A Multimodal Imaging Approach for Longitudinal Evaluation of Bladder Tumor Development in an Orthotopic Murine Model

RESEARCH ARTICLE A Multimodal Imaging Approach for Longitudinal Evaluation of Bladder Tumor Development in an Orthotopic Murine Model Chantal Scheepbouwer1, Sandra Meyer2, Maroeska J. Burggraaf1,3, Jithin Jose2, Carla F. M. Molthoff1* a11111 1 Department of Radiology & Nuclear Medicine, VU University Medical Center, Amsterdam, The Netherlands, 2 FUJIFILM VisualSonics Inc., Amsterdam, The Netherlands, 3 Department of Medical Microbiology and Infection control, Amsterdam, The Netherlands * Abstract OPEN ACCESS Citation: Scheepbouwer C, Meyer S, Burggraaf MJ, Jose J, Molthoff CFM (2016) A Multimodal Imaging Approach for Longitudinal Evaluation of Bladder Tumor Development in an Orthotopic Murine Model. PLoS ONE 11(8): e0161284. doi:10.1371/journal. pone.0161284 Editor: Robert M. Hoffman, AntiCancer Inc., UNITED STATES Received: January 28, 2016 Accepted: June 28, 2016 Published: August 17, 2016 Copyright: © 2016 Scheepbouwer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Bladder cancer is the fourth most common malignancy amongst men in Western industrialized countries with an initial response rate of 70% for the non-muscle invasive type, and improving therapy efficacy is highly needed. For this, an appropriate, reliable animal model is essential to gain insight into mechanisms of tumor growth for use in response monitoring of (new) agents. Several animal models have been described in previous studies, but so far success has been hampered due to the absence of imaging methods to follow tumor growth non-invasively over time. Recent developments of multimodal imaging methods for use in animal research have substantially strengthened these options of in vivo visualization of tumor growth. In the present study, a multimodal imaging approach was addressed to investigate bladder tumor proliferation longitudinally. The complementary abilities of Bioluminescence, High Resolution Ultrasound and Photo-acoustic Imaging permit a better understanding of bladder tumor development. Hybrid imaging modalities allow the integration of individual strengths to enable sensitive and improved quantification and understanding of tumor biology, and ultimately, can aid in the discovery and development of new therapeutics. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Introduction Funding: Fujifilm VisualSonics Inc. did not play a role in the study design, decision to publish, or preparation of the manuscript. FujiFilm VisualSonics Inc. did not provide any salary to the authors CS, MJB and CFMM. FUJIFILM VisualSonics Inc. only provided access and use of their equipment and both co-author’s (SM, JJ) helped with operating the system, explaining the system, how to collect the data without further interest related to the outcome of the data. The funder provided support in the form of Bladder cancer is the fourth most common malignancy amongst men in Western industrialized countries [1]. As the incidence increases with age, the risk of developing bladder cancer is highest in men above 60 years of age with cigarette smoking recognized as the most important risk factor in developing bladder cancer [2]. Even though there is a high variety in the natural course of the disease, approximately 75–85% of bladder tumors are presented as non-muscle invasive limited to the mucosa or submucosa at time of diagnosis [3]. Standard therapy of high-grade non-muscle invasive bladder cancer consists of transurethral resection of the bladder tumor (TURBT), followed by intravesical immunotherapy with mycobacterium bovis BCG. This approach is superior to surgical treatment alone with an initial complete response rate of PLOS ONE | DOI:10.1371/journal.pone.0161284 August 17, 2016 1 / 15 Multimodality Imaging of Bladder Cancer salaries for authors SM and JJ, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: SM & JJ are employees of FujiFilm VisualSonics Inc. This does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials. 70% [3–5]. As bladder cancer is a heterogeneous disease, the immune response and tolerance to BCG therapy can vary greatly among patients, so predicting a specific therapeutic outcome has proven to be challenging. Consequently, intensive monitoring is needed, which is reflected in high costs, making bladder cancer one of the most expensive cancer types [6]. Enhancing immunostimulatory properties would be an important step forward in promoting efficacy of BCG therapy for non-muscle invasive bladder cancer [7]. A reliable, simple, and reproducible animal model is essential to gain insight into mechanisms of tumor growth and spread, so these improved therapeutic strategies can be developed for human use [8]. Orthotopic implantation of bladder cancer cells in syngeneic immunocompetent animals has been extensively used in the preclinical testing of altered BCG therapies and research focused on the development of bladder cancer [9, 10]. MB49-luc cells, a carcinogen induced transitional cell carcinoma derived from C57BL/6 mice, are widely used to study these bladder tumors and replicate human urothelial carcinoma cell lines in many molecular and phenotypical responses to BCG in vitro [11]. Within this model, several tumor cell implantation procedures exist to establish in vivo tumor growth. Still challenging is to longitudinally monitor these bladder tumors, as this requires accurate visualization of tumor growth. Standard imaging methods in animal studies include radiography (i.e. X-rays), abdominal ultrasound, PET-CT and MRI, however sensitivity for bladder tumor detection is limited [12]. Specifically in radiographic imaging, resolution remains low and limits their utility. PET-CT and MRI greatly improve resolution and can provide 3-dimensional anatomic information [13], but still malignant lesions can be easily missed or misinterpreted. Over the last years new modalities based on Optical and Ultrasound are emerging for research purposes due to the shortcomings in standard bladder cancer imaging [14, 15]. Fluorescence Imaging (FLI) is one of these new modalities, commonly used to monitor in vivo processes with reporters such as green fluorescent protein (GFP), red fluorescent protein (RFP) and near-infrared proteins [16]. Complexity and lower sensitivity due to autofluorescence are the main disadvantages of these reporters [17]. Bioluminescence (BLI) is another commonly used optical imaging modality for both in vitro and in vivo measurements of molecular and cellular processes. Unlike fluorescence, BLI does not require excitation, thereby avoiding the autofluorescence background signal [18]. BLI is b (...truncated)