Characterization of Pancreatic Cancer‐Targeting Peptides MCA1 and MCA2

Abstract

Pancreatic cancer is the fourth leading cause of cancer deaths in the U.S., characterized by a 5‐year survival rate of 3%. The dire prognosis is due to inadequate detection methods. However, peptides offer advantages regarding in vivo molecular targeting due to their small size and ample binding affinity. Thus, peptides may be used in imaging of pancreatic cancer for the detection of the disease. Bacteriophage (phage) display technology using a 15‐mer fUSE5 peptide library (1013 virions) was previously performed by an initial negative selection round against 106 normal human pancreatic (hTERT‐HPNE) cells. Unbound phage were amplified in E. coli K91BK and isolated using PEG/NaCl precipitation. Phage (1013 virions) were then subjected to four subsequent rounds of positive selection against 106 human pancreatic cancer (Mia Paca‐2) cells. Following the last round, amplicons were produced by PCR and submitted to next‐generation sequencing. The results identified two clones, pMCA1 and pMCA2, which were comparatively prevalent in the fourth positive round. In order to evaluate the pancreatic cancer binding properties of phage‐displayed peptides (MCA1 and MCA2), the ligands were synthesized with an N‐terminal GSG spacer and a biotin group. A modified ELISA and fluorescent microscopy were performed, in which 10 μM MCA1, MCA2, J18 (negative control), or DMSO (vehicle) was incubated with Mia Paca‐2, hTERT‐HPNE, ovarian adenocarcinoma SKOV‐3, prostate cancer LNCaP, or immortalized human embryonic kidney HEK 293 cells for 1 h. For the modified ELISA, bound peptides were probed by HRP‐conjugated streptavidin, and detected spectrophotometrically at 405 nm after the addition of ABTS. For fluorescent microscopy, bound peptides were detected by FITC‐conjugated streptavidin. Results showed that MCA1 and MCA2 exhibited significantly (p<0.01) higher binding to Mia Paca‐2 cells, compared to J18 and DMSO, while no binding was detected to hTERT‐HPNE, SKOV‐3, LNCaP, or HEK 293 cells. Fluorescent microscopy validated these results by showing significantly (p<0.0001) increased fluorescent intensity of MCA1 and MCA2 to Mia Paca‐2. However, only minimal fluorescent intensity was observed to hTERT‐HPNE, SKOV‐3, LNCaP, or HEK 293 cells. This study indicates that peptides MCA1 and MCA2 specifically target pancreatic cancer cells. Hence, these ligands may be utilized in further studies to improve the detection of pancreatic cancer.