[CANCER RESEARCH 63, 7232–7240, November 1, 2003] An Integrated Approach to Measuring Tumor Oxygen Status Using Human Melanoma Xenografts as a Model 1 Chandrakala Menon, 2 Glenn M. Polin, 2 Indira Prabakaran, Alex Hsi, Cecil Cheung, Joseph P. Culver, James F. Pingpank, Chandra S. Sehgal, Arjun G. Yodh, Donald G. Buerk, and Douglas L. Fraker 3 Departments of Surgery [C. M., G. M. P., I. P., J. F. P., D. L. F.], Radiation Oncology [A. H.], Physics and Astronomy [C. C., J. P. C., A. G. Y.], Radiology [C. S. S.], and Physiology [D. G. B.], University of Pennsylvania, Philadelphia, Pennsylvania 19104 ABSTRACT Tumor oxygen status is a reliable prognostic marker that impacts malignant progression and outcome of tumor therapy. However, tumor oxygenation is heterogeneous and cannot be sufficiently described by a single parameter. It is influenced by several factors including microvessel density (MVD), blood flow (BF), blood volume (BV), blood oxygen satu- ration, tissue pO 2 , oxygen consumption rate, and hypoxic fraction. The goal of this investigation was to integrate these measurements to obtain a comprehensive profile of tumor oxygenation. Platelet/endothelial cell ad- hesion molecule immunohistochemistry, the recessed oxygen microelec- trode, color and power Doppler ultrasound (DUS), and diffuse light spectroscopy (DLS) were used to measure tumor oxygen status using vascular endothelial growth factor (VEGF)-transfected hypervascular hu- man melanoma xenografts and their nontransfected counterparts as a model. NIH1286 human melanoma cells were transfected with a retroviral vector  a 720-bp fragment of human VEGF 121 . High VEGF-producing clones were selected by ELISA. Oxygen consumption rate was measured in NIH1286/VEGF [VEGF-transfected cells (VEGF cells)] and NIH1286/Vec cells [cells transfected with vector alone (Vec cells)] using a standard Clark oxygen electrode. Athymic nude 6 – 8-week-old mice re- ceived s.c. injection in the right flank with 5  10 6 VEGF or Vec cells. When tumors were 10 –14 mm in maximum dimension, serum was ana- lyzed for VEGF by ELISA. Cryopreserved tumor tissue sections were immunostained for platelet/endothelial cell adhesion molecule, and MVD measurements were made. Tumor-bearing mice were anesthetized, and pO 2 measurements were made using Eppendorf pO 2 histograph or the recessed oxygen microelectrode. Tumor BF and BV were measured by quantitative analysis of DUS images. DLS was used to measure tumor BF and blood oxygen saturation variation. VEGF cell supernatants had 15,500 pg/ml VEGF, and Vec cells had 10 pg/ml. VEGF and Vec cells had equivalent oxygen consumption rates. VEGF tumors had a faster growth rate than Vec tumors. Serum from VEGF tumor-bearing mice showed 4,211 pg/ml VEGF, whereas VEGF was undetectable in the serum of control mice. MVD values were 74  11 in VEGF tumors and 39  4 in control tumors at 200 magnification/0.95-mm 2 area. The median pO 2 values were 3.5-fold higher in VEGF tumors than in Vec tumors by the recessed oxygen microelectrode and 18-fold higher by Eppendorf pO 2 histograph. DUS showed a 3.3-fold higher mean BF and a 5.5-fold higher BV in VEGF tumors than in Vec tumors. DLS showed a 3.2-fold higher mean BF and 1.7-fold higher oxygen saturation in the hypervascular tumors as compared with the control tumor type, consistent with in- creased BF and BV data by DUS. An integrated approach that yields a comprehensive and consistent profile of oxygen status in tumors could potentially provide critical information for prognosis and treatment. INTRODUCTION The physiology of solid tumors differs from that of normal tissues primarily because of consistent differences between tumor and normal microvasculature. Compared with the regular, ordered vasculature of normal tissues, blood vessels in tumors are often highly abnormal, with distended capillaries, leaky walls, and sluggish flow (1). Hypoxic regions are thus typical of virtually all solid tumors. There is signif- icant intertumoral and intratumoral variability in the extent of hy- poxia. In addition, local recurrences have been known to have a higher hypoxic fraction than primary tumors (2). Tumor oxygen status ap- pears to be strongly associated with tumor growth, malignant progres- sion, and resistance to various therapies including radiotherapy, pho- todynamic therapy, and chemotherapy (3). It can also influence angiogenesis, cytokine production, cell cycle position of tumor cells, and the development of apoptosis/necrosis (3). Tumor oxygen status plays a central role in tumor physiology and cancer treatment and is therefore a powerful independent prognostic factor of overall and disease-free survival. The routine evaluation of the pretherapeutic tumor oxygenation status may, in fact, facilitate the establishment of individual therapeutic strategies, independent of other oncologic parameters (4). Another area of importance in which measurement of tumor oxygen status can serve as an important end point is in the assessment of the efficacy of agents classified as antiangiogenic compounds, agents that target the tumor vasculature rather than cause direct cyotoxicity to malignant cells (5). Investiga- tors have argued that the standard measure of response in terms of tumor size is not suitable when evaluating these agents and that studies should use parameters associated with the tumor vasculature, especially those related to tumor oxygenation status, as the end point. Many independent studies involving different techniques have measured one or more parameters to define tumor oxygen status. These techniques include phosphorescence lifetime (quench) imaging (6) to measure oxygen diffusion distances between tumor microves- sels (7) and evaluate longitudinal tissue gradients of oxygen (8), magnetic resonance imaging to monitor vascular oxygenation and BF 4 (9), cryospectrophotometry to measure hemoglobin saturation (10), single-photon emission computed tomography and positron-emission tomography assays to measure perfusion and mark hypoxic areas (11–13), injection of Hoechst 33342, a DNA-binding dye that marks the presence of functional vessels (14), and the use of hypoxia markers to identify hypoxic tumor regions (15). Whereas these studies have provided important information, they have also shown that tumor oxygenation is quite heterogeneous, so that an assessment combining several different methods is desirable to define the oxygen profile of a tumor for diagnostic and prognostic purposes. Also, such a combi- nation of techniques will provide validation of the measurements while giving a more accurate overall tumor oxygenation profile. The clinically relevant oxygen status of a tumor is defined by a number of related parameters such as MVD, BF, total BV, blood OS, tumor tissue pO 2 , hypoxic fraction, oxygen consumption rate, and oxygen diffusion distance. This work has adopted an integrated approach to Received 2/11/03; revised 8/7/03; accepted 8/18/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by the Georgene S. Harmelin Endowment Fund. 2 Both authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Hospital of the University of Pennsylanvia, 4 Silverstein Building, 3400 Spruce Street, Philadelphia, PA 19104. Phone: (215) 662-7866; Fax: (215) 614-0765; E-mail: Frakerd@uphs.upenn.edu. 4 The abbreviations used are: BF, blood flow; MVD, microvessel density; BV, blood volume; OS, oxygen saturation; PECAM, platelet/endothelial cell adhesion molecule; DUS, Doppler ultrasound; DLS, diffuse light spectroscopy; VEGF, vascular endothelial growth factor; hVEGF, human VEGF; VD, vascular density; MCL, mean color level; CWFA, color-weighted fractional area; NIR, near-infrared. 7232