Identification of Metastasis-Associated Metabolic Profiles of Tumors by (1)H-HR-MAS-MRS.

Tumors develop an abnormal microenvironment during growth, and similar to the metastatic phenotype, the metabolic phenotype of cancer cells is tightly linked to characteristics of the tumor microenvironment (TME). In this study, we explored relationships between metabolic profile, metastatic propensity, and hypoxia in experimental tumors in an attempt to identify metastasis-associated metabolic profiles. Two human melanoma xenograft lines (A-07, R-18) showing different TMEs were used as cancer models. Metabolic profile was assessed by proton high resolution magic angle spinning magnetic resonance spectroscopy ((1)H-HR-MAS-MRS). Tumor hypoxia was detected in immunostained histological preparations by using pimonidazole as a hypoxia marker. Twenty-four samples from 10 A-07 tumors and 28 samples from 10 R-18 tumors were analyzed. Metastasis was associated with hypoxia in both A-07 and R-18 tumors, and (1)H-HR-MAS-MRS discriminated between tissue samples with and tissue samples without hypoxic regions in both models, primarily because hypoxia was associated with high lactate resonance peaks in A-07 tumors and with low lactate resonance peaks in R-18 tumors. Similarly, metastatic and non-metastatic R-18 tumors showed significantly different metabolic profiles, but not metastatic and non-metastatic A-07 tumors, probably because some samples from the metastatic A-07 tumors were derived from tumor regions without hypoxic tissue. This study suggests that (1)H-HR-MAS-MRS may be a valuable tool for evaluating the role of hypoxia and lactate in tumor metastasis as well as for identification of metastasis-associated metabolic profiles.

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.

DCE-MRI of the hypoxic fraction, radioresponsiveness, and metastatic propensity of cervical carcinoma xenografts.

BACKGROUND AND PURPOSE: Locoregional treatment failure and poor survival rates are associated with extensive hypoxia in the primary tumor in advanced cervical carcinoma. The potential of gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the hypoxic fraction, radioresponsiveness, and metastatic propensity of cervical carcinomas was investigated in this preclinical study. MATERIALS AND METHODS: CK-160 and TS-415 cervical carcinoma xenografts were used as tumor models. DCE-MRI was carried out at 1.5 T, and parametric images of K(trans) and v(e) were produced by pharmacokinetic analysis of the DCE-MRI series. Pimonidazole was used as a hypoxia marker. Tumor radioresponsiveness was determined by irradiating tumors with five fractions of 4 Gy in 48 h and measuring cell survival in vitro. Metastatic propensity was determined by examining host mice for tumor growth in lymph nodes. RESULTS: Low values of K(trans) were associated with extensive hypoxia and radiation resistance in tumors of both lines and with high incidence of metastases in CK-160 tumors. Associations between ve and hypoxia, radioresponsiveness, or metastatic propensity were not found in any of the tumor lines. CONCLUSION: K(trans) is a potentially useful biomarker of tumor hypoxia, radiation resistance, and metastatic growth in advanced cervical carcinoma.

Microenvironment-associated lymph node metastasis of human cervical carcinoma xenografts.

BACKGROUND: The prognosis is particularly poor for patients with advanced squamous cell carcinoma of the uterine cervix when the primary tumor has developed severe physiological abnormalities. The impact of the physiological microenvironment of the primary tumor on lymph node metastasis was investigated in this preclinical study. MATERIAL AND METHODS: Xenografted tumors of two human cervical carcinoma lines (CK-160 and TS-415) transplanted into BALB/c nu/nu mice were included in the study. The fraction of radiobiologically hypoxic cells (HF(Rad)), interstitial fluid pressure (IFP), and extracellular pH (pH(e)) were measured in 22 CK-160 tumors and 16 TS-415 tumors and related to the metastatic status of the host mice. RESULTS: In CK-160, HF(Rad) was significantly higher in the metastatic than in the nonmetastatic tumors, whereas the metastatic and nonmetastatic tumors did not differ significantly in IFP or pH(e). In TS-415, IFP was significantly higher in the tumors that metastasized than in those that did not metastasize, whereas the tumors of the metastasis-positive and metastasis-negative mice did not differ significantly in HF(Rad) or pH(e). CONCLUSION: Lymph node metastasis is associated with abnormalities in the physiological microenvironment of the primary tumor in cervical carcinoma xenografts, and tumor line-specific mechanisms are probably involved.

pO2 fluctuation pattern and cycling hypoxia in human cervical carcinoma and melanoma xenografts.

PURPOSE: Blood perfusion in tumors is spatially and temporally heterogeneous, resulting in local fluctuations in tissue oxygen tension (pO(2)) and tissue regions showing cycling hypoxia. In this study, we investigated whether the pO(2) fluctuation pattern and the extent of cycling hypoxia differ between tumor types showing high (e.g., cervical carcinoma xenograft) and low (e.g., melanoma xenograft) fractions of connective tissue-associated blood vessels. METHODS AND MATERIALS: Two cervical carcinoma lines (CK-160 and TS-415) and two melanoma lines (A-07 and R-18) transplanted into BALB/c nu/nu mice were included in the study. Tissue pO(2) was measured simultaneously in two positions in each tumor by using a two-channel OxyLite fiber-optic oxygen-sensing device. The extent of acute and chronic hypoxia was assessed by combining a radiobiological and a pimonidazole-based immunohistochemical assay of tumor hypoxia. RESULTS: The proportion of tumor regions showing pO(2) fluctuations, the pO(2) fluctuation frequency in these regions, and the relative amplitude of the pO(2) fluctuations were significantly higher in the melanoma xenografts than in the cervical carcinoma xenografts. Cervical carcinoma and melanoma xenografts did not differ significantly in the fraction of acutely hypoxic cells or the fraction of chronically hypoxic cells. However, the ratio between fraction of acutely hypoxic cells and fraction of chronically hypoxic cells was significantly higher in melanoma than in cervical carcinoma xenografts. CONCLUSIONS: Temporal heterogeneity in blood flow and tissue pO(2) in tumors may depend on tumor histology. Connective tissue surrounding microvessels may stabilize blood flow and pO(2) and, thus, protect tumor tissue from cycling hypoxia.

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 K(trans) (the volume transfer constant of gadolinium diethylene-triamine penta-acetic acid) and v(e) (the fractional distribution volume of gadolinium diethylene-triamine penta-acetic acid) were produced by pharmacokinetic analysis of the dynamic contrast-enhanced-MRI data, and K(trans) and v(e) frequency distributions of the non-necrotic tumor tissue were established and related to the extent of hypoxia. Tumors showing high K(trans) values and high v(e) values had low fractions of hypoxic cells, whereas tumors showing both low K(trans) values and low v(e) values had high hypoxic fractions. K(trans) differentiated better between tumors with low and high hypoxic fractions than did v(e). 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.

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.

Assessment of tumor radioresponsiveness and metastatic potential by dynamic contrast-enhanced magnetic resonance imaging.

PURPOSE: It has been suggested that gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) may provide clinically useful biomarkers for personalized cancer treatment. In this preclinical study, we investigated the potential of DCE-MRI as a noninvasive method for assessing the radioresponsiveness and metastatic potential of tumors. METHODS AND MATERIALS: R-18 melanoma xenografts growing in BALB/c nu/nu mice were used as experimental tumor models. Fifty tumors were subjected to DCE-MRI, and parametric images of Ktrans (the volume transfer constant of Gd-DTPA) and ve (the fractional distribution volume of Gd-DTPA) were produced by pharmacokinetic analysis of the DCE-MRI series. The tumors were irradiated after the DCE-MRI, either with a single dose of 10 Gy for detection of radiobiological hypoxia (30 tumors) or with five fractions of 4 Gy in 48 h for assessment of radioresponsiveness (20 tumors). The host mice were then euthanized and examined for lymph node metastases, and the primary tumors were resected for measurement of cell survival in vitro. RESULTS: Tumors with hypoxic cells showed significantly lower Ktrans values than tumors without significant hypoxia (p<0.0001, n=30), and Ktrans decreased with increasing cell surviving fraction for tumors given fractionated radiation treatment (p<0.0001, n=20). Tumors in metastasis-positive mice had significantly lower Ktrans values than tumors in metastasis-negative mice (p<0.0001, n=50). Significant correlations between ve and tumor hypoxia, radioresponsiveness, or metastatic potential could not be detected. CONCLUSIONS: R-18 tumors with low Ktrans values are likely to be resistant to radiation treatment and have a high probability of developing lymph node metastases. The general validity of these observations should be investigated further by studying preclinical tumor models with biological properties different from those of the R-18 tumors.

Differentiation between hypoxic and non-hypoxic experimental tumors by dynamic contrast-enhanced magnetic resonance imaging.

BACKGROUND AND PURPOSE: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been suggested to be a useful method for detecting tumor hypoxia. In this study, we investigated whether DCE-MRI can differentiate between hypoxic and non-hypoxic experimental tumors. MATERIALS AND METHODS: Three tumor models with hypoxic tissue and three tumor models without hypoxic tissue were subjected to DCE-MRI. Parametric images of K(trans) (the volume transfer constant of Gd-DTPA) and v(e) (the fractional distribution volume of Gd-DTPA) were produced by pharmacokinetic analysis of the DCE-MRI series. Tumor oxygenation status was assessed by using a radiobiological assay and a pimonidazole-based immunohistochemical assay. Tumor response to fractionated irradiation (six fractions of 2Gy in 60h) was measured in vitro by using a clonogenic assay. RESULTS: Tumors with hypoxic regions were more resistant to radiation treatment than were tumors without hypoxia. K(trans) was significantly higher for radiation sensitive tumors without hypoxia than for radiation resistant tumors with hypoxic regions, whereas v(e) did not differ significantly between non-hypoxic and hypoxic tumors. CONCLUSION: This study supports the clinical attempts to establish DCE-MRI as a noninvasive method for providing useful biomarkers for personalized radiation therapy.

Metastasis in melanoma xenografts is associated with tumor microvascular density rather than extent of hypoxia.

The development of metastases has been shown to be associated with the microvascular density of the primary tumor in some clinical studies and with the extent of hypoxia in others. The aim of this study was to investigate the validity of these apparently inconsistent observations and to reveal possible links between them. Xenografted tumors of nine melanoma cell lines established from patients with diseases differing in aggressiveness were studied. The aggressiveness of the cell lines was assessed by measuring their lung colonization potential, invasiveness, angiogenic potential, and tumorigenicity. Spontaneous metastasis was assessed in untreated mice and mice treated with neutralizing antibody against vascular endothelial growth factor A (VEGF-A) or interleukin 8 (IL-8). Microvascular density was scored in histologic preparations. Hypoxic fractions were measured by using a radiobiologic assay and a pimonidazole-based immunohistochemical assay. The aggressiveness of the melanoma lines reflected the aggressiveness of the donor patients' tumors. The metastatic propensity was associated with the microvascular density but not with the hypoxic fraction. Anti-VEGF-A and anti-IL-8 treatments resulted in decreased microvascular density and reduced incidence of metastases in all lines. Large hypoxic fractions were not a secondary effect of high cellular aggressiveness, whereas the microvascular density was associated with the cellular aggressiveness. The metastatic propensity was governed by the angiogenic potential of the tumor cells. The differences in microvascular density among the lines were most likely a consequence of differences in the constitutive angiogenic potential rather than differences in hypoxia-induced angiogenesis. VEGF-A and IL-8 may be important therapeutic targets for melanoma.

Quantitative assessment of hypoxia in melanoma xenografts by dynamic contrast-enhanced magnetic resonance imaging: intradermal versus intramuscular tumors.

BACKGROUND AND PURPOSE: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been suggested to be a useful method for assessing the extent of hypoxia in tumors. In this study, we investigated whether differences in hypoxic fraction between tumors caused by the site of growth can be detected by DCE-MRI. MATERIALS AND METHODS: Intradermal and intramuscular A-07 tumors were subjected to DCE-MRI, histological analysis of microvascular characteristics, and measurement of hypoxic cell fractions using a radiobiological assay and a pimonidazole-based immunohistochemical assay. Parametric images of E·F (blood perfusion) and v(e) (extracellular volume fraction) were produced by pharmacokinetic analysis of the DCE-MRI series. RESULTS: The intramuscular tumors had 3-4-fold higher hypoxic fractions than the intradermal tumors, owing to a lower microvascular density. This difference in extent of hypoxia was not detectable in the parametric MR images. Most likely, larger vessel diameters compensated for the lower vessel density in the intramuscular tumors, resulting in E·F images that were similar to those of the intradermal tumors. CONCLUSION: Quantitative assessment of hypoxic fractions from parametric MR images may require tumor site-specific translational criteria.

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% O(2) in N(2) 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 mm(3). 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.

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 (TCD(50)), 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: TCD(50) 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.

Radiocurability is associated with interstitial fluid pressure in human tumor xenografts.

Interstitial fluid pressure (IFP) has been shown to be an independent prognostic parameter for disease-free survival in cervical carcinoma patients treated with radiation therapy. However, the underlying mechanisms are not fully understood. The main aims of this study were to investigate whether tumor radiocurability may be associated with IFP and, if so, to identify possible mechanisms. Human melanoma xenografts transplanted intradermally or in window chamber preparations in BALB/c nu/nu mice were used as preclinical tumor models. Radiation dose resulting in 50% local tumor control was higher by a factor of 1.19 +/- 0.06 in tumors with IFP > or = 9 mm Hg than in tumors with IFP < or = 7 mm Hg. Tumor IFP was positively correlated to vessel segment length and vessel tortuosity and was inversely correlated to vessel density. Compared with tumors with low IFP, tumors with high IFP showed high resistance to blood flow, high frequency of Po(2) fluctuations, and high fractions of acutely hypoxic cells, whereas the fraction of radiobiologically hypoxic cells and the fraction of chronically hypoxic cells did not differ between tumors with high and tumors with low IFP. IFP showed a significant correlation to the fraction of acutely hypoxic cells, probably because both parameters were determined primarily by the microvascular resistance to blood flow. Therefore, the observed association between tumor radiocurability and IFP was most likely an indirect consequence of a strong relationship between IFP and the fraction of acutely hypoxic cells.

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.

Limitations of dynamic contrast-enhanced MRI in monitoring radiation-induced changes in the fraction of radiobiologically hypoxic cells in human melanoma xenografts.

PURPOSE: To investigate the potential of gadopentetate dimeglumine (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in detecting radiation-induced changes in the fraction of radiobiologically hypoxic cells in A-07 human melanoma xenografts. MATERIALS AND METHODS: A-07 tumors were randomly assigned to an unirradiated control group or a group given a single radiation dose of 20 Gy. DCE-MRI and measurement of fraction of hypoxic cells were performed immediately before and 24 h after the radiation exposure. Tumor images of E . F (E is the initial extraction fraction of Gd-DTPA and F is blood perfusion) and lambda (lambda is proportional to extracellular volume fraction) were produced by subjecting DCE-MRI series to Kety analysis. Fraction of hypoxic cells was measured by using a radiobiological assay based on the paired survival curve method. RESULTS: Fraction of radiobiologically hypoxic cells was higher in irradiated tumors (26.2+/-5.8%) than in unirradiated tumors (7.5+/-2.7%) by a factor of 3.5+/-1.5 (P=0.0093), whereas only minor radiation-induced changes in E . F and lambda could be detected. CONCLUSION: DCE-MRI does not seem to offer insight into the changes in fraction of radiobiologically hypoxic cells occurring in A-07 tumors within 24 h after irradiation with 20 Gy.

Detection of different hypoxic cell subpopulations in human melanoma xenografts by pimonidazole immunohistochemistry.

This study aimed at developing immunohistochemical assays for different subpopulations of hypoxic cells in tumors. BALB/c-nu/nu mice bearing A-07 or R-18 tumors were given a single dose of 90 mg/kg body weight or three doses (3 h apart) of 30 mg/kg body weight of pimonidazole hydrochloride intravenously. The fraction of pimonidazole-labeled cells was assessed in paraffin-embedded and frozen tumor sections and compared with the fraction of radiobiologically hypoxic cells. The staining pattern in paraffin-embedded sections indicated selective staining of chronically hypoxic cells. Frozen sections showed a staining pattern consistent with staining of both chronically and acutely/repetitively hypoxic cells. Fraction of pimonidazole-labeled cells in paraffin-embedded sections was lower than the fraction of radiobiologically hypoxic cells (single-dose and triple-dose experiment). In frozen sections, fraction of pimonidazole-labeled cells was similar to (single-dose experiment) or higher than (triple-dose experiment) fraction of radiobiologically hypoxic cells. Three different subpopulations of hypoxic cells could be quantified by pimonidazole immunohistochemistry: the fraction of cells that are hypoxic because of limitations in oxygen diffusion, the fraction of cells that are hypoxic simultaneously because of fluctuations in blood perfusion, and the fraction of cells that are exposed to one or more periods of hypoxia during their lifetime because of fluctuations in blood perfusion.

Assessment of fraction of hypoxic cells in human tumor xenografts with necrotic regions by dynamic contrast-enhanced MRI.

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 mm(3)) and large (500-1000 mm(3)) 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 lambda (lambda is proportional to extracellular volume fraction) were produced by subjecting the DCE-MRI data to Kety analysis. Necrotic tissue could be identified in lambda images but not in E.F images of the tumors. Most voxels in viable tissue showed lambda values of 0.15-0.70, whereas the lambda values of most voxels in necrotic tissue were either <0.15 or >0.70. The E.F and lambda frequency distributions of the viable tissue, but not the E.F and lambda 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 lambda 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.

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

PURPOSE: 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. MATERIALS AND METHODS: A-07 and R-18 human melanoma xenografts were used as preclinical tumor models. DCE-MRI was performed at a voxel size of 0.23 x 0.47 x 2.0 mm(3). Tumor images of E . F (E is the initial extraction fraction of Gd-DTPA and F is blood perfusion) and lambda (the partition coefficient of Gd-DTPA) were produced by subjecting DCE-MRI series to Kety analysis. Microvascular density and extracellular volume fraction (ECVF) were determined by analysis of histological preparations. The fraction of radiobiologically hypoxic cells was measured by the paired survival curve method. RESULTS: E . F correlated with microvascular density, and lambda correlated with ECVF. The fraction of hypoxic cells was approximately 6.5-fold higher in R-18 tumors than in A-07 tumors, consistent with the observation that A-07 tumors showed higher values for E . F and microvascular density and lower cell density (i.e., higher values for lambda and ECVF) than R-18 tumors. CONCLUSION: E . F and lambda images obtained by Kety analysis of DCE-MRI series contain information that may be utilized to estimate the extent of radiobiological hypoxia in tumors.

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 (HF(Rad)) was measured in individual primary tumors by using a radiobiological assay based on the paired survival curve method. Fraction of immunohistochemically hypoxic cells (HF(Imm)) was assessed in the same tumors by using a pimonidazole-based immunohistochemical assay optimized with respect to achieving selective staining of chronically hypoxic cells. HF(Imm) and the difference between HF(Rad) and HF(Imm), HF(Rad) - HF(Imm), 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.