Jon Vidar Gaustad

Tumors exposed to acute cyclic hypoxic stress show enhanced angiogenesis, perfusion and metastatic dissemination

Clinical studies have shown that patients with highly hypoxic primary tumors may have poor disease‐free and overall survival rates. Studies of experimental tumors have revealed that acutely hypoxic cells may be more metastatic than normoxic or chronically hypoxic cells. In the present work, causal relations between acute cyclic hypoxia and metastasis were studied by periodically exposing BALB/c nu/nu mice bearing A‐07 human melanoma xenografts to a low oxygen atmosphere. The hypoxia treatment consisted of 12 cycles of 10 min of 8% O2 in N2 followed by 10 min of air for a total of 4 hr, began on the first day after tumor cell inoculation and was given daily until the tumors reached a volume of 100 mm3. Twenty‐four hours after the last hypoxia exposure, the primary tumors were subjected to dynamic contrast‐enhanced magnetic resonance imaging for assessment of blood perfusion before being resected and processed for immunohistochemical examinations of microvascular density and expression of proangiogenic factors. Mice exposed to acute cyclic hypoxia showed increased incidence of pulmonary metastases, and the primary tumors of these mice showed increased blood perfusion, microvascular density and vascular endothelial growth factor‐A (VEGF‐A) expression; whereas, the expression of interleukin‐8, platelet‐derived endothelial cell growth factor and basic fibroblast growth factor was unchanged. The increased pulmonary metastasis was most likely a consequence of hypoxia‐induced VEGF‐A upregulation, which resulted in increased angiogenic activity and blood perfusion in the primary tumor and thus facilitated tumor cell intravasation and hematogenous transport into the general circulation.

Photochemical Internalization of Bleomycin is Superior to Photodynamic Therapy Due to the Therapeutic Effect in the Tumor Periphery

Photochemical internalization (PCI) is under development for clinical use in treatment of soft tissue sarcomas and other solid tumors. PCI may release endocytosed bleomycin (BLM) into the cytosol by photochemical rupture of the endocytic vesicles. In this study, the human fibrosarcoma xenograft HT1080 was transplanted into the leg muscle of athymic mice. The photosensitizer disulfonated aluminum phthalocyanine (AlPcS2a) and BLM were systemically administrated 48 h and 30 min, respectively, prior to light exposure at 670 nm (30 J cm−2). The purposes of this study were to evaluate the treatment response to AlPcS2a‐photodynamic therapy (PDT) and AlPcS2a‐PDT in combination with BLM (i.e. PCI of BLM) in an orthotopic, invasive and clinically relevant tumor model and to explore the underlying response mechanisms caused by PDT and PCI of BLM. The treatment response was evaluated by measuring tumor growth, contrast‐enhanced magnetic resonance imaging (CE‐MRI), histology and fluorescence microscopy. The results show that PCI of BLM is superior to PDT in inducing tumor growth retardation and acts synergistically as compared to the individual treatment modalities. The CE‐MRI analyses 2 h after AlPcS2a‐PDT and PCI of BLM identified a treatment‐induced nonperfused central zone of the tumor and a well‐perfused peripheral zone. While there were no differences in the vascular response between PDT and PCI, the histological analyses showed that PDT caused necrosis in the tumor center and viable tumor cells were found in the tumor periphery. PCI caused larger necrotic areas and the regrowth in the peripheral zone was almost completely inhibited after PCI. The results indicate that PDT is less efficient in the tumor periphery than in the tumor center and that the treatment effect of PCI is superior to PDT in the tumor periphery.

Dynamic contrast-enhanced-MRI of tumor hypoxia

Patients with highly hypoxic primary tumors show increased frequency of locoregional treatment failure and poor survival rates and may benefit from particularly aggressive treatment. The potential of gadolinium diethylene‐triamine penta‐acetic acid‐based dynamic contrast‐enhanced‐MRI in assessing tumor hypoxia was investigated in this preclinical study. Xenografted tumors of eight human melanoma lines were subjected to dynamic contrast‐enhanced‐MRI and measurement of the fraction of radiobiologically hypoxic cells and the fraction of pimonidazole‐positive hypoxic cells. Tumor images of Ktrans (the volume transfer constant of gadolinium diethylene‐triamine penta‐acetic acid) and ve (the fractional distribution volume of gadolinium diethylene‐triamine penta‐acetic acid) were produced by pharmacokinetic analysis of the dynamic contrast‐enhanced‐MRI data, and Ktrans and ve frequency distributions of the non‐necrotic tumor tissue were established and related to the extent of hypoxia. Tumors showing high Ktrans values and high ve values had low fractions of hypoxic cells, whereas tumors showing both low Ktrans values and low ve values had high hypoxic fractions. Ktrans differentiated better between tumors with low and high hypoxic fractions than did ve. This study supports the current attempts to establish dynamic contrast‐enhanced‐MRI as a method for assessing the extent of hypoxia in human tumors, and it provides guidelines for the clinical development of valid assays. Magn Reson Med, 2012.

Assessment of fraction of radiobiologically hypoxic cells in human melanoma xenografts by dynamic contrast-enhanced MRI

A noninvasive method for assessment of the extent of hypoxia in experimental and human tumors is highly needed. In this study, the potential usefulness of dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI) was investigated, using gadopentetate dimeglumine (Gd‐DTPA) as contrast agent and A‐07 human melanoma xenografts as tumor model. DCE‐MRI was performed at a voxel size of 0.3 × 0.6 × 2.0 mm3 with spoiled gradient‐recalled sequences. Images of E · F (E is the initial extraction fraction of Gd‐DTPA and F is perfusion) and λ (the partition coefficient of Gd‐DTPA, which is proportional to extracellular volume fraction) were obtained by Kety analysis of DCE‐MRI data. The study was based on the hypothesis that hypoxic tissue would have low E · F (i.e., poor oxygen supply) and/or low λ (i.e., high cell density and, hence, high oxygen consumption rate). Twenty‐two tumors were first subjected to DCE‐MRI and then to measurement of fraction of hypoxic cells, using a radiobiological assay. E · F was found to be strongly correlated to fraction of hypoxic cells (P < 0.000001), whereas significant correlation between λ and fraction of hypoxic cells could not be detected. It is thus possible that E · F may be a useful parameter for the extent of hypoxia in experimental and human tumors with physiologic properties similar to those of A‐07 tumors. This possibility warrants further studies involving experimental tumors of several lines, as well as human tumors. Magn Reson Med, 2006.

Dynamic contrast-enhanced magnetic resonance imaging of tumors: preclinical validation of parametric images.

Abstract Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been suggested to be a valuable method for characterizing the physiological microenvironment of tumors and thus a promising method for individualizing cancer treatment. The aim of this study was to test the hypothesis that valid parametric images of the tumor microenvironment can be obtained by pharmacokinetic analysis of DCE-MRI series. Cells of four human melanoma xenograft lines (A-07, D-12, R-18 and T-22) were used as preclinical models of human cancer. DCE-MRI was performed at 1.5 T at a spatial resolution of 0.23 × 0.47 × 2.0 mm3 and a time resolution of 14 s. Gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA) was used as contrast agent. The DCE-MRI data were analyzed on a voxel-by-voxel basis by using a pharmacokinetic model recommended for analysis of clinical DCE-MRI series. Parametric DCE-MR images were compared with tumor blood perfusion measured by the 86Rb uptake method, and fractional volume of the extravascular extracellular space assessed by analysis of histological preparations. Parametric images reflecting tumor blood perfusion and fractional volume of the extravascular extracellular space were obtained. The numerical values of the DCE-MRI-derived parameters were not significantly different from the absolute values of tumor blood perfusion or fractional volume of the extravascular extracellular space in any of the tumor lines. This study shows that DCE-MRI can provide valid quantitative parametric images of the tumor microenvironment in preclinical cancer models and thus supports the suggestion that DCE-MRI may be developed to be a clinically useful method for individualization of microenvironment-based cancer treatment, a possibility that merits increased clinical interest.

Intratumor heterogeneity in blood perfusion in orthotopic human melanoma xenografts assessed by dynamic contrast-enhanced magnetic resonance imaging

To determine the intratumor heterogeneity in blood perfusion of orthotopic human melanoma xenografts by use of gadopentetate dimeglumine (Gd‐DTPA)‐based dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI).

Magnetic resonance imaging of tumor necrosis

Abstract Background. The prognostic and predictive value of magnetic resonance (MR) investigations in clinical oncology may be improved by implementing strategies for discriminating between viable and necrotic tissue in tumors. The purpose of this preclinical study was to investigate whether the extent of necrosis in tumors can be assessed by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and/or T2-weighted MR imaging. Material and methods. Three amelanotic human melanoma xenograft lines differing substantially in tumor necrotic fraction, necrotic pattern, extracellular volume fraction, and blood perfusion were used as experimental models of human cancer. MRI was performed at 1.5 T and a spatial resolution of 0.23 × 0.47 × 2.0 mm3. Gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA) was used as contrast agent. Plots of Gd-DTPA concentration versus time were generated for each voxel, and three parameters were calculated for each curve: the extracellular volume fraction (νe), the final slope (a), and the Gd-DTPA concentration at one minute after the contrast administration (C1min). Parametric images of νe, a, C1min, and the signal intensity in T2-weighted images (SIT2W) were compared with the histology of the imaged tissue. Results. The νe, a, and C1min frequency distributions were significantly different for necrotic and viable tissue in all three tumor lines. By using adequate values of νe, a, and C1min to discriminate between necrotic and viable tissue, significant correlations were found between the fraction of necrotic tissue assessed by MRI and the fraction of necrotic tissue assessed by image analysis of histological preparations. On the other hand, the SIT2W frequency distributions did not differ significantly between necrotic and viable tissue in two of the three tumor lines. Conclusion. Necrotic regions in tumor tissue can be identified in parametric images derived from DCE-MRI series, whereas T2-weighted images are unsuitable for detection of tumor necrosis.

Assessment of microvascular density, extracellular volume fraction, and radiobiological hypoxia in human melanoma xenografts by dynamic contrast-enhanced MRI

To investigate whether gadopentetate dimeglumine (Gd‐DTPA)‐based dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI) may be a useful method for assessing fraction of radiobiologically hypoxic cells in tumors.

Assessment of Hypoxia in Human Cervical Carcinoma Xenografts by Dynamic Contrast-Enhanced Magnetic Resonance Imaging

PURPOSE Patients with advanced cervical cancer and highly hypoxic primary tumors show increased frequency of locoregional treatment failure and poor disease-free and overall survival rates. The potential usefulness of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing tumor hypoxia noninvasively was investigated in the present preclinical study. METHODS AND MATERIALS CK-160 and TS-415 human cervical carcinoma xenografts transplanted intramuscularly (i.m.) or subcutaneously (s.c.) in BALB/c nu/nu mice were subjected to DCE-MRI and measurement of fraction of radiobiologically hypoxic cells. Tumor images of K(trans) (the volume transfer constant of Gd-DTPA) and v(e) (the extracellular volume fraction of the imaged tissue) were produced by pharmacokinetic analysis of the DCE-MRI data. Fraction of radiobiologically hypoxic cells was measured by using the paired survival curve method. RESULTS Fraction of radiobiologically hypoxic cells differed significantly among the four tumor groups. The mean values +/- SE were determined to be 44% +/- 7% (i.m. CK-160), 77% +/- 10% (s.c. CK-160), 23% +/- 5% (i.m. TS-415), and 52% +/- 6% (s.c. TS-415). The four tumor groups differed significantly also in K(trans), and there was an unambiguous inverse relationship between K(trans) and fraction of radiobiologically hypoxic cells. On the other hand, significant differences among the groups in v(e) could not be detected. CONCLUSIONS The study supports the clinical development of DCE-MRI as a method for assessing the extent of hypoxia in carcinoma of the cervix.

Assessment of Fraction of Hypoxic Cells in Human Tumor Xenografts with Necrotic Regions by Dynamic Contrast-Enhanced MRI

Abstract Egeland, T. A. M., Gaustad, J-V., Benjaminsen, I. C., Hedalen, K., Mathiesen, B. and Rofstad, E. K. Assessment of Fraction of Hypoxic Cells in Human Tumor Xenografts with Necrotic Regions by Dynamic Contrast-Enhanced MRI. Radiat. Res. 169, 689–699 (2008). The potential usefulness of gadopentetate dimeglumine (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) for assessing hypoxia in tumors with significant necrosis was investigated. Small (100–350 mm3) and large (500–1000 mm3) D-12 and U-25 tumors were subjected to DCE-MRI, measurement of the fraction of necrotic tissue, and measurement of the fraction of radiobiologically hypoxic cells. Images of E·F (E is the initial extraction fraction of Gd-DTPA and F is perfusion) and λ (λ is proportional to extracellular volume fraction) were produced by subjecting the DCE-MRI data to Kety analysis. Necrotic tissue could be identified in λ images but not in E·F images of the tumors. Most voxels in viable tissue showed λ values of 0.15–0.70, whereas the λ values of most voxels in necrotic tissue were either <0.15 or >0.70. The E·F and λ frequency distributions of the viable tissue, but not the E·F and λ frequency distributions of the whole tissue, were consistent with the observation that the four groups of tumors showed similar fractions of radiobiologically hypoxic cells. E·F and λ images may thus provide useful information on the extent of hypoxia in tumors provided that voxels in necrotic tumor regions are identified and excluded from the images.