Longitudinal Imaging Studies of Tumor Microenvironment in Mice Treated with the mTOR Inhibitor Rapamycin Keita Saito 1 , Shingo Matsumoto 1 , Hironobu Yasui 1,2 , Nallathamby Devasahayam 1 , Sankaran Subramanian 1 , Jeeva P. Munasinghe 3 , Vyomesh Patel 4 , J. Silvio Gutkind 4 , James B. Mitchell 1 , Murali C. Krishna 1 * 1 Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America, 2 Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan, 3 National Institute of Neurological Disorder and Stroke, National Institutes of Health, Bethesda, Maryland, United States of America, 4 National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America Abstract Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII) by electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500–750 mm 3 ) and measurements of tumor pO 2 and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO 2 levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO 2 ,10 mm Hg) in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy. Citation: Saito K, Matsumoto S, Yasui H, Devasahayam N, Subramanian S, et al. (2012) Longitudinal Imaging Studies of Tumor Microenvironment in Mice Treated with the mTOR Inhibitor Rapamycin. PLoS ONE 7(11): e49456. doi:10.1371/journal.pone.0049456 Editor: Kwan Man, The University of Hong Kong, Hong Kong Received June 19, 2012; Accepted October 9, 2012; Published November 20, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by the Intramural Research Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: murali@helix.nih.gov Introduction Multiple genetic and epigenetic events are known to result in the dysregulation of several signaling pathways that have an impact on neoplastic disease progression, such as squamous cell carcinomas (SCC) [1,2]. One such pathway, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway is frequently activated in many cancers, and controls cellular metabolism, growth, and proliferation [3–6]. The mammalian target of rapamycin (mTOR) is an atypical serine/ threonine kinase, which acts downstream of PI3K/Akt and, therefore has become an attractive therapeutic target [7–10]. It follows that inhibitors of mTOR, such as rapamycin and its derivatives are currently being evaluated for molecular targeted therapy of neoplastic diseases [9]. The inhibition of mTOR with its specific allosteric inhibitor, rapamycin, provokes a rapid death of squamous xenografts, resulting in tumor regression [11]. The molecular basis of this is currently an active area of research [12]. For example, a recent study using a reverse-pharmacology approach, which involved the expression of a rapamycin-insensitive form of mTOR in squamous cancer cells, showed that cancer cells are the primary targets of rapamycin in vivo, and that mTOR controls the expression of hypoxia-inducible factor-1a (HIF-1a), a key transcription factor that orchestrates the cellular response to hypoxic stress, including the regulation of the expression of angiogenic factors, thus providing a likely mechanism by which rapamycin exerts its tumor suppressive and antiangiogenic effects [13]. Blocking mTOR pathway in SCC tumors was also shown to prevent accumulation of HIF-1a resulting in inhibition of processes involved in glucose metabolism as well as decrease in pro- angiogenic factors such as vascular endothelial growth factor (VEGF) [13]. Recent studies using magnetic resonance imaging (MRI) showed that treatment with mTOR inhibitors results in strong PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49456