Targeted noninvasive imaging of EGFR-expressing orthotopic pancreatic cancer using multispectral optoacoustic tomography

Abstract

Detection of orthotopic xenograft tumors is difficult due to poor spatial resolution and reduced image fidelity with traditional optical imaging modalities. In particular, light scattering and attenuation in tissue at depths beyond subcutaneous implantation hinder adequate visualization. We evaluate the use of multispectral optoacoustic tomography (MSOT) to detect upregulated epidermal growth factor (EGF) receptor in orthotopic pancreatic xenografts using a near-infrared EGF-conjugated CF-750 fluorescent probe. MSOT is based on the photoacoustic effect and thus not limited by photon scattering, resulting in high-resolution tomographic images. Pancreatic tumor-bearing mice with luciferase-transduced S2VP10L tumors were intravenously injected with EGF-750 probe before MSOT imaging. We characterized probe specificity and bioactivity via immunoblotting, immunocytochemistry, and flow cytometric analysis. In vitro data along with optical bioluminescence/fluorescence imaging were used to validate acquired MSOT in vivo images of probe biodistribution. Indocyanine green dye was used as a nonspecific control to define specificity of EGF-probe accumulation. Maximum accumulation occurred at 6 hours postinjection, demonstrating specific intratumoral probe uptake and minimal liver and kidney off-target accumulation. Optical bioluminescence and fluorescence imaging confirmed tumor-specific probe accumulation consistent with MSOT images. These studies demonstrate the utility of MSOT to obtain volumetric images of ligand probe biodistribution in vivo to detect orthotopic pancreatic tumor lesions through active targeting of the EGF receptor.

Figure 1

EGF-750 Probe Characterization. A, UV-Visible spectra of EGF-750 probe (red) and CF-750 dye (blue) measured to confirm that the absorbance signature of the dye is not significantly altered by conjugation. B, bioactivity of EGF-750 was verified by its ability to phosphorylate EGFR receptor tyrosine kinase in HeLa cells, measured by immunodetection of phosphorylated EGFR via Western blot with pY1068 antibody. Cells treated with either unlabeled EGF (EGF) or EGF-750 (Labeled EGF) at a concentration of 10 ng/ml showed similar levels of tyrosine kinase activation of EGFR, indicating ligand conjugation had nominal effect on probe bioactivity.

Figure 2

Expression of EGFR in Pancreatic Cancer Cell Lines. A, expression of EGFR was confirmed in S2VP10, S2CP9, MiaPaCa-2, PANC-1, and SKOV3.ip1 (EGFR positive control) cell lines; minimal expression was detected in NIH/3T3 (EGFR negative control) and MCF7 cells. B, dosimetry was performed using band intensity values for EGFR in comparison to actin. Values demonstrate the relative abundance of EGFR in each cell line.

Figure 3

Uptake of EGF-750 Probe detected via Immunocytochemistry. To demonstrate retention of biopotency of EGF in a validated fluorescent probe complex, Texas Red-EGF was incubated with three pancreatic cancer cell lines and analyzed at various time points (0, 10, and 30 min). S2VP10 and MiaPaCa-2 cell lines show internalization of labeled EGFR-Texas Red-EGF complex (red), while MCF7 (low EGFR-expressing) cells exhibit significantly reduced receptor-ligand internalization even with similar levels of endocytosis activity, apparent via labeled EEA1 antibody (green).

Figure 4

Extracellular accumulation of EGF-750 probe was assessed using flow cytometry. A, percentages represent proportion of APC-Cy7-positive tumor cells within gated area; values are representative of the mean of two experimental replicates. B, Wilcoxon signed-rank test determined differences in MFI was not significant among treatments (p=0.0613).

Figure 5

Optical Imaging of Pancreatic Adenocarcinoma. Multispectral optoacoustic detection of pancreatic adenocarcinoma from a mouse at seven days post-implantation, with EGF-750 biodistribution shown at 6 h post injection. High-resolution (75 μm) backprojection reconstruction and multispectral processing using linear regression in MATLAB was used to create reconstructed image. A, serial images of pancreatic tumor mass ranging from 38–43 mm illustrate capability to precisely define region of probe distribution. B, maximum intensity projection of reconstructed image to determine 3D conformation of tumor in xyz-space, enabling measurement of total tumor volume. C, upper panels show fluorescence imaging of EGF-750 probe-labelled pancreatic tumor; as a control, unconjugated inert CF-750 dye was shown to not accumulate preferentially in the tumor. In the lower panels, detection of pancreatic tumor was confirmed using S2VP10L cells (Luc positive) through bioluminescence imaging on the AMI. D, in vivo MSOT images were further validated through fluorescence imaging-detection of EGF-750 probe in pancreatic tumor and liver tissues analyzed ex vivo on the AMI; ex vivo scan of organs confirmed the accumulation in the pancreas and lack of fluorescent EGF-750 probe distribution in the liver, corresponding to MSOT signal quantified for the liver and kidney at <10 MSOT signal units (a.u.).