High Interstitial Fluid Pressure Is Associated with Tumor-Line Specific Vascular Abnormalities in Human Melanoma Xenografts

PURPOSE: Interstitial fluid pressure (IFP) is highly elevated in many solid tumors. High IFP has been associated with low radiocurability and high metastatic frequency in human melanoma xenografts and with poor survival after radiation therapy in cervical cancer patients. Abnormalities in tumor vascular networks have been identified as an important cause of elevated tumor IFP. The aim of this study was to investigate the relationship between tumor IFP and the functional and morphological properties of tumor vascular networks. MATERIALS AND METHODS: A-07-GFP and R-18-GFP human melanomas growing in dorsal window chambers in BALB/c nu/nu mice were used as preclinical tumor models. Functional and morphological parameters of the vascular network were assessed from first-pass imaging movies and vascular maps recorded after intravenous bolus injection of 155-kDa tetramethylrhodamine isothiocyanate-labeled dextran. IFP was measured in the center of the tumors using a Millar catheter. Angiogenic profiles of A-07-GFP and R-18-GFP cells were obtained with a quantitative PCR array. RESULTS: High IFP was associated with low growth rate and low vascular density in A-07-GFP tumors, and with high growth rate and high vascular density in R-18-GFP tumors. A-07-GFP tumors showed chaotic and highly disorganized vascular networks, while R-18-GFP tumors showed more organized vascular networks with supplying arterioles in the tumor center and draining venules in the tumor periphery. Furthermore, A-07-GFP and R-18-GFP cells differed substantially in angiogenic profiles. A-07-GFP tumors with high IFP showed high geometric resistance to blood flow due to high vessel tortuosity. R-18-GFP tumors with high IFP showed high geometric resistance to blood flow due to a large number of narrow tumor capillaries. CONCLUSIONS: High IFP in A-07-GFP and R-18-GFP human melanoma xenografts was primarily a consequence of high blood flow resistance caused by tumor-line specific vascular abnormalities

Sunitinib treatment does not improve blood supply but induces hypoxia in human melanoma xenografts

Abstract Background Antiangiogenic agents that disrupt the vascular endothelial growth factor pathway have been demonstrated to normalize tumor vasculature and improve tumor oxygenation in some studies and to induce hypoxia in others. The aim of this preclinical study was to investigate the effect of sunitinib treatment on the morphology and function of tumor vasculature and on tumor oxygenation. Methods A-07-GFP and R-18-GFP human melanoma xenografts grown in dorsal window chambers were used as preclinical tumor models. Morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and tumor blood supply time was assessed from first-pass imaging movies recorded after a bolus of 155 kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Tumor hypoxia was assessed from immunohistochemical preparations of the imaged tissue by use of pimonidazole as a hypoxia marker. Results Sunitinib treatment reduced vessel densities, increased vessel segment lengths, did not affect blood supply times, and increased hypoxic area fractions. Conclusion Sunitinib treatment did not improve vascular function but induced hypoxia in A-07-GFP and R-18-GFP tumors.</p

IFP and growth time in individual A-07-GFP and R-18-GFP tumors.

<p><b>A</b>) IFP-values measured in the center of individual A-07-GFP and R-18-GFP tumors. <b>B–C</b>) IFP versus growth time for A-07-GFP (<b>B</b>) and R-18-GFP (<b>C</b>) tumors. Points represent individual A-07-GFP (•) and R-18-GFP (Δ) tumors. Horizontal bars represent mean values (A). Lines represent linear regression curves (B<b>–</b>C).</p

IFP and vascular density.

<p>Fluorescence images showing vascular networks of A-07-GFP (<b>A</b>) and R-18-GFP (<b>B</b>) tumors representing low (upper panel), intermediate (middle panel), and high (lower panel) IFP values. Scale bars, 1 mm. <b>C–E</b>) IFP versus vascular density calculated as vascular area fraction (<b>C</b>), total vessel density (<b>D</b>), and density of large vessels (density of vessels with diameter >20 µm) (<b>E</b>). Points represent individual A-07-GFP (•) and R-18-GFP (Δ) tumors. Lines represent linear regression curves. Solid lines, P<0.05; dotted lines, P>0.05.</p

IFP and vascular function.

<p>Blood supply time (BST) images and BST frequency distributions of A-07-GFP (<b>A</b>) and R-18-GFP (<b>B</b>) tumors representing low (upper panel), intermediate (middle panel), and high (lower panel) IFP values. Scale bars, 1 mm; color bars, BST scale in seconds. Vertical lines in BST frequency distributions indicate median BST. <b>C</b>) IFP versus median BST (left) and variance BST (right). <b>D</b>) Plasma velocity in tumor arterioles (TAs; red symbols) and tumor venules (TVs; blue symbols), and IFP versus ratio of TA plasma velocity to TV plasma velocity. <b>E</b>) IFP versus TA plasma velocity (left panel), TV plasma velocity (middle panel), and TV diameter (right panel). Points represent individual A-07-GFP (•) and R-18-GFP (Δ) tumors. Horizontal bars represent mean values. Lines represent linear regression curves.</p

IFP and intratumor heterogeneity in R-18-GFP tumors.

<p><b>A</b>) Illustration of concentric regions of interest: ROI#1, ROI#2, and ROI#3. Upper panel: BST-images. Lower panel: morphology images. <b>B</b>) Intertumor heterogeneity in BST (upper panel) and vascular density (lower panel) in A-07-GFP and R-18-GFP tumors calculated as median BST and total vessel density in ROI#2 and in ROI#3 relative to ROI#1 (ratio ROI#2/ROI#1 and ratio ROI#3/ROI#1). <b>C</b>) IFP versus BST ratio ROI#3/ROI#1 and IFP versus total vessel density ratio ROI#2/ROI#1 in R-18-GFP tumors. <b>D</b>) Vessel density ratio ROI#2/ROI#1 for small vessels (diameter <20 µm) and for large vessels (diameter >20 µm) in R-18-GFP tumors. Points represent individual A-07-GFP (•) and R-18-GFP (Δ) tumors. Horizontal bars represent mean values. Lines represent linear regression curves. <b>E</b>) Schematic illustration of blood flow pattern and vessel distribution in R-18-GFP tumors with low IFP and R-18-GFP tumors with high IFP. Red color indicates early blood supply; blue color indicates late blood supply.</p

Growth and histology of window chamber tumors.

<p><b>A</b>) Schematic illustration of a transversal section through a tumor growing in the dorsal skin fold window chamber. Dark grey areas represent normal tissue, light grey areas represent tumor tissue, red areas represent vasculature, and the light blue area represents the plastic window. Two planes parallel to the window are indicated. Plane 1 represents the plane imaged with intravital microscopy while plane 2 represents a plane through the tumor center, where IFP was measured. <b>B-C</b>) Histological sections stained for the hypoxia marker pimonidazole corresponding to the planes indicated in A) for R-18-GFP tumors showing peripheral staining (<b>B</b>) and scattered staining (<b>C</b>). Scale bars, 1 mm.</p