Hypoxic Tumor Environments Exhibit Disrupted Collagen I Fibers and Low Macromolecular Transport Samata M. Kakkad 1,3 , Marie-France Penet 1 , Alireza Akhbardeh 1 , Arvind P. Pathak 1,2 , Meiyappan Solaiyappan 1 , Venu Raman 1,2 , Dieter Leibfritz 3 , Kristine Glunde 1,2 , Zaver M. Bhujwalla 1,2 * 1 JHU ICMIC Program, Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 2 Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 3 Universita ¨t Bremen, Fachbereich 2, Bremen, Germany Abstract Hypoxic tumor microenvironments result in an aggressive phenotype and resistance to therapy that lead to tumor progression, recurrence, and metastasis. While poor vascularization and the resultant inadequate drug delivery are known to contribute to drug resistance, the effect of hypoxia on molecular transport through the interstitium, and the role of the extracellular matrix (ECM) in mediating this transport are unexplored. The dense mesh of fibers present in the ECM can especially influence the movement of macromolecules. Collagen 1 (Col1) fibers form a key component of the ECM in breast cancers. Here we characterized the influence of hypoxia on macromolecular transport in tumors, and the role of Col1 fibers in mediating this transport using an MDA-MB-231 breast cancer xenograft model engineered to express red fluorescent protein under hypoxia. Magnetic resonance imaging of macromolecular transport was combined with second harmonic generation microscopy of Col1 fibers. Hypoxic tumor regions displayed significantly decreased Col1 fiber density and volume, as well as significantly lower macromolecular draining and pooling rates, than normoxic regions. Regions adjacent to severely hypoxic areas revealed higher deposition of Col1 fibers and increased macromolecular transport. These data suggest that Col1 fibers may facilitate macromolecular transport in tumors, and their reduction in hypoxic regions may reduce this transport. Decreased macromolecular transport in hypoxic regions may also contribute to poor drug delivery and tumor recurrence in hypoxic regions. High Col1 fiber density observed around hypoxic regions may facilitate the escape of aggressive cancer cells from hypoxic regions. Citation: Kakkad SM, Penet M-F, Akhbardeh A, Pathak AP, Solaiyappan M, et al. (2013) Hypoxic Tumor Environments Exhibit Disrupted Collagen I Fibers and Low Macromolecular Transport. PLoS ONE 8(12): e81869. doi:10.1371/journal.pone.0081869 Editor: Patrick A. Singleton, University of Chicago, Department of Medicine, United States of America Received July 19, 2013; Accepted October 20, 2013; Published December 12, 2013 Copyright: ß 2013 Kakkad 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. Funding: This work was supported by NIH R01CA136576, R01CA138515, R01CA73850, R01CA82337, P50CA103175, and P30CA006973. 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: zaver@mri.jhu.edu Introduction Tumors display abnormal physiological environments such as hypoxia, which primarily arise from their abnormal and chaotic vasculature [1]. Hypoxia is associated with increased resistance to radiation and chemotherapy, and with a more aggressive phenotype [1]. The discovery of the hypoxia inducible factor (HIF), and the identification of hypoxia response elements (HREs) as transcriptional controls in multiple genes [2], is continuing to unravel the critical role of hypoxia in influencing cancer progression and metastasis. The molecular mechanisms underly- ing the cascade of changes induced by hypoxia and the HIF-axis have attracted significant attention [2], but the functional impact of hypoxia on the tumor extracellular matrix (ECM) and on the transport of macromolecules in the tumor interstitium is relatively unexplored. Our purpose here was to investigate the role of hypoxia in altering the ECM, particularly the collagen 1 (Col1) fiber distribution, and its effect on macromolecular transport. To study the relationship between hypoxia, macromolecular trans- port, and Col1 fiber distribution, we combined dynamic magnetic resonance imaging (MRI) of the macromolecular contrast agent (MMCA) albumin-Gd-diethylenetriaminepentaacetate (albumin- GdDTPA) to detect interstitial macromolecular transport, with second harmonic generation (SHG) microscopy to measure Col1 fibers morphology and distribution. SHG is a nonlinear optical process that requires a molecular environment without a center of symmetry, such as an interfacial region, to produce a signal that can be used to image endogenous structural proteins such as Col1 [3]. These studies were performed with MDA-MB-231 human breast cancer xenografts genetically engineered to express tdTomato red fluorescent protein (RFP) under control of HRE [4]. As an abundant stromal component, Col1 fibers form a major part of the breast tumor ECM [5–8]. Malignant breast cancers are characterized by significantly higher Col1 fiber density and altered Col1 fiber architecture [8]. High mammary Col1 density was shown to cause mammary tumor initiation, progression, and metastasis [8]. We have previously shown that hypoxic regions in breast and prostate tumor xenografts contain significantly lower Col1 fiber density and volume compared to normoxic tumor regions [9]. Hypoxia stimulates the gene expression of a cluster of hydroxylases necessary for Col1 fiber formation [10]. Hypoxic environments in tumors may lead to abnormal collagen deposition either by cancer cells or fibroblasts present within the tumor stroma [8,11]. In normal tissue, Col1 fibers direct interstitial fluid into lymphatic channels [12]. In tumors, these fibers may not be PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e81869