Ovarian Cancer Targeting Phage Clones for In Vivo Near‐Infrared Optical Imaging

Abstract

Ovarian cancer is the fifth leading cause of cancer deaths in women, which is directly related to inadequate diagnostic methods. Phage display technology may be used to develop peptide‐displaying filamentous phage as cancer detection and imaging agents. Such modalities offer advantages in regard to the high specificity and affinity of peptides, as well as increased avidity by displaying multiple peptides per virion. Additionally, the large size of filamentous phage (0.9 μm) allows for signal amplification by labeling with multiple imaging moieties.Phage display technology was previously used to select phage clones with specific binding to human ovarian cancer cells (SKOV‐3). In short, a 15‐mer fUSE5 phage display library was injected into the tail vein of a nude mouse without tumors, and unbound phage were collected after 15 min of circulation. This pre‐cleared library was then subjected to four rounds of selection in nude mice carrying xenografted SKOV‐3 tumors. During each round, phage were allowed to circulate for 1 h, after which the mouse was sacrificed and the tumor was excised. Tumor‐bound phage were collected by 3‐[(3‐cholamideopropyl) dimethylammonio]‐1‐propanesulfonate (CHAPS) elution, and displayed peptides were identified by DNA sequencing. From the last round of selection, 31 phage clones were identified, and each was subjected to micropanning experiments, in which the binding ratio for SKOV‐3‐to‐normal human ovarian (HS‐832) cells were evaluated. These results showed that phage clones pJ18 and pJ24 exhibited specificity for ovarian cancer cells, exhibiting SKOV‐3‐to‐HS‐832 ratios of 6.57 and 3.55, respectively.To evaluate the tumor targeting capabilities, pJ18, pJ24, and WT phage were labeled with a near‐infrared fluorophore (AF680), and employed in biodistribution and optical imaging studies in mice bearing xenografted SKOV‐3 tumors. In brief, mice were injected with 1012 virions of phage, and the animals were sacrificed after 4 h. The tumor, organs and tissues were excised and fluorescent intensity was measured using a Xenogen IVIS 200 Imaging System. Tumor uptake of pJ24 was significantly higher compared to WT phage. For all phage (pJ18, pJ24, and WT), the liver showed increased fluorescent uptake compared to other organs, indicating that the phage were excreted through the reticuloendothelial system. Next, mice carrying xenografted SKOV‐3 tumors were injected with 1012 virions of AF680‐labeled phage. The animals were imaged pre‐injection (0 h), and after 2 h and 4 h of circulation. The results showed that the ovarian tumors were easily localized and exhibited ample tumor‐to‐background contrast. Further, the tumor fluorescent intensity of pJ18 and pJ24, was significantly higher (p<0.001, p<0.05, respectively) in comparison to WT after 2 h. The fluorescent intensity was lower for both clones after 4 h, however, the uptake of pJ18 remained significantly higher (p<0.01) compared to WT.Taken together, these results indicate that phage clones pJ18 and pJ24 target and image ovarian SKOV‐3 tumors in vivo. This demonstrates the potential us of these phage clones as ovarian cancer detection and imaging agents.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.