Berit Mathiesen

Acidic Extracellular pH Promotes Experimental Metastasis of Human Melanoma Cells in Athymic Nude Mice

Extracellular pH (pH(e)) is lower in many tumors than in the corresponding normal tissue. The significance of acidic pH(e) in the development of metastatic disease was investigated in the present work. Human melanoma cells (A-07, D-12, and T-22) were cultured in vitro at pH(e) 6.8 or 7.4 (control) before being inoculated into the tail vein of BALB/c nu/nu mice for formation of experimental pulmonary metastases. Cell invasiveness was studied in vitro by using Matrigel invasion chambers and angiogenesis was studied in vivo by using an intradermal assay. Protein secretion was measured by ELISA and immunocapture assays. Cells cultured at acidic pH(e) showed increased secretion of proteinases and proangiogenic factors, enhanced invasive and angiogenic potential, and enhanced potential to develop experimental metastases. Acidity-induced metastasis was inhibited by treatment with the general matrix metalloproteinase (MMP) inhibitor GM6001, the general cysteine proteinase inhibitor E-64, or blocking antibody against vascular endothelial growth factor-A (VEGF-A) or interleukin-8 (IL-8). Our study indicates that acidic pH(e) promotes experimental pulmonary metastasis in A-07, D-12, and T-22 human melanoma cells by a common mechanism involving acidity-induced up-regulation of the proteolytic enzymes MMP-2, MMP-9, cathepsin B, and cathepsin L and acidity-induced up-regulation of the proangiogenic factors VEGF-A and IL-8. One consequence of this observation is that treatment strategies involving deliberate tumor acidification to improve the efficacy of chemotherapy, photodynamic therapy, and hyperthermia should be avoided. Moreover, the possibility that the pH(e) of the primary tumor may be an important prognostic parameter for melanoma patients merits clinical investigation.

Fluctuating and Diffusion-Limited Hypoxia in Hypoxia-Induced Metastasis

Purpose: Most tumors develop regions with hypoxic cells during growth, owing to permanent limitations in oxygen diffusion (chronic or diffusion-limited hypoxia) and/or transient limitations in blood perfusion (acute or fluctuating hypoxia). The aim of this study was to investigate the relative significance of chronic and acute hypoxia in the development of metastatic disease. Experimental Design: D-12 and R-18 human melanoma xenografts were used as models of human cancer. D-12 tumors metastasize to the lungs, whereas R-18 tumors develop lymph node metastases. Fraction of radiobiologically hypoxic cells (HFRad) was measured in individual primary tumors by using a radiobiological assay based on the paired survival curve method. Fraction of immunohistochemically hypoxic cells (HFImm) was assessed in the same tumors by using a pimonidazole-based immunohistochemical assay optimized with respect to achieving selective staining of chronically hypoxic cells. HFImm and the difference between HFRad and HFImm, HFRad − HFImm, were verified to be adequate variables for fraction of chronically hypoxic cells and fraction of acutely hypoxic cells, respectively. Results: Chronic as well as acute hypoxia were found to promote spontaneous metastasis of D-12 and R-18 tumors. Acute hypoxia influenced metastasis to a greater extent than chronic hypoxia, partly because the fraction of acutely hypoxic cells was larger than the fraction of chronically hypoxic cells in most tumors and partly because acutely hypoxic cells showed a higher metastatic potential than chronically hypoxic cells. Conclusions: It may be beneficial to focus on fluctuating hypoxia rather than diffusion-limited hypoxia when searching for hypoxia-related prognostic variables and predictive assays.

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.

The Tumor Bed Effect: Increased Metastatic Dissemination from Hypoxia-Induced Up-regulation of Metastasis-Promoting Gene Products

Cancer patients with recurrent local disease after radiation therapy have increased probability of developing regional and distant metastases. The mechanisms behind this observation were studied in the present work by using D-12 and R-18 human melanoma xenografts growing in preirradiated beds in BALB/c-nu/nu mice as preclinical models of recurrent primary tumors in humans. D-12 tumors metastasize to the lungs, whereas R-18 tumors develop lymph node metastases. Based on earlier studies, we hypothesized that metastasis was governed primarily by the proangiogenic factor interleukin-8 (IL-8) in D-12 tumors and by the invasive growth-promoting receptor urokinase-type plasminogen activator receptor (uPAR) in R-18 tumors. Pimonidazole was used as a hypoxia marker, and hypoxia, microvascular hotspots, and the expression of IL-8 and uPAR were studied by immunohistochemistry. The metastatic frequency was significantly higher in tumors in preirradiated beds than in control tumors in unirradiated beds, and it increased with the preirradiation dose. D-12 tumors showed increased fraction of hypoxic cells, increased fraction of IL-8-positive cells, and increased density of microvascular hotspots in preirradiated beds, and R-18 tumors showed increased fraction of hypoxic cells and increased fraction of uPAR-positive cells in preirradiated beds. Strong correlations were found between these parameters and metastatic frequency. IL-8 was up-regulated in hypoxic regions of D-12 tumors, and uPAR was up-regulated in hypoxic regions of R-18 tumors. Daily treatment with anti-IL-8 antibody (D-12) or anti-uPAR antibody (R-18) suppressed metastasis significantly. Our preclinical study suggests that primary tumors recurring after inadequate radiation therapy may show increased metastatic propensity because of increased fraction of hypoxic cells and hypoxia-induced up-regulation of metastasis-promoting gene products. Two possible mechanisms were identified: hypoxia may enhance metastasis by inducing neoangiogenesis facilitating hematogenous spread and by promoting invasive growth facilitating lymphogenous spread. The aggressive behavior of postirradiation local recurrences suggests that they should be subjected to curative treatment as early as possible to prevent further metastatic dissemination. Moreover, the possibility that patients with a high probability of developing local recurrences after radiation therapy may benefit from postirradiation treatment with antiangiogenic and/or anti-invasive agents merits clinical investigation.

Increased metastatic dissemination in human melanoma xenografts after subcurative radiation treatment: radiation-induced increase in fraction of hypoxic cells and hypoxia-induced up-regulation of urokinase-type plasminogen activator receptor.

Cancer patients showing local failure after radiation treatment have increased probability for developing metastatic disease. The mechanisms behind this observation have not been identified. In the present work, metastatic spread after inadequate radiation therapy was studied by using R-18 human melanoma xenografts as models of cancer in humans. Pimonidazole was used as a hypoxia marker, and hypoxia and urokinase-type plasminogen activator receptor (uPAR) expression were detected by immunohistochemistry. R-18 tumors regrowing after subcurative irradiation showed a higher frequency of lymph node metastasis than unirradiated tumors. The expression of uPAR was up-regulated in hypoxic tumor regions, and the fractions of hypoxic and uPAR-positive cells were ∼2-fold higher in regrowing irradiated tumors than in untreated tumors. Treatment with anti-uPAR antibody blocked metastasis almost completely in irradiated as well as unirradiated tumors. The metastatic frequency was higher in tumors regrowing after irradiation than in unirradiated tumors because the irradiation induced tumor hypoxia, and tumor hypoxia induced up-regulation of uPAR.

Associations between Radiocurability and Interstitial Fluid Pressure in Human Tumor Xenografts without Hypoxic Tissue

Purpose: The interstitial fluid pressure (IFP) of the primary tumor is an independent prognostic parameter for cervical cancer patients treated with radiation therapy. The aim of this preclinical study was to investigate whether tumor radiocurability may be associated with IFP through hypoxia-independent mechanisms. Experimental Design: Small A-07 and R-18 melanoma xenografts without hypoxic tissue were used as preclinical tumor models. IFP was measured by using the wick-in-needle method. Radiation dose resulting in 50% local tumor control (TCD50), cell density, cell tumorigenicity, plating efficiency in vitro, mitotic index, fraction of Ki67-positive cells, vascular endothelial growth factor-A (VEGF-A) concentration, and radiation-induced endothelial cell apoptosis were assessed in tumors with low and high IFP. Results: TCD50 was found to be higher for tumors with high IFP than for tumors with low IFP by factors of 1.13 ± 0.03 (A-07; P < 0.0001) and 1.10 ± 0.03 (R-18; P < 0.0001). In the A-07 line, tumors with high IFP showed a larger number of clonogenic cells and a higher rate of cell proliferation than tumors with low IFP. In the R-18 line, tumors with high IFP showed a higher concentration of VEGF-A and a lower endothelial cell apoptotic index after irradiation than tumors with low IFP. Conclusions: The radiation resistance of normoxic tumor tissue with highly elevated IFP may be an indirect consequence of increased tumor cell clonogenicity as well as increased VEGF-A expression, possibly caused by hypertension-induced modifications of signaling pathways regulating cell proliferation, cell survival, and/or angiogenesis. Clin Cancer Res; 16(3); 936–45

Tumor Interstitial Fluid Pressure—A Link between Tumor Hypoxia, Microvascular Density, and Lymph Node Metastasis

High microvascular density (MVD) in the primary tumor has been shown to be associated with increased incidence of lymph node metastases and poor clinical outcome. Other investigations have revealed that a large fraction of hypoxic tissue in the primary tumor is associated with metastatic disease and impaired survival. These data are apparently incompatible because tumor hypoxia is primarily a consequence of poor oxygen supply caused by an inadequate vasculature with increased intervessel distances. Here, we provide an explanation of these observations. Human melanoma xenografts were used as preclinical cancer models. Tumors that metastasized to lymph nodes showed higher interstitial fluid pressure (IFP) than those that did not metastasize, and compared with tumors with low IFP, tumors with high IFP showed large hypoxic fractions centrally, high MVD in the periphery, high peritumoral density of lymphatics, and elevated expression of vascular endothelial growth factor A (VEGF-A) and VEGF-C. Significant correlations were found between peripheral MVD and central hypoxia, and lymph node metastasis was associated with high values of both parameters. These findings suggest that the outcome of cancer may be associated with both high MVD and extensive hypoxia in the primary tumor. We propose that proangiogenic factors are upregulated in the tumor center and that the outward interstitial fluid flow caused by the elevated IFP transports these factors to the tumor surface where they evoke hemangiogenesis and lymphangiogenesis, and consequently, that the IFP serves as a link between tumor hypoxia, peripheral tumor hemangiogenesis, peritumoral lymphangiogenesis, and lymph node metastasis.

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.

Assessment of hypoxia and radiation response in intramuscular experimental tumors by dynamic contrast-enhanced magnetic resonance imaging

BACKGROUND AND PURPOSE Studies of intradermal melanoma xenografts have suggested that dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) may be a useful method for assessing the extent of hypoxia in tumors. Because the microvascular network of tumors is influenced significantly by the site of growth, we challenged this possibility in the present work by studying relationships between DCE-MRI-derived parameters and hypoxia in intramuscular melanoma xenografts. MATERIALS AND METHODS Intramuscular R-18, U-25, and V-27 tumors were subjected to DCE-MRI and measurement of the fraction of radiobiologically hypoxic cells (HF(Rad)). Parametric images of K(trans) and v(e) were produced by pharmacokinetic analysis, and K(trans) and v(e) were related to HF(Rad) in individual tumors. RESULTS K(trans) decreased with increasing HF(Rad). The correlations between K(trans) and HF(Rad) were similar for the three tumor lines and were highly significant (P<0.00001). There was no correlation between v(e) and HF(Rad). However, v(e) decreased significantly with increasing cell survival after single dose irradiation. CONCLUSION Intramuscular melanoma xenografts show similar inverse correlations between K(trans) and HF(Rad) as intradermal tumors, which support the current clinical attempts to establish DCE-MRI as a method for detecting hypoxia and defining therapeutic targets in tumors.