High Resolution Ultrasound and Photoacoustic Imaging of Orthotopic Lung Cancer in Mice: New Perspectives for Onco-Pharmacology

RESEARCH ARTICLE High Resolution Ultrasound and Photoacoustic Imaging of Orthotopic Lung Cancer in Mice: New Perspectives for OncoPharmacology Florian Raes1*, Julien Sobilo1, Marilyne Le Mée1, Stéphanie Rétif1, Sharuja Natkunarajah1, Stéphanie Lerondel1, Alain Le Pape1,2 1 PHENOMIN-TAAM-UPS44, CIPA (Centre d’Imagerie du Petit Animal), CNRS Orléans, France, 2 INSERM U1100, CEPR, University of Tours, France * a11111 Abstract Objectives OPEN ACCESS Citation: Raes F, Sobilo J, Le Mée M, Rétif S, Natkunarajah S, Lerondel S, et al. (2016) High Resolution Ultrasound and Photoacoustic Imaging of Orthotopic Lung Cancer in Mice: New Perspectives for Onco-Pharmacology. PLoS ONE 11(4): e0153532. doi:10.1371/journal.pone.0153532 Editor: Bernhard Ryffel, French National Centre for Scientific Research, FRANCE Received: February 2, 2016 We have developed a relevant preclinical model associated with a specific imaging protocol dedicated to onco-pharmacology studies in mice. Materials and Methods We optimized both the animal model and an ultrasound imaging procedure to follow up longitudinally the lung tumor growth in mice. Moreover we proposed to measure by photoacoustic imaging the intratumoral hypoxia, which is a crucial parameter responsible for resistance to therapies. Finally, we compared ultrasound data to x-ray micro computed tomography and volumetric measurements to validate the relevance of this approach on the NCI-H460 human orthotopic lung tumor. Accepted: March 30, 2016 Published: April 12, 2016 Copyright: © 2016 Raes 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. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Results This study demonstrates the ability of ultrasound imaging to detect and monitor the in vivo orthotopic lung tumor growth by high resolution ultrasound imaging. This approach enabled us to characterize key biological parameters such as oxygenation, perfusion status and vascularization of tumors. Conclusion Such an experimental approach has never been reported previously and it would provide a nonradiative tool for assessment of anticancer therapeutic efficacy in mice. Considering the absence of ultrasound propagation through the lung parenchyma, this strategy requires the implantation of tumors strictly located in the superficial posterior part of the lung. PLOS ONE | DOI:10.1371/journal.pone.0153532 April 12, 2016 1 / 15 High Resolution Ultrasound and Photoacoustic Imaging of Orthotopic Lung Cancer in Mice 1. Introduction Because lung cancer still remains the leading cause of cancer-related death, there is a need to develop more accurate and predictive preclinical protocols and relevant cancer models. Orthotopic lung cancer models have the advantage of being more predictive regarding clinical relevance, including the ability of primary tumors to develop spontaneous metastasis but also more predictive regarding the therapeutic response. The implementation and exploration of such orthotopic models allows us to improve our understanding of the biology of cancer to interpret preclinical in vivo results in humans, especially for the potential therapeutic response of anticancer agents. Studies taking into account more representative parameters from clinical situations, particularly hypoxia, are of great interest to boost innovation for new anticancer treatments [1–3]. One important parameter in oncology is tumor volume assessment before but also during treatments [4]. In a clinical setting, the pulmonary tumor measurements are predominantly performed with X-ray computed tomography (CT) imaging [5]. For pulmonary preclinical oncology, imaging objectives are to improve the accuracy for determining volumes, without irradiation effects or interferences with the anti-tumor response. Thanks to technological developments for both X-ray sources and detectors, CT dedicated to small animal imaging provides a sub-millimetric resolution making this tool efficient for the characterization of lung tumor volumes. However, the radiation dose delivered to tumors remains a limitation, especially when a study requires repeated exams [6]. Bioluminescence imaging (BLI) brought about a revolution in preclinical oncology research but this method provides quantitative information about tumor proliferation without any possible sizing. Moreover, since BLI is dependent upon metabolism, it is not reliable when tumors become hypoxic [7]. In clinical practices, lung ultrasound (US) has been gaining in popularity among clinicians and has become an essential tool in critically ill management [8,9]. However regarding human pulmonary oncology, there is no possible use of US except for invasive endoscopy of cancer nodules and lymph nodes [10,11]. The main limitation of endoscopy and ultrasound is the detection of these nodules if proximity with the probe is not close enough. This access limitation is due to the absence of US propagation through the lung parenchyma because of air. On the contrary, preclinical high resolution US and photoacoustic imaging (PAI) are promising modalities to investigate lung tumor progression and hypoxia respectively but considering the specific constraints of US, the implantation of tumors in the superficial posterior lung region is required. The large cell NCI-H460 orthotopic lung carcinoma model that we chose to improve, is based on a study by Gagnadoux et al. [12], leading to the growth of a solitary intrapulmonary nodule located near the posterior diaphragmatic surface. Here we recommend refining such an onco-pharmacology protocol in a translational approach while overcoming physical US limitations allowing lung tumor exploration. In this longitudinal study we assessed orthotopic lung tumor volumes in mice by in vivo 3D US and also hypoxic tumor status by PAI. Furthermore, we compared our data to different imaging methods with the aim to validate this new approach. 2. Materials and Methods 2.1 Ethics Statement All procedures on animals were performed in accordance with European ethical guidelines (European directives 2010/63/EU) and were approved by the Regional Committee for Animal PLOS ONE | DOI:10.1371/journal.pone.0153532 April 12, 2016 2 / 15 High Resolution Ultrasound and Photoacoustic Imaging of Orthotopic Lung Cancer in Mice Care and Ethics in Animal Experiments (C2EA-03 Comité d’éthique en expérimentation animale Campus CNRS d’Orléans). 2.2 Cell Culture The NCI-H460-luc2 human lung cancer cell line was obtained from Perkin Elmer (France). This cancer cell line was maintained according to the supplier’s instructions. 2.3 Animals Pathogen-free 6 to (...truncated)