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. 2008 Apr 15;5(4):e85.
doi: 10.1371/journal.pmed.0050085.

Targeted nanoparticles for imaging incipient pancreatic ductal adenocarcinoma

Kimberly A Kelly  1 , Nabeel Bardeesy, Rajesh Anbazhagan, Sushma Gurumurthy, Justin Berger, Herlen Alencar, Ronald A Depinho, Umar Mahmood, Ralph Weissleder
Affiliations

Targeted nanoparticles for imaging incipient pancreatic ductal adenocarcinoma

Kimberly A Kelly et al. PLoS Med. .

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) carries an extremely poor prognosis, typically presenting with metastasis at the time of diagnosis and exhibiting profound resistance to existing therapies. The development of molecular markers and imaging probes for incipient PDAC would enable earlier detection and guide the development of interventive therapies. Here we sought to identify novel molecular markers and to test their potential as targeted imaging agents.

Methods and findings: Here, a phage display approach was used in a mouse model of PDAC to screen for peptides that specifically bind to cell surface antigens on PDAC cells. These screens yielded a motif that distinguishes PDAC cells from normal pancreatic duct cells in vitro, which, upon proteomics analysis, identified plectin-1 as a novel biomarker of PDAC. To assess their utility for in vivo imaging, the plectin-1 targeted peptides (PTP) were conjugated to magnetofluorescent nanoparticles. In conjunction with intravital confocal microscopy and MRI, these nanoparticles enabled detection of small PDAC and precursor lesions in engineered mouse models.

Conclusions: Our approach exploited a well-defined model of PDAC, enabling rapid identification and validation of PTP. The developed specific imaging probe, along with the discovery of plectin-1 as a novel biomarker, may have clinical utility in the diagnosis and management of PDAC in humans.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Isolation and Validation of PDAC-Specific Peptides
(A) After selection and subtraction, 30 individual phage clones were picked, amplified, and analyzed for affinity and specificity via ELISA. The heat map depicts affinity (mean absorbance values of indicated clones in ELISA assay) and specificity (ratio of clones' affinity to tumor cells versus normal ductal cells). Data are displayed in terms of higher rankings (green) to lower rankings (red). (B) FITC-labeled clone 27 binds specifically to mouse PDAC cells. FITC-27 was incubated with PDAC or normal mouse ductal cells then analyzed via FACS. (C) FITC-27 or FITC unrelated phage clone (negative control) were incubated with the indicated cells then analyzed via FACS. Data plotted are the mean fluorescence units obtained from FACS analysis.
Figure 2
Figure 2. Clone 27 Detects Human PDAC
FITC-labeled clone 27 (PDAC-27) (middle row) or wild-type phage (no peptide insert; bottom row) were incubated with frozen sections of the indicated tissue. H&E staining of the same tissues is shown in the top row. Results shown are representative images.
Figure 3
Figure 3. In Vivo Validation of Clone-27
(A) Wild type, (B) 29-wk-old Kras/p16+/−, and (C) 12-wk-old Kras/p53L/+ mice were injected with Cy5.5-labeled phage clone 27 (PDAC-27) and SYTOX green (nonspecific cell-labeling agent) then imaged via intravital confocal microscopy. Correlative Histology: Pancreata from intravital imaging experiments were embedded in OCT, frozen, and stained with HE or anti-M13 antibody. Black boxes correspond to regions of PanIN (B) or PDAC (C) and are magnified in the anti-M13 photomicrograph. Red boxes correspond to uninvolved adjacent regions (HE and inset in anti-M13 photomicrograph).
Figure 4
Figure 4. Identification and Validation of Plectin-1 Binding to Clone-27
Mouse PDAC cells were incubated with either sulfo-SAED and biotin-modified phage clone-27 or control phage, exposed to light, and lysates were incubated with streptavidin-coated beads. (A) Precipitated protein was eluted with dithiothreitol (DTT) then run on an SDS-PAGE gel and silver stained (left). Far Western (right): PDAC lysates were loaded onto an SDS-PAGE gel then transferred and analyzed with clone-27 or control (no insert) biotinylated phage. (B) A band corresponding to clone-27 affinity-purified protein from (A) was cut from the gel, digested with trypsin, and analyzed via mass spectroscopy. (C) Affinity-purified protein from (A) was run on a SDS-PAGE gel, transferred to PVDF, and analyzed for the presence of plectin-1 with antibody to plectin-1. (D) 293T cells, human umbilical vein endothelial cells (HUVECs), mouse PDAC cells, mouse normal duct cells, Paca-2 cells (human PDAC), and normal human duct cells were subcellularly fractionated, and the components probed for the presence of plectin-1. (E) Pancreata from wild-type (left), 29-wk old Kras/p16+/− (center), and 12-wk old Kras/p53 (right) mice were embedded in OCT, frozen, and stained with anti-plectin-1 antibody. (F) Competition experiment: mouse PDAC cells were incubated with FITC-labeled clone 27 and either plectin-1 antibody or vehicle then analyzed via FACS for the ability of clone 27 to bind.
Figure 5
Figure 5. Fluorescence Imaging of PDAC Using PTP-NP or Control-NP
(A) Schematic of conjugation of PTP to NP. Control-NP is synthesized the same way with substitution of control peptide for PTP. (B) Intravital confocal microscopy of early pancreatic lesions imaged using PTP-NP (red, top) or control-NP (red, bottom) and AF750-labeled bloodpool agent (blue). (C) Low-magnification view of pancreatic fluorescence shows distribution of PTP-NP in distinct areas of the pancreas. White light overlay provides anatomic correlation (left). Dotted line outlines the pancreas. (D) Biodistribution of PTP-NP and control-NP.
Figure 6
Figure 6. MRI and Correlative Histology
(A and B) Three adjacent slices (A) from an ex vivo MRI of the pancreas from a 9-wk-old Kras/p53L/+ mouse demonstrates focal nanoparticle uptake (one example, yellow arrow), which corresponds to tumor seen on correlated HE sections (B), but not to regions of ductal metaplasia or normal pancreas (labeled). (C) Fluorescence microscopy of adjacent sections demonstrate uptake of Cy5.5-labeled PTP-NP in regions of tumor (left) but not in adjacent tissue (right).
See this image and copyright information in PMC

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