• KMID: 2453256
• DOI: http://doi.org/10.4166/kjg.2019.74.1.30

Abstract

BACKGROUND/AIMS
Pancreatic cancer has a very poor prognosis, and early diagnosis is a way to increase the survival rate of patients. The purpose of this study was to develop pancreatic cancer-specific peptides for imaging studies.
METHODS
Three pancreatic cancer cell lines, MIA PaCa-2, UACC-462, and BxPC-3, and a control cell line, CCD841, were used. Biopannings were performed on MIA PaCa-2 using a phage display library. After this, the peptides were synthesized and labeled with fluorescein isothiocyanate (FITC). Immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell sorter (FACS) were performed to examine the specific binding. To examine its therapeutic applications, a photosensitizer, chlorin e6 (Ce6), was conjugated on the peptide and photodynamic therapy was performed. Cell survival was investigated using a [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay.
RESULTS
After three biopannings, the phages were amplified from 1.4×104 to 3.2×105 plaque-forming units. The most strongly binding phage was selected from the ELISA and ICC results. FITC-labeled peptide, M5, in the three pancreatic cancer cell lines showed significantly higher immunofluorescence in the ICC experiments than that of CCD841. The higher binding ability to MIA PaCa-2 cells was confirmed from FACS analysis, which showed a right shift compared to CCD841. M5 bound to Ce6 showed a significantly lower cell survival rate than that of Ce6 alone in photodynamic therapy, which was observed consistently as a change in the tumor size and fluorescence intensity in MIA PaCa-2 cell-implanted animal models.
CONCLUSIONS
This study showed that the noble peptide, M5, binds specifically to the pancreatic cancer cell line, MIA PaCa-2. The M5 peptide has potential use in future optical diagnostic and therapeutic purposes.

Keyword

Bacteriophages; Pancreatic neoplasms; Peptides; Photochemotherapy

MeSH Terms

Bacteriophages*
Cell Line
Cell Survival
Early Diagnosis
Enzyme-Linked Immunosorbent Assay
Fluorescein
Fluorescence
Fluorescent Antibody Technique
Humans
Immunohistochemistry
Models, Animal
Pancreatic Neoplasms*
Peptides
Photochemotherapy
Prognosis
Survival Rate
Fluorescein
Peptides

Figure

• 

  Fig. 1 ELISA assays were performed to select a phage that adhered specifically to MIA PaCa-2 cells among nine randomly selected phage candidate groups. ELISA, enzyme-linked immunosorbent assay.

• 

  Fig. 2 Immunohistochemical staining was performed using Alexa 488 to confirm the binding affinity of the phage clone to pancreatic cancer (A) phage 2; (B) phage 3; (C) phage 4; (D) phage 5. The binding site of the phage expresses as green fluorescence; and (E) CCD841, MIA PaCa-2, UACC-462, and BxPC-3 cells, which were subjected to immunofluorescence staining to confirm the cell binding ability of the M5 peptide. The M5 peptide was labeled with FITC, and the cells were stained with DAPI (Magnification ×200). FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole.

• 

  Fig. 3 Structure of the peptide and peptide HPLC profile. (A) Structure of the M5 peptide, (B) structure of the peptide M5 coupled with the photosensitizer Ce6, (C) HPLC analysis of peptide M5, (D) HPLC analysis of peptide M5 and the Ce6 conjugate peptide. Ce6, chlorin e6; HPLC, high-performance liquid chromatography.

• 

  Fig. 4 (A–D) Fluorescence activated cell sorter of the CCD841 and MIA PaCa-2, UACC-462, BxPC-3 cells bound with the control and M5 peptide: blue line-control, and red line-M5 peptide. FITC, fluorescein isothiocyanate.

• 

  Fig. 5 Cellular uptake of the photosensitizer, Ce6, in the Ce6-treated group, in which the Ce6-treated group and M5-peptide were combined, was confirmed in the control cell CCD841 and the pancreatic cancer cell line MIA PaCa-2. 4 µg/mL and 12 µg/mL, respectively. (A) CCD841 cell Ce6 alone and M5-Ce6 treatment group, (B) MIA PaCa-2 cell Ce6 alone and M5-Ce6 treatment group. DAPI, 4′,6-diamidino-2-phenylindole; Ce6, chlorin e6.

• 

  Fig. 6 MTT cytotoxicity assay confirmed the laser irradiation effect of the M5 peptide and M5-Ce6. (A) Laser irradiation in CCD841 cells, (B) laser non-irradiation in CCD841 cells. (C) Laser irradiation in MIA PaCa-2 cells, (D) laser non-irradiation in MIA PaCa-2 cells. Ce6, chlorin e6; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide.

• 

  Fig. 7 In vivo study using nude mice implanted with MIA PaCa-2 cells. (A) Comparison of the fluorescence intensity between chlorin e6 (Ce6) group (left) and M5-conjugated chlorin e6 (M5-Ce6) group (right) by the optical in vivo imaging system-IVIS. Ce6 and M5-Ce6 were injected into the tumors with a volume of 150 µL (4 µM). In vivo fluorescence just after the injection of the photosensitizers (middle) and after one hour (bottom). (B) Fluorescence intensity ratio of the tumors between the two groups (each n=5). (C) Change in tumor size before and 48 hours after photodynamic therapy of the two groups (each n=3). (D) Microscopic findings. M5-Ce6 group showed more necrosis (right top, H&E, ×100) than the Ce6-pretreated group (left top, H&E, ×100). Cleaved caspase-3 positive cells were also observed abundantly in the M5-Ce6 group (right bottom, Cleaved Cas-3, ×100) than the Ce6 group (left bottom, Cleaved Cas-3, ×100).

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