Detection of pancreatic ductal adenocarcinoma in mice by ultrasound imaging of thymocyte differentiation antigen 1

Abstract

Background & aims: Early detection of pancreatic ductal adenocarcinoma (PDAC) allows for surgical resection and increases patient survival times. Imaging agents that bind and amplify the signal of neovascular proteins in neoplasms can be detected by ultrasound, enabling accurate detection of small lesions. We searched for new markers of neovasculature in PDAC and assessed their potential for tumor detection by ultrasound molecular imaging.

Methods: Thymocyte differentiation antigen 1 (Thy1) was identified as a specific biomarker of PDAC neovasculature by proteomic analysis. Up-regulation in PDAC was validated by immunohistochemical analysis of pancreatic tissue samples from 28 healthy individuals, 15 with primary chronic pancreatitis tissues, and 196 with PDAC. Binding of Thy1-targeted contrast microbubbles was assessed in cultured cells, in mice with orthotopic PDAC xenograft tumors expressing human Thy1 on the neovasculature, and on the neovasculature of a genetic mouse model of PDAC.

Results: Based on immunohistochemical analyses, levels of Thy1 were significantly higher in the vascular of human PDAC than chronic pancreatitis (P = .007) or normal tissue samples (P < .0001). In mice, ultrasound imaging accurately detected human Thy1-positive PDAC xenografts, as well as PDACs that express endogenous Thy1 in genetic mouse models of PDAC.

Conclusions: We have identified and validated Thy1 as a marker of PDAC that can be detected by ultrasound molecular imaging in mice. The development of a specific imaging agent and identification of Thy1 as a new biomarker could aid in the diagnosis of this cancer and management of patients.

Keywords: CD90; Diagnostic Test; IHC; MB; Molecular Imaging; PDAC; Pancreatic Cancer; Thy1; WT; immunohistochemistry; microbubble; pancreatic ductal adenocarcinoma; thymocyte differentiation antigen 1; wild-type.

Figure 1

Overview of study design, from identification of novel human PDAC-neovasculature-associated imaging biomarker Thy1, to target validation, creation of human Thy1-targeted ultrasound contrast agent, generation of a novel orthotopic PDAC xenograft model expressing human Thy1 on its neovasculature, and in vivo testing of imaging properties of Thy1-targeted ultrasound contrast agent.

Figure 2

Immunohistochemistry (IHC) analysis of Thy1 staining in human pancreatic tissue samples. Examples of normal pancreas (A) and primary chronic pancreatitis (B) with no Thy1 staining on vasculature. (C). Example of positive Thy1-staining on vessels associated with PDAC. D. Summary of IHC scores on Thy1 stained tissues from normal pancreas (normal), chronic pancreatitis (CP), and pancreatic cancer (PC).

Figure 3

Evaluation of Thy1 expression on vascular endothelial cells. (A) Stably transfected cells and wild-type cells were assessed for human Thy1 expression by immunofluorescence staining. Clone 1 showed strong Thy1 staining, clone 2 showed low staining, and wild-type cells showed no Thy1 staining. (B) Human Thy1 expression levels on different cell types were quantitatively assessed by FACS analysis. Histogram overlay of signals from cells with different levels of Thy1 expression is shown. (C) Mean fluorescence intensity values are shown in bar graph. Error bars are ± standard deviations.

Figure 4

Dynamic cell culture binding assay of microbubbles (MB) in a parallel plate flow chamber setting. Phase-contrast bright-field micrographs show binding of MB_Thy1 and MB_Control (white spheres, arrowheads) to different cell types; binding could be substantially blocked by incubation of cells beforehand with an anti-human Thy1 antibody (quantitative date in Table 1).

Figure 5

In vivo ultrasound molecular imaging of orthotopic PDAC xenografts in mice and corresponding ex vivo immunofluorescence analysis. Transverse ultrasound images obtained in contrast mode following intravenous injection of MB_Thy1 and MB_Control show strong imaging signal in human Thy1-positive tumor and background signal in both types of control tumors (scale bar = 5 mm; color coded scale is shown for ultrasound molecular imaging signal in arbitrary units, a.u.). Note low imaging signal following MB_Control in all tumor types (green circles, region of interest). Corresponding immunofluorescence micrographs of merged double-stained sections (murine CD31, red; human thy1, green) confirm human Thy1 expression on neovasculature in Thy1-positive tumors (yellow) while both negative control tumors did not show human Thy1 staining on the neovasculature (scale bar = 50 µm).

Figure 6

In vivo ultrasound molecular imaging of a transgenic pancreatic tumor, chronic pancreatitis and normal pancreas tissue and corresponding ex vivo immunofluorescence analysis. A) Transverse US images obtained in contrast mode following intravenous injection of murine Thy1-targeted MB show strong imaging signal in pancreatic tumor and a low signal following MB_Control. Note low imaging signal following MB_Thy1 or MB_Control in chronic pancreatitis and normal pancreas tissue (scale bar = 5mm; color coded scale is shown for ultrasound molecular imaging signal in arbitrary units, a.u.). B) Tiled confocal micrographs of murine Thy1 expression in a transgenic pancreatic tumor (top), chronic pancreatitis (middle), and WT pancreas (bottom) with murine Thy1 in red and CD31 in green show expression of murine Thy1 on the tumor vasculature but no expression on the vasculature in chronic pancreatitis and normal WT tumor.