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An Integrated Approach to Measuring Tumor Oxygen Status Using Human Melanoma Xenografts as a Model¹

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

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 saturation, tissue pO₂, 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 adhesion molecule immunohistochemistry, the recessed oxygen microelectrode, 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 human 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 VEGF₁₂₁. 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 received s.c. injection in the right flank with 5 x 10⁶ VEGF+ or Vec cells. When tumors were 10–14 mm in maximum dimension, serum was analyzed 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 pO₂ measurements were made using Eppendorf pO₂ 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 x200 magnification/0.95-mm² area. The median pO₂ values were 3.5-fold higher in VEGF+ tumors than in Vec tumors by the recessed oxygen microelectrode and 18-fold higher by Eppendorf pO₂ 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 increased 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.

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