Oncology/Endocrine
Orthotopic pancreatic tumors detected by optoacoustic tomography using Syndecan-1

https://doi.org/10.1016/j.jss.2014.06.045Get rights and content

Abstract

Background

Advances in small animal imaging have improved the detection and monitoring of cancer in vivo; although with orthotopic models, precise localization of tumors remains a challenge. In this study, we evaluated multispectral optoacoustic tomography (MSOT) as an imaging modality to detect pancreatic adenocarcinoma in an orthotopic murine model.

Methods

In vitro binding of Syndecan-1 probe to the human pancreatic cancer cell line S2VP10 was evaluated on flow cytometry. For in vivo testing, S2VP10 cells were orthotopically implanted into the pancreas of severe combined immunodeficiency mice. At 7 d after implantation, the mice were intravenously injected with Syndecan-1 probe, and tumor uptake was evaluated with MSOT at multiple time points. Comparison was made with a free-dye control, indocyanine green (ICG). Probe uptake was verified ex vivo with fluorescent imaging.

Results

Syndecan-1 probe demonstrated partial binding to S2VP10 cells in vitro. In vivo, Syndecan-1 probe preferentially accumulated in the pancreas tumor (480 MSOT a.u.) compared with off-target organs, including the liver (67 MSOT a.u.) and kidney (96 MSOT a.u.). Syndecan-1 probe accumulation peaked at 6 h (480 MSOT a.u.), whereas the ICG control dye failed to demonstrate similar retention within the tumor bed (0.0003 MSOT a.u.). At peak accumulation, signal intensity was 480 MSOT a.u., resulting in several times greater signal in the tumor bed than in the kidney or liver. Ex vivo fluorescent imaging comparing tumor signal with that within off-target organs confirmed the in vivo results.

Conclusions

MSOT demonstrates successful accumulation of Syndecan-1 probe within pancreatic tumors, and provides high-resolution images, which allow noninvasive, real-time comparison of signal within individual organs. Syndecan-1 probe preferentially accumulates within a pancreatic adenocarcinoma model, with minimal off-target effects.

Introduction

Improving the diagnosis, treatment, and monitoring of cancer remains a primary focus of research today, and significant breakthroughs have been made with new developments in targeted therapy and preclinical molecular imaging. Advances in imaging have contributed by providing real-time information with a high degree of spatial resolution and anatomic detail in vivo [1], [2]. The addition of targeted fluorescent probes allows visualization of individual tissues or cellular processes, including tumors [3]. By conjugating fluorescent proteins to tumor-specific ligands, fluorescent probes can specifically bind and identify tumor tissue [4].

However, depending on the imaging modality, precise characterization of a tumor's location and features can be difficult using fluorescent probes in vivo. In particular, two-dimensional planar imaging is limited by light scattering and signal attenuation, which results in a lack of specificity between fluorescent signal and the internal anatomy [5]. Multispectral optoacoustic tomography (MSOT) is an emerging technology that offers high-resolution in vivo imaging of tumors that addresses the limitations inherent in the traditional planar imaging [6]. MSOT takes advantage of the photoacoustic effect, a process whereby particles excited by pulsed light emit an acoustic signal detectable by ultrasound. Using a 5 MHz tomographic ultrasound array, the MSOT system detects a wide spectrum of sound waves that are emitted after tissue becomes excited by wavelengths of pulsed near-infrared light. Individual tissues will absorb light and emit sound waves best at different wavelengths, and integrating the acoustic signals that result from multiple excitation wavelengths yield a high-resolution ultrasound image. Cross-sectional images with resolution up to 100–150 μm can be obtained, and the distribution of fluorescent molecules can be isolated from the background signal [7]. In this way, fluorescent probes serve as contrast agents, and tumor-targeted probes can give precise images that relate the tumor to the surrounding internal anatomy [8]. Prior studies have used MSOT and fluorescent markers to evaluate subcutaneous tumors, glioblastoma, and renal perfusion in small animals [8], [9], [10].

Although MSOT has been used preclinically for several tumor models, there is little experience with its use to identify abdominal tumors at depth, such as orthotopic pancreatic cancer xenografts. For this study, we used both MSOT and traditional planar imaging to evaluate the in vivo binding of a fluorescent probe targeted to a murine orthotopic pancreatic tumor. Our primary goal was to compare imaging modalities and evaluate probe accumulation, including distribution within off-target organs, with the underlying hypothesis that MSOT would provide superior tumor imaging. As a secondary goal, we evaluated recombinant Syndecan-1 as the ligand of a targeted probe for pancreatic tumors. Syndecan-1 is upregulated in pancreatic adenocarcinoma, and recombinant syndecan-1 has been shown to bind overexpressed αvβ3 integrin on tumor cells [11], [12]. To demonstrate tumor targeting, we compared Syndecan-1 with a nontargeted indocyanine green (ICG) reference dye. Syndecan-1 probe binding within the pancreas tumor compared with off-target organs was further confirmed using ex vivo imaging.

Section snippets

Cell culture

The human pancreatic cancer cell line S2VP10 was a gift from M. Hollingsworth (University of Nebraska, Omaha, NE). The human glioma cell line U251-MG was originally obtained from Dr D.D. Bigner (Duke University, Durham, NC) to serve as the positive control for Syndecan-1 binding [13]. Cells were grown at 37°C and 5% CO2 in Dulbecco modified Eagle medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 1% L-glutamine

Results

Successful in vitro probe binding was assessed via flow cytometry. Probe demonstrated partial binding over control cells, and the 100 nM probe concentration yielded 23.9% binding in vitro. Partial in vitro binding (48.6%) was also observed with the positive control U251 cells, which are known to express high levels of αvβ3 (Fig. 1) [13]. In vivo binding was then assessed with traditional fluorescent imaging. Focal accumulation of Syndecan-1 probe was evident early after injection, but was

Discussion

This study is one of the first to demonstrate the feasibility of using MSOT for the detection and monitoring of orthotopic pancreatic tumors in vivo. MSOT offers sufficient resolution at greater depth to overcome traditional imaging limitations and distinguishes accumulation within the tumor bed from off-target organs [17]. As our orthogonal images suggest, accumulation throughout the tumor parenchyma (Fig. 6) could be seen despite the intra-abdominal location of the tumor, allowing for precise

Acknowledgment

This work was supported by NIH grant CA139050.

C.W.K., S.H., and A.K., contributed to the data collection. C.W.K. wrote the article. M.E.E. contributed to the design. M.E.E. and L.R.M. did the revision of the article. LRM contributed to the concept of the article.

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