Publications

PURPOSE: To investigate the combined use of hyperoxia-induced ΔR(2) * and ΔR(1) as a noninvasive imaging biomarker of tumor hypoxia. MATERIALS AND METHODS: MRI was performed on rat GH3 prolactinomas (n = 6) and human PC3 prostate xenografts (n = 6) propagated in nude mice. multiple gradient echo and inversion recovery truefisp images were acquired from identical transverse slices to quantify tumor R(2) * and R(1) before and during carbogen (95% O(2) /5% CO(2) ) challenge, and correlates of ΔR(2) * and ΔR(1) assessed. RESULTS: Mean baseline R(2) * and R(1) were 119 ± 7 s(-1) and 0.6 ± 0.03 s(-1) for GH3 prolactinomas and 77 ± 12 s(-1) and 0.7 ± 0.02 s(-1) for PC3 xenografts, respectively. During carbogen breathing, mean ΔR(2) * and ΔR(1) were -20 ± 8 s(-1) and 0.08 ± 0.03 s(-1) for GH3 and -0.5 ± 1 s(-1) and 0.2 ± 0.08 s(-1) for the PC3 tumors, respectively. A pronounced relationship between ΔR(2) * and ΔR(1) was revealed. CONCLUSION: Considering the blood oxygen-hemoglobin dissociation curve, fast R(2) * suggested that GH3 prolactinomas were more hypoxic at baseline, and their carbogen response dominated by increased hemoglobin oxygenation, evidenced by highly negative ΔR(2) *. PC3 tumors were less hypoxic at baseline, and their response to carbogen dominated by increased dissolved oxygen, evidenced by highly positive ΔR(1) . Because the two biomarkers are sensitive to different oxygenation ranges, the combination of ΔR(2) * and ΔR(1) may better characterize tumor hypoxia than each alone. J. Magn. Reson. Imaging 2013;. © 2013 Wiley Periodicals, Inc.

Vessel size index (R(v), μm) has been proposed as a quantitative magnetic resonance imaging (MRI) derived imaging biomarker in oncology, for the non-invasive assessment of tumour blood vessel architecture and vascular targeted therapies. Appropriate pre-clinical evaluation of R(v) in animal tumour models will improve the interpretation and guide the introduction of the biomarker into clinical studies. The objective of this study was to compare R(v) measured in vivo with vessel size measurements from high-resolution X-ray computed tomography (μCT) of vascular corrosion casts measured post mortem from the same tumours, with and without vascular targeted therapy. MRI measurements were first acquired from subcutaneous SW1222 colorectal xenografts in mice following treatment with 0 (n=6), 30 (n=6) or 200 mg/kg (n=3) of the vascular disrupting agent ZD6126. The mice were then immediately infused with a low viscosity resin and, following polymerisation and maceration of surrounding tissues, the resulting tumour vascular casts were dissected and subsequently imaged using an optimised μCT imaging approach. Vessel diameters were not measurable by μCT in the 200 mg/kg group as the high dose of ZD6126 precluded delivery of the resin to the tumour vascular bed. The mean R(v) for the three treatment groups was 24, 23 and 23.5 μm respectively; the corresponding μCT measurements from corrosion casts from the 0 and 30 mg/kg cohorts were 25 and 28 μm. The strong association between the in vivo MRI and post mortem μCT values supports the use of R(v) as an imaging biomarker in clinical trials of investigational vascular targeted therapies.

Many tumors exhibit defective cell-cycle checkpoint control and increased replicative stress. CHK1 is critically involved in the DNA damage response and maintenance of replication fork stability. We have therefore discovered a novel potent, highly selective, orally active ATP-competitive CHK1 inhibitor, CCT244747, and present its preclinical pharmacology and therapeutic activity.

Non-invasive quantitative imaging biomarkers are essential for the evaluation of novel targeted therapeutics. Before deployment in clinical trials, such imaging biomarkers require qualification, typically through pre-clinical identification of imaging-pathology correlates.

Hypoxia-inducible factor-1 (HIF-1) mediates the transcriptional response to hypoxic stress, promoting tumour progression and survival. This study investigated the acute effects of the small-molecule HIF-pathway inhibitor NSC-134754.

We have previously demonstrated an increased DNA copy number and expression of IGF1R to be associated with poor outcome in Wilms tumors. We have now tested whether inhibiting this receptor may be a useful therapeutic strategy by using a panel of Wilms tumor cell lines. Both genetic and pharmacological targeting resulted in inhibition of downstream signaling through PI3 and MAP kinases, G(1) cell cycle arrest, and cell death, with drug efficacy dependent on the levels of phosphorylated IGF1R. These effects were further associated with specific gene expression signatures reflecting pathway inhibition, and conferred synergistic chemosensitisation to doxorubicin and topotecan. In the in vivo setting, s.c. xenografts of WiT49 cells resembled malignant rhabdoid tumors rather than Wilms tumors. Treatment with an IGF1R inhibitor (NVP-AEW541) showed no discernable antitumor activity and no downstream pathway inactivation. By contrast, Wilms tumor cells established orthotopically within the kidney were histologically accurate and exhibited significantly elevated insulin-like growth factor-mediated signaling, and growth was significantly reduced on treatment with NVP-AEW541 in parallel with signaling pathway ablation. As a result of the paracrine effects of enhanced IGF2 expression in Wilms tumor, this disease may be acutely dependent on signaling through the IGF1 receptor, and thus treatment strategies aimed at its inhibition may be useful in the clinic. Such efficacy may be missed if only standard ectopic models are considered as a result of an imperfect recapitulation of the specific tumor microenvironment.

The recently described combined carbogen USPIO (CUSPIO) magnetic resonance imaging (MRI) method uses spatial correlations in independent imaging biomarkers to assess specific components of tumor vascular structure and function. Our study aimed to evaluate CUSPIO biomarkers for the assessment of tumor response to antiangiogenic therapy. CUSPIO imaging was performed in subcutaneous rat C6 gliomas before and 2 days after treatment with the potent VEGF-signaling inhibitor cediranib (n = 12), or vehicle (n = 12). Histological validation of Hoechst 33342 uptake (perfusion), smooth muscle actin staining (maturation), pimonidazole adduct formation (hypoxia) and necrosis were sought. Following treatment, there was a significant decrease in fractional blood volume (-43%, p < 0.01) and a significant increase in hemodynamic vascular functionality (treatment altered ΔR(2) *(carbogen) from 1.2 to -0.2 s(-1) , p < 0.05). CUSPIO imaging revealed an overall significant decrease in plasma perfusion (-27%, p < 0.05) following cediranib treatment, that was associated with selective effects on immature blood vessels. The CUSPIO responses were associated with a significant 15% reduction in Hoechst 33342 uptake (p < 0.05), but no significant difference in vascular maturation or necrosis. Additionally, treatment with cediranib resulted in a significant 40% increase in tumor hypoxia (p < 0.05). The CUSPIO imaging method provides novel and more specific biomarkers of tumor vessel maturity and vascular hemodynamics, and their response to VEGF-signaling inhibition, compared to current MR imaging biomarkers utilized in the clinic. Such biomarkers may prove effective in longitudinally monitoring tumor vascular remodeling and/or evasive resistance in response to antiangiogenic therapy.

Susceptibility contrast magnetic resonance imaging (MRI), utilising ultrasmall superparamagnetic iron oxide (USPIO) particles, was evaluated for the quantitation of vessel size index (Rv, μm), a weighted average measure of tumour blood vessel calibre, and fractional tumour blood volume (fBV, %), in orthotopically propagated murine PC3 prostate tumour xenografts. Tumour vascular architecture was assessed in vivo by MRI prior to and 24 hr after treatment with 200 mg/kg of the vascular disrupting agent ZD6126. A Bayesian hierarchical model (BHM) was used to reduce the uncertainty associated with quantitation of Rv and fBV. Quantitative histological analyses of the uptake of Hoechst 33342 for perfused vasculature, and haematoxylin and eosin staining for necrosis, were also performed to qualify the MRI data. A relatively large median Rv of 40.3 μm (90% confidence interval (CI90) = 37.4, 44.0 μm) and a high fBV of 5.4% (CI90 = 5.3, 5.5%) were determined in control tumours, which agreed with histologically determined vessel size index. Treatment with ZD6126 significantly (p < 0.01) reduced tumour Rv (34.2 μm, CI90 = 31.2, 38.0 μm) and fBV (3.9%, CI90 = 3.8, 4.1%), which were validated against histologically determined significant reductions in perfusion and vessel size, and increased necrosis. Together these data (i) highlight the use of a BHM to optimise the inferential power available from susceptibility contrast MRI data, (ii) provide strong evaluation and qualification of R(v) and fBV as non-invasive imaging biomarkers of tumour vascular morphology, (iii) reveal the presence of a different vascular phenotype and (iv) demonstrate that ZD6126 exhibits good anti-vascular activity against orthotopic prostate tumours.

The pseudomonad protein, carboxypeptidase G2 (CPG2), is a prodrug-activating enzyme utilized in the targeted chemotherapy strategies of antibody- and gene-directed enzyme prodrug therapy (ADEPT and GDEPT). We have developed a noninvasive imaging approach to monitor CPG2 activity in vivo that will facilitate the preclinical and clinical development of CPG2-based ADEPT and GDEPT strategies. Cleavage of the novel reporter probe, 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu), by CPG2, in human colon adenocarcinoma WiDr xenografts engineered to stably express CPG2, was monitored using (19)F MRSI. The high signal-to-noise ratio afforded by the two MR-equivalent (19)F nuclei of 3,5-DFBGlu, and the 1.4 ppm (19)F chemical shift difference on CPG2-mediated cleavage, enabled the dynamics and quantification of the apparent pharmacokinetics of 3,5-DFBGlu and its CPG2-mediated cleavage in the tumor to be evaluated. In addition, the apparent rate of increase of 3,5-difluorobenzoic acid concentration could also provide a biomarker of CPG2 activity levels in tumors of patients undergoing CPG2-based therapies, as well as a biomarker of treatment response. The addition of in vivo reporter probes, such as 3,5-DFBGlu, to the armamentarium of prodrugs cleaved by CPG2 affords new applications for CPG2 as a gene reporter of transgene expression.

The spatial distribution of apparent diffusion coefficient (ADC) estimates in tumors is typically heterogeneous, although this observed variability is composed of both true regional differences and random measurement uncertainty. In this study, an adaptive Bayesian adaptive smoothing (BAS) model for estimating ADC values is developed and applied to data acquired in two murine tumor models in vivo. BAS models have previously been shown to reduce parameter uncertainty through the use of a Markov random field. Here, diffusion data acquired with four averages was used as an empirical gold standard for evaluating the BAS model. ADC estimates using BAS displayed a significantly closer accordance with the gold standard data and, following analysis of uncertainty estimates, appeared to even outperform the gold standard. These observations were also reflected in simulations. These results have strong implications for clinical studies, as it suggests that the BAS postprocessing technique can be used to improve ADC estimates without the need to compromise on spatial resolution or signal-to-noise or for the adaptation of acquisition hardware. A novel measure of tumor ADC heterogeneity was also defined, which identified differences between tumors derived from different cell lines, which were reflected in histological variations within the tissue microenvironment.

A combined carbogen ultrasmall superparamagnetic iron oxide (USPIO) imaging protocol was developed and applied in vivo in two murine colorectal tumor xenograft models, HCT116 and SW1222, with established disparate vascular morphology, to investigate whether additional information could be extracted from the combination of two susceptibility MRI biomarkers. Tumors were imaged before and during carbogen breathing and subsequently following intravenous administration of USPIO particles. A novel segmentation method was applied to the image data, from which six categories of R(2)* response were identified, and compared with histological analysis of the vasculature. In particular, a strong association between a negative ΔR(2)*(carbogen) followed by positive ΔR(2)*(USPIO) with the uptake of the perfusion marker Hoechst 33342 was determined. Regions of tumor tissue where there was a significant ΔR(2)*(carbogen) but no significant ΔR(2)*(USPIO) were also identified, suggesting these regions became temporally isolated from the vascular supply during the experimental timecourse. These areas correlated with regions of tumor tissue where there was CD31 staining but no Hoechst 33342 uptake. Significantly, different combined carbogen USPIO responses were determined between the two tumor models. Combining ΔR(2)*(carbogen) and ΔR(2)*(USPIO) with a novel segmentation scheme can facilitate the interpretation of susceptibility contrast MRI data and enable a deeper interrogation of tumor vascular function and architecture.

BACKGROUND: Progressive tumour growth is dependent on the development of a functional tumour vasculature and highly regulated by growth factors and cytokines. Nitric oxide (NO) is a free radical, produced both by tumour and host cells, and functions as a signalling molecule downstream of several angiogenic factors. Both pro-and antitumourigenic properties have been attributed to NO.METHODS: The expression of the inducible isoform of NO synthase (iNOS) was knocked down in the C6 glioma cell line using constitutive expression of antisense RNA, and the effect of tumour-derived NO on tumour progression and angiogenesis was investigated.RESULTS: Tumours in which iNOS expression was decreased displayed significantly reduced growth rates compared with tumours derived from parental C6 cells. Quantitative non-invasive magnetic resonance imaging and fluorescence microscopy of tumour uptake of Hoechst 33342, and haematoxylin and eosin staining, revealed significantly impaired vascular development and function in antisense iNOS tumours compared with control in vivo, primarily associated with the more necrotic tumour core. Decreased iNOS expression had no effect on tumour VEGF expression.CONCLUSION: Nitric oxide derived from tumour iNOS is an important modulator of tumour progression and angiogenesis in C6 gliomas and further supports the therapeutic strategy of inhibiting iNOS for the treatment of cancer. British Journal of Cancer (2011) 104, 83-90. doi:10.1038/sj.bjc.6606034 www.bjcancer.com Published online 7 December 2010 (C) 2011 Cancer Research UK

Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes the endogenous inhibitor of nitric oxide synthesis, asymmetric dimethylarginine (ADMA). Constitutive over-expression of DDAH1, the isoform primarily associated with neuronal nitric oxide synthase (nNOS) results in increased tumour growth and vascularization, and elevated VEGF secretion. To address whether DDAH1-mediated tumour growth is reliant upon the enzymatic activity of DDAH1, cell lines expressing an active site mutant of DDAH1 incapable of metabolizing ADMA were created. Xenografts derived from these cell lines grew significantly faster than those derived from control cells, yet not as fast as those over-expressing wild-type DDAH1. VEGF expression in DDAH1 mutant-expressing tumours did not differ from control tumours but was significantly lower than that of wild-type DDAH1-over-expressing tumours. Fluorescence microscopy for CD31 and pimonidazole adduct formation demonstrated that DDAH1 mutant-expressing tumours had a lower endothelial content and demonstrated less hypoxia, respectively, than wild-type DDAH1-expressing tumours. However, there was no difference in uptake of the perfusion marker Hoechst 33342. Non-invasive multiparametric quantitative MRI, including the measurement of native T(1) and T(2) relaxation times and apparent water diffusion coefficient, was indicative of higher cellularity in DDAH1-expressing xenografts, which was confirmed by histological quantification of necrosis. C6 xenografts expressing active site mutant DDAH1 displayed an intermediate phenotype between tumours over-expressing wild-type DDAH1 and control tumours. These data suggest that enhanced VEGF expression downstream of DDAH1 was dependent upon ADMA metabolism, but that the DDAH1-mediated increase in tumour growth was only partially dependent upon its enzymatic activity, and therefore must involve an as-yet unidentified mechanism. DDAH1 is an important mediator of tumour progression, but appears to have addition roles independent of its metabolism of ADMA, which need to be considered in therapeutic strategies targeted against the NO/DDAH pathway in cancer.

Hypoxia is thought to be critical in regulating physiological processes within the female reproductive system, including ovulation, composition of the fluid in the oviductal/uterine lumens and ovarian follicle development. This study examined the localisation of exogenous (pimonidazole) and endogenous [hypoxia inducible factor 1 alpha and 2 alpha (HIF1 alpha, -2 alpha), glucose transporter type 1 (GLUT1) and carbonic anhydrase 9 (CAIX)] hypoxia-related antigens within the oviduct and uterus of the rat reproductive tract. The extent to which each endogenous antigen co-compartmentalised with pimonidazole was also assessed. Female Wistar Furth rats (n = 10) were injected intraperitoneally with pimonidazole (60 mg/kg) 1 h prior to death. Reproductive tissues were removed immediately following death and fixed in 4% paraformaldehyde before being embedded in paraffin. Serial sections were cut (6-7 mu m thick) and antigens of interest identified using standard immunohistochemical procedures. The mucosal epithelia of the ampulla, isthmus and uterus were immunopositive for pimonidazole in most sections. Co-compartmentalisation of pimonidazole with HIF1 alpha was only expressed in the mucosa of the uterus whilst co-compartmentalisation with HIF2 alpha was observed in the mucosa of the ampulla, isthmus and uterus. Both GLUT1 and CAIX were co-compartmentalised with pimonidazole in mucosa of the isthmus and uterus. This study confirms that mucosal regions of the rat oviduct and uterus frequently experience severe hypoxia and there are compartment specific variations in expression of endogenous hypoxia-related antigens, including the HIF isoforms. The latter observation may relate to target gene specificity of HIF isoforms or perhaps HIF2 alpha's responsiveness to non-hypoxic stimuli such as hypoglycaemia independently of HIF1 alpha.

To investigate relationships between magnetic resonance (MR) imaging measurements of R2* and carbogen-induced DeltaR2* in vivo with subsequent histologic assessment of grade, hypoxia, fibrosis, and necrosis in a chemically induced rat mammary tumor model.

Although the biasing of R(2)* estimates by assuming magnitude MR data to be normally distributed has been described, the effect on changes in R(2)* (DeltaR(2)*), such as induced by a paramagnetic contrast agent, has not been reported. In this study, two versions of a novel Bayesian maximum a posteriori approach for estimating DeltaR(2)* are described and evaluated: one that assumes normally distributed data and the other, Rice-distributed data. The approach enables the robust, voxelwise determination of the uncertainty in DeltaR(2)* estimates and provides a useful statistical framework for quantifying the probability that a pixel has been significantly enhanced. This technique was evaluated in vivo, using ultrasmall superparamagnetic iron oxide particles in orthotopic murine prostate tumors. It is shown that assuming magnitude data to be normally distributed causes DeltaR(2)* to be underestimated when signal-to-noise ratio is modest. However, the biasing effect is less than is found in R(2)* estimates, implying that the simplifying assumption of normally distributed noise is more justifiable when evaluating DeltaR(2)* compared with when evaluating precontrast R(2)* values.

The spatial distribution of apparent diffusion coefficient (ADC) estimates in tumors is typically heterogeneous, although this observed variability is composed of both true regional differences and random measurement uncertainty. In this study, an adaptive Bayesian adaptive smoothing (BAS) model for estimating ADC values is developed and applied to data acquired in two murine tumor models in vivo. BAS models have previously been shown to reduce parameter uncertainty through the use of a Markov random field. Here, diffusion data acquired with four averages was used as an empirical gold standard for evaluating the BAS model. ADC estimates using BAS displayed a significantly closer accordance with the gold standard data and, following analysis of uncertainty estimates, appeared to even outperform the gold standard. These observations were also reflected in simulations. These results have strong implications for clinical studies, as it suggests that the BAS postprocessing technique can be used to improve ADC estimates without the need to compromise on spatial resolution or signal-to-noise or for the adaptation of acquisition hardware. A novel measure of tumor ADC heterogeneity was also defined, which identified differences between tumors derived from different cell lines, which were reflected in histological variations within the tissue microenvironment. Magn Reson Med, 2010. © 2010 Wiley-Liss, Inc.

The least-squares algorithm is known to bias apparent diffusion coefficient (ADC) values estimated from magnitude MR data, although this effect is commonly assumed to be negligible. In this study the effect of this bias on tumor ADC estimates was evaluated in vivo and was shown to introduce a consistent and significant underestimation of ADC, relative to those given by a robust maximum likelihood approach (on average, a 23.4 +/- 12% underestimation). Monte Carlo simulations revealed that the magnitude of the bias increased with ADC and decreasing signal-to-noise ratio (SNR). In vivo, this resulted in a much-reduced ability to resolve necrotic regions from surrounding viable tumor tissue compared with a maximum likelihood approach. This has significant implications for the evaluation of diffusion MR data in vivo, in particular in heterogeneous tumor tissue, when evaluating bi- and multiexponential tumor diffusion models for the modeling of data acquired with larger b-values (b > 1000 s/mm(2)) and for data with modest SNR. Use of a robust approach to modeling magnitude MR data from tumors is therefore recommended over the least-squares approach when evaluating data from heterogeneous tumors.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme that is currently employed in a range of targeted cancer chemotherapy strategies such as gene-directed enzyme prodrug therapy (GDEPT). Employing dynamic nuclear polarization (DNP) and natural abundance (13)C magnetic resonance spectroscopy (MRS), we observed the CPG2-mediated conversion of a novel hyperpolarized reporter probe 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu) to 3,5-difluorobenzoic acid (3,5-DFBA) and L-glutamic acid (L-Glu) in vitro. Isotopic labeling of the relevant nuclei with (13)C in 3,5-DFBGlu or related substrates will yield a further factor of 100 increase in the signal-to-noise. We discuss the feasibility of translating these experiments to generate metabolic images of CPG2 activity in vivo.

Platelet-derived growth factors (PDGF) play a major role in pericyte recruitment in tumor capillaries. Pericytes are required for proper vessel development, and contribute to tumor angiogenesis by promoting stabilization and maturation of newly formed vessels. To investigate the effects of pericyte coverage on tumor vessel morphology and function in vivo, tumors derived from B16 melanoma cells transfected with either control plasmid (B16/ctr) or plasmid encoding full-length PDGF-BB (B16/PDGF), the latter previously shown to have enhanced blood vessel pericyte coverage and an increased tumor growth rate, were assessed using histopathological methods, Hoechst 33342-based perfusion analyses, and two noninvasive susceptibility magnetic resonance imaging (MRI) methods. Susceptibility-contrast MRI, incorporating the use of ultrasmall superparamagnetic iron oxide particles, revealed a significant (p < 0.05) reduction in vessel size index (R-nu) of B16/PDGF tumors, and which was validated histologically by the presence of significantly smaller (p < 0.001), more punctate blood vessels identified by fluorescence microscopy of the perfusion marker Hoechst 33342. Intrinsic-susceptibility MRI was used to measure the transverse MRI relaxation rate R-2*, sensitive to changes in endogenous paramagnetic [deoxyhaemoglobin], and used to probe for vascular maturation and function. Hypercapnia (5% CO2/95% air) induced a negligible Delta R-2* response in the B16/ctr and B16/PDGF tumors. In contrast, hyperoxia (5% CO2/95% O-2) induced a significantly greater R2* reduction in the B16/PDGF tumors (p < 0.02). Together the susceptibility MRI-derived biomarkers reveal novel pericyte-dependent changes in the morphology and function of the perfused tumor vasculature in vivo. (c) 2007 Wiley-Liss, Inc.

Purpose: Vascular disrupting agents are anticancer agents that typically produce a cytostatic tumor response. Vessel size index magnetic resonance imaging (MRI) allows for the estimation of the fractional blood volume (fBV) and blood vessel size (Rv). We assessed whether the vessel size index parameters provided imaging biomarkers for detecting early tumor response to a vascular disrupting agent.Methods and Materials: GH3 prolactinomas were grown subcutaneously in 12 rats. Vessel size index MRI was performed with Sinerem, an ultrasmall superparamagnetic iron oxide intravascular contrast agent, to determine the tumor fBV and Rv. MRI was performed before and at 24 h after treatment with either the vascular disrupting agent, 5,6-dimethylxanthenone 4-acetic acid (DMXAA) (n = 6) or with the drug vehicle (n = 6). After treatment, the tumors were analyzed histologically and correlates with the MRI findings sought.Results: Histogram analysis showed non-normal distributions of Rv and fBV. The 25th percentiles of the fBV and Rv were significantly reduced (p < 0.01) after treatment with DMXAA, with an increase in the regions of low-measured fBV. For the treated and control tumors, the fraction of tumor with an fBV of <= 1% correlated with the histologically determined percentage of necrosis (r = 0.77, p < 0.005). The fraction of tumor with an fBV of <= 1% in treated tumors was significantly increased compared with before treatment (p < 0.05) and with that in the controls (p < 0.05).Conclusion: The vessel size index results were consistent with the known action of DMXAA to cause vascular collapse, with histogram analysis of the fBV providing the most sensitive indicator of response. In particular, the parameter, the fraction of tumor with an fBV of <= 1% is a potential biomarker that correlates with the histopathologic measure of tumor necrosis. (C) 2008 Elsevier Inc.

Purpose: To study the impact of Gd-DTPA-BMA on choline signals of HT29 colon carcinomas determined by localized H-1 MRS in vivo at 4.7T.Materials and Methods: PRESS H-1 MR spectra (2-second repetition time and echo times of 20-272 msec) were acquired from HT29 xenografts prior to and following intravenous administration of 0.1 or 0.2 mmol/kg Gd-DTPA-BMA. The magnetic resonance spectroscopy (MRS) data were analyzed by 1) normalizing choline and water peak areas to their precontrast values; and 2) estimating absolute choline concentration relative to tissue water.Results: Changes in the T-1 and T-2 of choline and water were apparent following administration of Gd-DTPA-BMA. Administration of 0.1 mmol/kg Gd-DTPA-BMA induced significant increases in the choline peak area, concomitant with enhancements of the water peak area, whereas 0.2 mmol/kg Gd-DTPA-BMA induced no enhancement of choline peak area but significant increases in water peak area at short echo times.Conclusion: The effect of Gd-DTPA-BMA on estimation of tumor choline concentration varied with the dose of contrast agent, the echo time, and the time after contrast agent administration. These data highlight the potential pitfalls associated with the modulation of choline and water signals post-Gd-DTPA-BMA and may account for the apparently contradictory results previously reported.

Purpose: To characterize longitudinal tumor progression in a murine orthotopic model of liver metastasis using susceptibility contrast magnetic resonance imaging (MRI).Materials and Methods: Nude mice were inoculated intra-splenically with LS174T colorectal carcinoma cells 24 hours postadministration of 2.5 mgFe/kg of the ultrasmall superparamagnetic iron oxide particle preparation feruglose. Contiguous T2 and T2*-weighted multislice MR images were acquired 10, 15, 20, 25, 30, and 35 days postinoculation to longitudinally evaluate metastatic progression. Functional tumor vasculature and hypoxia were histologically evaluated at the final timepoint using Hoechst 33342 uptake, pimonidazole and hematoxylin and eosin staining. A parallel cohort of subcutaneous tumors was included for comparison.Results: All intrasplenically inoculated mice developed liver metastases, evident in both T2*- and T2-weighted images as high-signal deposits, compared to feruglose-nulled normal liver. Small lesions were detected as early as day 10 and all mice exhibited progressing lesions over 35 days. Liver metastases took longer to establish, but exhibited a similar volume doubling time to the subcutaneously propagated tumors of approximate to 2-3 days. Different functional tumor vascular architectures between the two growth sites were apparent.Conclusion: Susceptibility-contrast MRI using a single dose of feruglose can be used to easily detect and longitudinally monitor orthotopically propagated liver metastases in vivo.

Purpose: To assess tumor fractional blood volume (xi), determined in vivo by susceptibility contrast magnetic resonance imaging (MRI) as a noninvasive imaging biomarker of tumor response to the vascular disrupting agent ZD6126.Methods and Materials: The transverse MRI relaxation rate R-2(*) of rat GH3 prolactinomas was quantified prior to and following injection of 2.5 mgFe/kg feruglose, an ultrasmall superparamagnetic iron oxide intravascular contrast agent, and xi (%) was determined from the change in R-2(*). The rats were then treated with either saline or 50 mg/kg ZD6126, and xi measured again 24 hours later. Following posttreatment MRI, Hoechst 33342 (15 mg/kg) was administered to the rats and histological correlates from composite images of tumor perfusion and necrosis sought.Results: Irrespective of treatment, tumor volume significantly increased over 24 hours. Saline-treated tumors showed no statistically significant change in xi, whereas a significant (p = 0.002) 70% reduction in xi of the ZD6126-treated cohort was determined. Hoechst 33342 uptake was associated with viable tumor tissue and was significantly (p = 0.004) reduced and restricted to the rim of the ZD6126-treated tumors. A significant positive correlation between posttreatment xi and Hoechst 33342 uptake was obtained (r = 0.83, p = 0.002), providing validation of the MRI-derived measurements of fractional tumor blood volume.Conclusions: These data clearly highlight the potential of susceptibility contrast MRI with ultrasmall superparamagnetic iron oxide contrast agents to provide quantitative imaging biomarkers of fractional tumor blood volume at high spatial resolution to assess tumor vascular status and response to vascular disrupting agents. (C) 2007 Elsevier Inc.

Purpose: To investigate the use of the transverse magnetic resonance imaging (MRI) relaxation rate R-2* (s(-1)) as a biomarker of tumor vascular response to monitor vascular disrupting agent (VDA) therapy.Methods and Materials: Multigradient echo MRI was used to quantify R-2* in rat GH3 prolactinomas. R-2* is a sensitive index of deoxyhemoglobin in the blood and can therefore be used to give an index of tissue oxygenation. Tumor R-2* was measured before and up to 35 min after treatment, and 24 h after treatment with either 350 mg/kg 5,6-dimethylxanthenone-4-acetic acid (DMXAA) or 100 mg/kg combretastatin-A4-phosphate (CA4P). After acquisition of the MRI data, functional tumor blood vessels remaining after VDA treatment were quantified using fluorescence microscopy of the perfusion marker Hoechst 33342.Results: DMXAA induced a transient, significant (p < 0.05) increase in tumor R-2* 7 min after treatment, whereas CA4P induced no significant changes in tumor R-2* over the first 35 min. Twenty-four hours after treatment, some DMXAA-treated tumors demonstrated a decrease in R2*, but overall, reduction in R-2* was not significant for this cohort. Tumors treated with CA4P showed a significant (p < 0.05) reduction in R2* 24 It after treatment. The degree of Hoechst 33342 uptake was associated with the degree of R-2* reduction at 24 h for both agents.Conclusions: The reduction in tumor R-2* or deoxyhemoglobin levels 24 h after VDA treatment was a result of reduced blood volume caused by prolonged vascular collapse. Our results suggest that DMXAA was less effective than CA4P in this rat tumor model. @ 2007 Elsevier Inc.

The response of radiation-induced fibrosarcoma 1 (RIF-1) tumors treated with the vascular-disrupting agent (VDA) ZD6126 was assessed by in vivo and ex vivo 1H magnetic resonance spectroscopy (MRS) methods. Tumors treated with 200 mg/kg ZD6126 showed a significant reduction in total choline (tCho) in vivo 24 hours after treatment, whereas control tumors showed a significant increase in tCho. This response was investigated further within both ex vivo unprocessed tumor tissues and tumor tissue metabolite extracts. Ex vivo high-resolution magic angle spinning (HRMAS) and 1H MRS of metabolite extracts revealed a significant reduction in phosphocholine and glycerophosphocholine in biopsies of ZD6126-treated tumors, confirming in vivo tCho response. ZD6126-induced reduction in choline compounds is consistent with a reduction in cell membrane turnover associated with necrosis and cell death following disruption of the tumor vasculature. In vivo tumor tissue water diffusion and lactate measurements showed no significant changes in response to ZD6126. Spin-spin relaxation times (T2) of water and metabolites also remained unchanged. Noninvasive 1H MRS measurement of tCho in vivo provides a potential biomarker of tumor response to VDAs in RIF-1 tumors.

The Cancer Imaging Program of the National Cancer Institute convened a workshop to assess the current status of hypoxia imaging, to assess what is known about the biology of hypoxia as it relates to cancer and cancer therapy, and to define clinical scenarios in which in vivo hypoxia imaging could prove valuable.

The dose-dependent effects of 5,6-dimethylxanthenone4- acetic acid ( DMXAA) on rat GH3 prolactinomas were investigated in vivo. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to assess tumor blood flow/permeability pretreatment and 24 hours posttreatment with 0, 100, 200, or 350 mg/kg DMXAA. DCE-MRI data were analyzed using K trans and the integrated area under the gadolinium time curve (IAUGC) as response biomarkers. High-performance liquid chromatography ( HPLC) was used to determine the plasma concentration of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) following treatment to provide an index of increased vessel permeability and vascular damage. Finally, tumor necrosis was assessed by grading hematoxylin and eosin-stained sections cut from the same tumors investigated by MRI. Both tumor K trans and IAUGC were significantly reduced 24 hours posttreatment with 350 mg/kg DMXAA only, with no evidence of dose response. HPLC demonstrated a significant increase in plasma 5-HIAA 24 hours posttreatment with 200 and 350 mg/kg DMXAA. Histologic analysis revealed some evidence of tumor necrosis following treatment with 100 or 200 mg/kg DMXAA, reaching significance with 350 mg/kg DMXAA. The absence of any reduction in K trans or IAUGC following treatment with 200 mg/kg, despite a significant increase in 5-HIAA, raises concerns about the utility of established DCE-MRI biomarkers to assess tumor response to DMXAA.

Tumours derived from DLD-1 colon adenocarcinoma cells, transfected to either overexpress inducible nitric oxide synthase (clone iNOS-19) or with empty vector (pBAN2R), were utilised to test the hypothesis that tumour expression of iNOS (a) increases tumour angiogenesis and (b) modulates the anti-tumour activity of the vascular disrupting agent ZD6126. Overexpression of iNOS by clone iNOS-19 cells and murine xenografts was confirmed by the Griess assay and western blot analysis respectively. Clone iNOS-19 tumours grew more rapidly than pBAN2R tumours. Tumour perfusion, assessed by Hoechst 33342 uptake, was significantly greater in the clone iNOS-19 tumours (P < 0.001). A significant reduction in the perfusion of only the pBAN2R tumours, compared with control, was obtained 24 h after treatment with an intermediate dose of 100 mg/kg ZD6126 (P < 0.001), whereas 200 mg/kg significantly reduced the perfusion of both tumour types (P < 0.001). Whilst pBAN2R tumour necrosis increased in a dose-dependent manner, significant at 100 and 200 mg/kg ZD6126 (P < 0.05), intermediate doses did not induce a similar degree of necrosis in clone iNOS-19 tumours. A significant reduction in splenic perfusion was found 24 h after treatment with 100 mg/kg ZD6126, primarily associated with the red pulp. Overexpression of iNOS increases tumour growth, the degree of functionally perfused vasculature and angiogenesis, and also confers resistance to the vascular disrupting agent ZD6126. (c) 2006 Elsevier Inc. All rights reserved.

Modulation of tumour oxygenation may be used to increase or decrease tumour hypoxia in order to improve the effect of radiotherapy or bioreductive drugs, respectively. Magnetic resonance imaging (MRI) and near infrared spectroscopy (NIRS) are techniques sensitive to blood deoxyhemoglobin concentration (Hb) that can be used to investigate tumour hypoxia indirectly via blood oxygenation levels. In this study we have used NIRS to determine absolute Hb and changes in deoxyhemoglobin and oxyhemoglobin (HbO) in subcutaneous rodent tumours for challenges that alter blood flow and oxygenation, with the aim to better interpret our MRI data. Both carbogen [95% O2 + 5% CO2] and 100% O2 breathing produced a similar and significant reduction in Hb and increase in HbO, but a negligible change in HbT (= Hb + HbO). In contrast, N2 breathing to terminal anoxia and intravenous hydralazine produced a negligible increase in Hb, but large reductions in HbO and HbT. HbT is proportional to blood volume, so our data suggests large blood volume decreases occur with challenges likely to cause reduced arterial blood pressure. Hence MRI techniques that measure the R2* relaxation rate, which varies linearly with total Hb, will underestimate the effects of hypotensive agents at increasing tumour hypoxia.

Purpose: We have shown previously that carbogen (95% 0(2), 5% CO2) breathing by rodents can increase uptake of anticancer drugs into tumours. The aim of this study was to extend these observations to other rodent models using the anticancer drug 5-fluorouracil (5FU). 5FU pharmacokinetics in tumour and plasma and physiological effects on the tumour by carbogen were investigated to determine the locus of carbogen action on augmenting tumour uptake of 5FU. Methods: Two different tumour models were used, rat GH3 prolactinomas xenografted s.c. into nude mice and rat H9618a hepatomas grown s.c. in syngeneic Buffalo rats. Uptake and metabolism of 5FU in both tumour models with or without host carbogen breathing was studied non-invasively using fluorine-19 magnetic resonance spectroscopy (F-19-MRS), while plasma samples from Buffalo rats were used to construct a NONMEM pharmacokinetic model. Physiological effects of carbogen on tumours were studied using P-31-MRS for energy status (NTP/Pi) and pH, and gradient-recalled echo magnetic resonance imaging (GRE-MRI) for blood flow and oxygenation. Results: In both tumour models, carbogan-induced GRE-MRI signal intensity increases of similar to 60% consistent with an increase in tumour blood oxygenation and/or flow. In GH3 xenografts, F-19-MRS showed that carbogen had no significant effect on 5FU uptake and metabolism by the tumours, and P-31-MRS showed there was no change in the NTP/Pi ratio. In H9618a hepatomas, F-19-MRS showed that carbogen had no effect on tumour 5FU uptake but significantly (p = 0.0003) increased 5FU elimination from the tumour (i.e. decreased the t(1/2)) and significantly (p = 0.029) increased (53%) the rate of metabolism to cytotoxic fluoronucleotides (FNuct). The pharmacokinetic analysis showed that carbogen increased the rate of tumour uptake of 5FU from the plasma but also increased the rate of removal. P-31-MRS showed there were significant (pless than or equal to 0.02) increases in the hepatoma NTP/Pi ratio of 49% and transmembrane pH gradient of 0.11 units. Conclusions: We suggest that carbogen can transiently increase tumour blood flow, but this effect alone may not increase uptake of anticancer drugs without a secondary mechanism operating. In the case of the hepatoma, the increase in tumour energy status and pH gradient may be sufficient to augment 5FU metabolism to cytotoxic FNuct, while in the GH3 xenografts this was not the case. Thus carbogen breathing does not universally lead to increased uptake of anticancer drugs.

The effective magnetic resonance imaging (MRI) transverse relaxation rate R(2)* was investigated as an early acute marker of the response of rat GH3 prolactinomas to the vascular-targeting agent, ZD6126. Multigradient echo (MGRE) MRI was used to quantify R(2)*, which is sensitive to tissue deoxyhemoglobin levels. Tumor R(2)* was measured prior to, and either immediately for up to 35 minutes, or 24 hours following administration of 50 mg/kg ZD6126. Following MRI, tumor perfusion was assessed by Hoechst 33342 uptake. Tumor R(2)* significantly increased to 116 +/- 4% of baseline 35 minutes after challenge, consistent with an ischemic insult induced by vascular collapse. A strong positive correlation between baseline R(2)* and the subsequent increase in R(2)* measured 35 minutes after treatment was obtained, suggesting that the baseline R(2)* is prognostic for the subsequent tumor response to ZD6126. In contrast, a significant decrease in tumor R(2)* was found 24 hours after administration of ZD6126. Both the 35-minute and 24-hour R(2)* responses to ZD6126 were associated with a decrease in Hoechst 33342 uptake. Interpretation of the R(2)* response is complex, yet changes in tumor R(2)* may provide a convenient and early MRI biomarker for detecting the antitumor activity of vascular-targeting agents.

Purpose: To use P-31 and H-1 magnetic resonance spectroscopy (MRS) to assess changes in tumor metabolic profile in vivo in response to 5,6-dimethylxanthenone-4-acetic acid (DMXAA) with a view to identifying biomarkers associated with tumor dose response.Experimental Design: In vivo P-31 and H-1 MRS measurements of (a) tumor bioenergetics [&beta;-nucleoside triphosphate/inorganic phosphate (&beta;-NTIP/Pi)], (b) the membrane-associated phosphodiesters and phosphomonoesters (PDE/PME), (c) choline (mmol/L), and (d) lactate/water ratio were made on murine HT29 colon carcinoma xenografts pretreatment and 6 or 24 hours posttreatment with increasing doses of DMXAA.. Following in vivo MRS, the tumors were excised and used for high-resolution P-31 and H-1 MRS of extracts to provide validation of the in vivo MRS data, histologic analysis of necrosis, and high-performance liquid chromatography.Results: Both &beta;-NTP/Pi and PDE/PME decreased in a dose-dependent manner 6 hours posttreatment with DMXAA, with significant decreases in &beta;-NTP/Pi with 15 mg/kg (P &LT; 0.001) and 21 mg/kg (P &LT; 0.01). A significant decrease in total choline in vivo was found 24 hours posttreatment with 21 mg/kg DMXAA (P &LT; 0.05); this was associated with a significant reduction in the concentration of the membrane degradation products glycerophosphoethanolamine and glycerophosphocholine measured in tissue extracts (P &LT; 0.05).Conclusions: The reduction in tumor energetics and membrane turnover is consistent with the vascular-disrupting activity of DMXAA, P-31 MRS revealed tumor response to DMXAA at doses below the maximum tolerated dose for mice. Both P-31 and H-1 MRS provide biomarkers of tumor response to DMXAA that could be used in clinical trials.

The thalidomide analogue and immunomodulatory drug (IMiD (R)) lenalidomide (CC-5013, REVLIMD (TM)) is emerging as a useful treatment for a number of cancers and has recently entered phase III trials for multiple myeloma. It has been suggested that the anti-tumor effect of lenalidomide is related to its anti-angiogenic potency. In this regard, we have previously shown that lenalidomide inhibits angiogenesis in both rat and human in vitro models but does not affect endothelial cell proliferation. We now show that oral administration of lenalidomide attenuates growth factor-induced angiogenesis in vivo; the rat mesenteric window assay was utilized to show that lenalidomide significantly inhibits vascularization in a dose-dependent manner. We also found that lenalidomide significantly inhibits growth factor-induced endothelial cell migration. This correlates with the inhibitory effect of lenalidomide on growth factor-induced Akt phosphorylation, thereby providing a potential mechanism for its anti-migratory and subsequent anti-angiogenic effects. These data further support the use of lenalidomide as an orally administered drug for the effective treatment of angiogenesis-dependent conditions, including cancer, and suggest a potential mechanism of action. (c) 2005 Elsevier Inc. All rights reserved.

Objective: The objective of this study was to investigate the duration of R-2* and R-2 enhancement in murine liver in vivo after administration of a single dose of 3 different iron oxide-based contrast agents.Materials and Methods: Murine liver R-2* and R-2 were quantified longitudinally postadministration of 2.5 mgFe/kg ferumoxides, 2.5 mgFe/kg ferumoxytol, 2.5 or 5 mgFe/kg feruglose, or saline over 50 days. Changes in R-2* and R-2 were evaluated histologically using Perl's staining and by atomic absorption spectrometry.Results: All 3 contrast agents significantly increased liver R-2* and R-2 4 hours after challenge. After 10 days, R-2* and R-2 for both the ferumoxides and ferumoxytol cohorts had recovered to saline control levels, whereas the faster R-2* and R-2 of the feruglose cohort was sustained and significantly faster than control at day 50. Histology revealed feruglose in both Kupffer and endothelial cells, whereas both ferumoxides and ferumoxytol were associated with the Kupffer cells.Conclusion: Compared with ferumosides and ferumoxytol, feruglose exhibits prolonged R-2* and R-2 enhancement of murine liver.

To test the prognostic potential of tumor R2* with respect to radiotherapeutic outcome. Blood oxygenation level dependent (BOLD) MRI images are sensitive to changes in deoxyhemoglobin concentration through the transverse MRI relaxation rate R2* of tissue water, hence the quantitative measurement of tumor R2* may be related to tissue oxygenation.

The oxygenation status of tumors derived from wild-type C6 glioma cells and clone D27 cells overexpressing dimethylarginine dimethylaminohydrolase (DDAH) was assessed in vivo using a variety of direct and indirect assays of hypoxia. Clone D27 tumors exhibit a more aggressive and better-vascularized phenotype compared to wild-type C6 gliomas. Immunohistochemical analyses using the 2-nitroimidazole hypoxia marker pimonidazole, fiber optic OxyLite measurements of tumor pO2, and localized 31P magnetic resonance spectroscopy measurements of tumor bioenergetic status and pH clearly demonstrated that the D27 tumors were more hypoxic compared to C6 wild type. In the tumor extracts, only glucose concentrations were significantly lower in the D27 tumors. Elevated Glut-1 expression, a reliable functional marker for hypoxia-inducible factor-1-mediated metabolic adaptation, was observed in the D27 tumors. Together, the data show that overexpression of DDAH results in C6 gliomas that are more hypoxic compared to wild-type tumors, and point strongly to an inverse relationship of tumor oxygenation and angiogenesis in vivo--a concept now being supported by the enhanced understanding of oxygen sensing at the molecular level.

The objective was to present a method for the repeated noninvasive measurement of tumor oxygenation (Po(2)) over the whole period of tumor growth.

Tumor vasculature is an attractive therapeutic target as it differs structurally from normal vasculature, and the destruction of a single vessel can lead to the death of many tumor cells. The effects of antivascular drugs are frequently short term, with regrowth beginning less than 24 hours posttreatment. This study investigated the duration of the response to the vascular targeting agent, ZD6126, of the GH3 prolactinoma, in which efficacy and dose-response have previously been demonstrated. GH3 prolactinomas were grown in the flanks of eight Wistar Furth rats. All animals were treated with 50 mg/kg ZD6126. The tumors were examined with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) 24 hours pretreatment and posttreatment, and at a single time between 48 and 96 hours posttreatment. No evidence of recovery of perfusion was observed even at the longest (96-hour) time point. Involvement of a statistician at the project planning stage and the use of DCE-MRI, which permits noninvasive quantitation of parameters related to blood flow in intact animals, allowed this highly significant result to be obtained using only eight rats.

To characterize spontaneously occurring c-neu/HER2 overexpressing tumours in oncomice and their response to herceptin by non-invasive magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). Oncomice were monitored by localized (31)p MRS during unperturbed growth and before and after treatment with 10 mg/kg herceptin (Hoffman La Roche) intraperitoneally for up to 21 days post-treatment. Vascular morphology and function was assessed by quantitation of tumour magnetic resonance (MR) relaxation rates R-2* and R-2 prior to and either during carbogen (95% O-2/5% CO2) breathing or following administration of the blood-pool contrast agent NC100150 (Clariscan, Amersham Health). lmmunohistochemistry showed strong membrane staining for HER2 protein overexpression. The P-31 MRS showed only a significant (p < 0.01) increase of phosphomonoester / total phosphate ratio over 21 days of growth. Herceptin increased the tumour volume doubling time compared to untreated tumours and significantly increased the phosphomonoester beta-nucleoside triphosphate ratio 2 days after treatment (p = 0.01). Tumours showed a highly heterogeneous yet significant (p < 0.01) decrease or increase in R2* in response to carbogen or NC100150 respectively The absence of a decline in tumour bioenergetics with growth, commonly seen in P-31 MRS studies of transplanted rodent tumour models, coupled with the heterogeneous blood volume revealed by 1 H MRI, suggest a metabolic and vascular phenotype similar to that found in human tumours.

It is now well established that uncontrolled proliferation of tumour cells together with the chaotic and poorly regulated blood supply of solid tumours result in tissue hypoxia, and that hypoxic regions of tumouts are resistant to radiotherapy and chemotherapy. The development and application of non-invasive methods to rapidly determine the degree and extent of tumour hypoxia in an individual tumour would clearly enhance cancer treatment strategies. This review describes the current status of two F-19 nuclear magnetic resonance (NMR) methodologies that have been exploited to investigate turnout hypoxia, namely: (i) F-19 NMR oximetry following administration of perfluorocarbons, from which tumour po(2) measurements can be made; and (ii) F-19 NMR measurements of the turnout retention of fluorinated 2-nitroimidazoles.

ZD6126 is a vascular targeting agent that disrupts the tubulin cytoskeleton of proliferating neo-endothelial cells. This leads to the selective destruction and congestion of tumour blood vessels in experimental tumours, resulting in extensive haemorrhagic necrosis. In this study, the dose-dependent activity of ZD6126 in rat GH3 prolactinomas and murine RIF-1 fibrosarcomas was assessed using two magnetic resonance imaging (MRI) methods. Dynamic contrast-enhanced (DCE) MRI, quantified by an initial area under the time-concentration product curve (IAUC) method, gives values related to tumour perfusion and vascular permeability. Multigradient recalled echo MRI measures the transverse relaxation rate T(2)*, which is sensitive to tissue (deoxyhaemoglobin). Tumour IAUC and R(2)* (=1/T(2)*) decreased post-treatment with ZD6126 in a dose-dependent manner. In the rat model, lower doses of ZD6126 reduced the IAUC close to zero within restricted areas of the tumour, typically in the centre, while the highest dose reduced the IAUC to zero over the majority of the tumour. A decrease in both MRI end points was associated with the induction of massive central tumour necrosis measured histologically, which increased in a dose-dependent manner. Magnetic resonance imaging may be of value in evaluation of the acute clinical effects of ZD6126 in solid tumours. In particular, measurement of IAUC by DCE MRI should provide an unambiguous measure of biological activity of antivascular therapies for clinical trial.

Intracellular factors that regulate nitric oxide (NO) synthesis represent important targets in tumor progression. Overexpression of dimethylarginine dimethylaminohydrolase (DDAH), which metabolizes the endogenous inhibitors of NO synthesis asymmetric dimethylarginine and N-monomethyl-L-arginine, results in C6 gliomas with enhanced growth rate compared with wild type. To investigate the effects of DDAH on tumor vascular morphogenesis in vivo, we have measured the transverse relaxation rates R(2)* and R(2) in clone D27 gliomas overexpressing DDAH and C6 wild-type gliomas using intrinsic susceptibility magnetic resonance imaging (MRI), sensitive to changes in endogenous [deoxyhemoglobin], and susceptibility contrast-enhanced MRI using the intravascular blood pool contrast agent NC100150, and we compared the results with fluorescence microscopy of the tumor uptake of the perfusion marker Hoechst 33342. The baseline R(2)* was significantly faster in the D27 tumors, consistent with a greater vascular development (P < 0.02, ANOVA). There was no significant difference between the response of the two tumor types to hypercapnia (5% CO(2)/95% air), used as a probe for vascular maturation, or hyperoxia (5% CO(2)/95% O(2)), used as a probe for vascular function. NC100150 increased the R(2)* and R(2) rates of both tumor types and demonstrated a significantly larger blood volume in the D27 tumors (P < 0.02, ANOVA). This correlated with a significantly greater uptake of Hoechst 33342 in the D27 tumors compared with C6 wild-type tumors (P < 0.02, ANOVA). Despite the increased tumor blood volume, the Delta R(2)*/Delta R(2) ratio, an index of microvessel size, showed that the capillaries in the two tumor types were of a similar caliber. The data highlight the potential of susceptibility MRI-derived quantitative end points to noninvasively assess tumor angiogenesis, and in this regard, the use of intravascular blood pool contrast agents such as NC100150 appears very promising. Overexpression of DDAH results in increased neovascularization of C6 gliomas in vivo. The lack of significant difference in hypercapnic/hyperoxic response between the C6 and D27 tumors and the similar vessel caliber are also consistent with a role for DDAH in the initial stages of vasculogenesis.

Tumor oxygenation determines the efficacy of radiotherapy, but there is no non-invasive way to image this parameter. Since gradient recalled echo (GRE) images are sensitive to blood deoxyhaemoglobin concentration ([dHb]) they could have a role in assessing tumor oxygenation. In brain, linear relationships have been demonstrated between brain tissue R2* relaxation rate and tissue [dHb] or oxygen saturation, but in tumors, vascular and tissue heterogeneity, and the presence of simultaneous oxidative and glycolytic metabolism, complicate the analysis. We have studied the effects of vascular challenge in a rat prolactinoma tumor model by MR imaging and spectroscopy and comment on the implications of these results for calibrating GRE images for blood or tissue pO2.

To investigate the physiological origins responsible for the varying blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) responses to carbogen (95% O(2)/5% CO(2)) breathing observed with different tumor types.

Previously, (31)P magnetic resonance spectroscopy (MRS) has been used to detect ifosfamide (IF) in vivo and to show that breathing carbogen (5% CO(2)/95% O(2)) enhances the uptake and increases the efficacy of IF in rat GH3 prolactinomas [Rodrigues LM, Maxwell RJ, McSheehy PMJ, Pinkerton CR, Robinson SP, Stubbs M, and Griffiths JR (1997). In vivo detection of ifosfamide by (31)P MRS in rat tumours; increased uptake and cytotoxicity induced by carbogen breathing in GH3 prolactinomas. Br J Cancer 75, 62-68]. We now show that other hypercapnic and/or hyperoxic (5% CO(2) in air, 2.5% CO(2) in O(2)) gas mixtures also increase the uptake of IF into tumors, measured by (31)P MRS. All gases caused an increased uptake (C(max)) of IF compared to air breathing, with carbogen inducing the largest increase (85% (P<.02) compared to 46% with 2.5% CO(2) in O(2) (P<.004) and 48% with 5% CO(2) in air (P<.004)). The T(max) (time of maximum concentration in tumor posintravenous injection of IF) was significantly (P<.04) later in the cohort that breathed 5% CO(2) in air. The increased uptake of IF with carbogen breathing was selective to tumor tissue and there were no significant increases in any of the normal tissues studied, suggesting that any host tissue toxicity would be minimal. Carbogen breathing by patients causes breathlessness. There was no significant difference in IF uptake between breathing carbogen and 2.5% CO(2) in O(2) and, therefore, the ability of 2.5% CO(2) in O(2) to also increase IF uptake may be clinically useful as it causes less patient discomfort.

Hypoxia-inducible factor-1 (HIF-1) regulates many pathways potentially important for tumor growth, including angiogenesis and glycolysis. Most attention has focused on its role in the response to hypoxia, but HIF-1 is also constitutively expressed in many tumors. To analyze the role of this pathway in vivo, we used magnetic resonance (MR) methods and complementary techniques to monitor metabolic changes in tumors derived from HEPA-1 mouse hepatoma lines that were either wild type (WT) or deficient in hypoxia-inducible transcription factor HIF-1beta (c4). The c4 tumors grew significantly more slowly than the WT tumors (P < 0.05), but were examined at a similar size (0.4-0.6 g). At the tumor size used in these studies, no differences in vascularity were observed, and MR parameters measured that related to tumor blood flow, vascularity, and oxygenation demonstrated no significant differences between the two tumor types. Unexpectedly, the ATP content of the c4 tumor was similar to5 times less than in the WT tumor [measured in tumor extracts (P < 0.001) and by metabolic imaging (P < 0.05)]. Noninvasive P-31 MR spectroscopy showed that the nucleoside triphosphate/P-i ratio of the two tumor types was similar, so the low ATP content of the c4 tumors was not caused by (or a cause of) impaired cellular bioenergetics. Rather, glycine, an essential precursor for de novo purine formation, was significantly lower in the c4 tumors (P < 0.05), suggesting that ATP synthesis was impaired in the mutant tumor cells. Supporting evidence for this hypothesis came from the significantly lower concentrations of betaine, phosphocholine, and choline in the c4 tumors (P < 0.05); these are intermediates in an alternative pathway for glycine synthesis. No significant differences were seen in lactate or glucose content. MR resonances from phosphodiesters, which relate to the metabolic turnover of phospholipid membranes, were significantly lower in the WT tumors than in the c4 tumors, both in vivo (P < 0.05) and in extracts (P < 0.01). We propose that loss of up-regulation of expression of the genes for glucose transporters and glycolytic enzymes in the c4 tumors decreased formation of glycine, an essential precursor of ATP synthesis, and thus caused the low ATP content of the c4 tumors. In summary, these data suggest that disruption of the HIF-1 pathway in these tumor cells impairs the supply of anabolic precursors required for cell synthesis. They suggest potential biochemical targets that may be modified by therapy blocking HIF-1 function.

Angiogenesis is a prerequisite for tumour progression and is highly regulated by growth factors and cytokines a number of which also stimulate the production of nitric oxide. Asymmetric dimethylarginine is an endogenous inhibitor of nitric oxide synthesis. Asymmetric dimethylarginine is metabolised by dimethylarginine dimethylaminohydrolase. To study the effect of dimethylarginine dimethylaminohydrolase on tumour growth and vascular development, the rat C6 glioma cell line was manipulated to overexpress the rat gene for dimethylarginine dimethylaminohydrolase 1. Enhanced expression of dimethylarginine dimethylaminohydrolase I increased nitric oxide synthesis (as indicated by a two-fold increase in the production of cGMP), expression and secretion of vascular endothelial cell growth factor, and induced angiogenesis in vitro. Tumours derived from these cells grew more rapidly in vivo than cells with normal dimethylarginine dimethylaminohydrolase I expression. Immunohistochemical and magnetic resonance imaging measurements were consistent with increased tumour vascular development Furthermore, dimethylarginine dimethylaminohydrolase activity was detected in a series of human tumours. This data demonstrates that dimethylarginine dimethylaminohydrolase plays a pivotal role in tumour growth and the development of the tumour vasculature by regulating the concentration of nitric oxide and altering vascular endothelial cell growth factor production. (C) 2002 Cancer Research UK.

The sensitivity of blood oxygenation level dependent (BOLD) contrast techniques to changes to tumour deoxyhaemoglobin concentration is of relevance to many strategies in cancer treatments. In the context of tumour studies, which frequently involve the use of agents to modify blood flow, there are underlying physiological changes different to those of BOLD in the brain. Hence we use the term, flow and oxygenation dependent (FLOOD) contrast, to emphasize this difference and the importance of flow effects. We have measured the R(2)* changes in a prolactinoma tumour model for a variety of vasoactive challenges [carbogen, 100% oxygen and 100% nitrogen as different breathing gases, and administration of tumour blood flow modifiers such as calcitonin gene related peptide (CGRP), hydralazine and nicotinamide]. In addition we have measured other relevant physiological parameters, such as bioenergetic status from (31)P MRS, and blood pH and glucose, that may change during a vasoactive challenge. Here we discuss how they relate to our understanding of FLOOD contrast in tumours. We frequently observe R(2)* changes that match the expected action of the vascular stimulus: R(2)* decreases with agents expected to improve tumour oxygenation and blood flow, and increases with agents designed to increase tumour hypoxia. Unlike most normal tissues, tumours have a chaotic and poorly regulated blood supply, and a mix of glycolytic and oxidative metabolism; thus the response to a vasoactive challenge is not predictable. Changes in blood volume can counteract the effect of blood oxygenation changes, and changes in blood pH and glucose levels can alter oxygen extraction. This can lead to R(2)* changes that are smaller or the reverse of those expected. To properly interpret FLOOD contrast changes these effects must be accounted for.

The hypercapnia induced by carbogen (95% O(2)/5% CO(2)) breathing, which is being re-evaluated as a clinical radiosensitiser, causes patient discomfort and hence poor compliance. Recent preclinical and clinical studies have indicated that the CO(2) content might be lowered without compromising increased tumour oxygenation and radiosensitisation. This preclinical study was designed to see if lower levels of hypercapnia could evoke similar decreases in the transverse relaxation rate R(2)* of rodent tumours to those seen with carbogen breathing. The response of rat GH3 prolactinomas to 1%, 212% and 5% CO(2) in oxygen, and 100% O(2) breathing, was monitored by non-invasive multi-gradient echo MRI to quantify R(2)*. As the oxygenation of haemoglobin is proportional to the blood p(a)O(2) and therefore in equilibrium with tissue pO(2), R(2)* is a sensitive indicator of tissue oxygenation. Hyperoxia alone decreased R(2)* by 13%, whilst all three hypercapnic hyperoxic gases decreased R(2)* by 29%. Breathing 1% CO(2) in oxygen evoked the same decrease in R(2)* as carbogen. The DeltaR(2)* response is primarily consistent with an increase in blood oxygenation, though localised increases in tumour blood flow were also identified in response to hypercapnia. The data support the concept that levels of hypercapnia can be reduced without loss of enhanced oxygenation and hence potential radiotherapeutic benefit.

A solid tumor presents a unique challenge as a system in which the dynamics of the relationship between vascularization, the physiological environment and metabolism are continually changing with growth and following treatment. Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) studies have demonstrated quantifiable linkages between the physiological environment, angiogenesis, vascularization and metabolism of tumors. The dynamics between these parameters continually change with tumor aggressiveness, tumor growth and during therapy and each of these can be monitored longitudinally, quantitatively and non-invasively with MRI and MRS. An important aspect of MRI and MRS studies is that techniques and findings are easily translated between systems. Hence, pre-clinical studies using cultured cells or experimental animals have a high connectivity to potential clinical utility. In the following review, leaders in the field of MR studies of basic tumor physiology using pre-clinical models have contributed individual sections according to their expertise and outlook. The following review is a cogent and timely overview of the current capabilities and state-of-the-art of MRI and MRS as applied to experimental cancers. A companion review deals with the application of MR methods to anticancer therapy.

Both host carbogen (95% oxygen/5% carbon dioxide) breathing and nicotinamide administration enhance tumour radiotherapeutic response and are being re-evaluated in the clinic. Non-invasive magnetic resonance imaging (MRI) and P-31 magnetic resonance spectroscopy (MRS) methods have been used to give information on the effects of nicotinamide alone and in combination with host carbogen breathing on transplanted rat GH3 prolactinomas. Gradient recalled echo (GRE) MRI, sensitive to blood oxygenation changes, and spin echo (SE) MRI, sensitive to perfusion/flow, showed large signal intensity increases with carbogen breathing. Nicotinamide, thought to act by suppressing the transient closure of small blood vessels that cause intermittent tumour hypoxia, induced a small increase in blood oxygenation but no detectable change in perfusion/flow. Carbogen combined with nicotinamide was no more effective than carbogen alone. Both carbogen and nicotinamide caused significant increases in the nucleoside triphosphate/inorganic phosphate (beta NTP/P-i) ratio, implying that the tumour cells normally receive sub-optimal substrate supply, and is consistent with either increased glycolysis and/or a switch to more oxidative metabolism. The most striking observation was the marked increase in blood glucose (twofold) induced by both nicotinamide and carbogen. Whether this may play a role in tumour radiosensitivity has yet to be determined. (C) 2000 Cancer Research Campaign.

Flow and oxygenation dependent (FLOOD) MR images of GH3 prolactinomas display large intensity increases in response to carbogen (5% CO2/95%O-2) breathing. To assess the relative contributions of carbon dioxide and oxygen to this response and the tumour oxygenation state, the response of GH3 prolactinomas to 5% CO2/95% air, carbogen and 100% O-2 was monitored by FLOOD MRI and pO(2) histography. A 10-30% image intensity increase was observed during 5% CO2/95% air breathing, consistent with an increase in tumour blood flow, as a result of CO2-induced vasodilation, reducing the concentration of deoxyhaemoglobin in the blood. Carbogen caused a further 40-50% signal enhancement, suggesting an additional improvement due to increase blood oxygenation. A small 5-10% increase was observed in response to 100% O-2, highlighting the dominance of CO2-induced vasodilation in the carbogen response. Despite the large FLOOD response, non-significant increases in tumour pO(2) were observed in response to the three gases. Tissue pO(2) is determined by the balance of oxygen supply and demand, hence increased blood flow/oxygenation may not necessarily produce a large increase in tissue pO(2). The FLOOD response is determined by the level of deoxygenation of blood, the size of this response relating to vascular density and the potential of high-oxygen content gases to improve the oxygen supply to tumour tissue. Copyright (C) 1999 John Wiley & Sons, Ltd.

Gradient recalled echo (GRE) images are sensitive to both paramagnetic deoxyhaemoglobin concentration (via T2*) and flow (via T1*). Large GRE signal intensity increases have been observed in subcutaneous tumors during carbogen (5% carbon dioxide, 95% oxygen) breathing. We term this combined effect flow and oxygenation-dependent (FLOOD) contrast. We have now used both spin echo (SE) and GRE images to evaluate how changes in relaxation times and flow contribute to image intensity contrast changes. T1-weighted images, with and without outer slice suppression, and calculated T2, T2* and "flow" maps, were obtained for subcutaneous GH3 prolactinomas in rats during air and carbogen breathing. T1-weighted images showed bright features that increased in size, intensity and number with carbogen breathing. H&E stained histological sections confirmed them to be large blood vessels. Apparent T1 and T2 images were fairly homogeneous with average relaxation times of 850 ms and 37 ms, respectively, during air breathing, with increases of 2% for T1 and 11% for T2 during carbogen breathing. The apparent T2* over all tumors was very heterogeneous, with values between 9 and 23 ms and localized increases of up to 75% during carbogen breathing. Synthesised "flow" maps also showed heterogeneity, and regions of maximum increase in flow did not always coincide with maximum increases in T2*. Carbogen breathing caused a threefold increase in arterial rat blood PaO2, and typically a 50% increase in tumor blood volume as measured by 51Cr-labelled RBC uptake. The T2* increase is therefore due to a decrease in blood deoxyhaemoglobin concentration with the magnitude of the FLOOD response being determined by the vascular density and responsiveness to blood flow modifiers. FLOOD contrast may therefore be of value in assessing the magnitude and heterogeneity of response of individual tumors to blood flow modifiers for both chemotherapy, antiangiogenesis therapy in particular, and radiotherapy.

The effects of hyperoxia (induced by host carbogen [95% oxygen/5% carbon dioxide breathing] and hypoxia (induced by host carbon monoxide [CO at 660 ppm] breathing) were compared by using noninvasive magnetic resonance (MR) methods to gain simultaneous information on blood flow/oxygenation and the bioenergetic status of rat Morris H9618a hepatomas. Both carbogen and CO breathing induced a 1.5- to 2-fold increase in signal intensity in blood oxygenation level dependent (BOLD) MR images. This was due to a decrease in deoxyhemoglobin (deoxyHb), which acts as an endogenous contrast agent, caused either by formation of oxyhemoglobin in the case of carbogen breathing, or carboxyhemoglobin with CO breathing. The results were confirmed by observation of similar changes in deoxyHb in arterial blood samples examined ex vivo after carbogen or CO breathing. There was no change in nucleoside triphosphates (NTP)/P(i) in either tumor or liver after CO breathing, whereas NTP/P(i) increased twofold in the hepatoma (but not in the liver) after carbogen breathing. No changes in tumor intracellular pH were seen after either treatment, whereas extracellular pH became more alkaline after CO breathing and more acid after carbogen breathing, respectively. This tumor type and the liver are unaffected by CO breathing at 660 ppm, which implies an adequate oxygen supply.

The purpose of this study was to examine the effect of carbogen gas (95% O-2-5% CO2) on uptake and metabolism of 5-fluorouracil (5FU) in murine RIF-1 tumors and their growth in vivo, In addition, we have explored the mechanisms by which carbogen can transiently affect the physiology of RIF-1 tumors.After i.p. injection of 1 mmol/kg 5FU into C3H mice, the uptake and metabolism of the drug by s.c. RIF-1 tumors was followed for 2 h noninvasively using F-19-magnetic resonance spectroscopy (MRS). In all animals, irrespective of tumor size, carbogen caused a significant increase in the half-life (t(1/2)) of the elimination of 5FU by the tumor and a significant increase in growth inhibition, In 2-3-g tumors (group II), carbogen also caused increased 5FU uptake and metabolism to the cytotoxic 5-fluoronucleotides, whereas in 0.8-1.5-g tumors (group I), only the t(1/2) was slightly increased, These results suggested that tumor size was an important factor in the effect of carbogen on tumor physiology.Measurements of RIF-1 tumor vascular and necrotic volume showed no significant differences between group I and group II tumors, However, H-1-MR images of RIF-1 tumors showed that carbogen caused a transient decrease in signal intensity, which correlated positively (P = 0.02) with tumor size, suggesting that larger tumors responded to carbogen by transiently increasing O-2 uptake from the blood. F-19-MRS was used to measure RIF-1 tumor retention of the fluorinated nitroimidazole SR-4554. These studies also showed a positive correlation (P = 0.001) with tumor size, implying greater hypoxia in larger tumors, We propose that carbogen may transiently open nonfunctional blood vessels in the tumor, allowing increased leakage of 5FU from the plasma into the extracellular space, 5FU transport is known to be pH dependent, Intra- and extracellular tumor pH was measured using P-31- and F-19-MRS, which showed that carbogen caused a significant decrease in the extracellular pH of 0.1 unit in group II tumors and a consequent increase in the negative pH gradient across the tumor plasma membrane, which can cause increased 5FU uptake, The pH gradient was unaffected in group I tumors.We conclude that carbogen breathing can increase tumor uptake of 5FU by two independent mechanisms involving changes in tumor blood pow and pH, which consequently cause increased formation of 5-fluoronucleotides and cytotoxicity. The effect seems more pronounced in hypoxic tumors, implying that carbogen would be a valuable aid in clinical chemotherapy.

Characteristics of the tumour metabolic profile play a role in both the tumour-host interaction and in resistance to treatment. Because carbogen (95% oxygen/5% carbon dioxide) breathing can both increase sensitivity to radiation and improve chemotherapeutic efficacy, we have studied its effects on the metabolic characteristics of Morris hepatoma 9618a. Host carbogen breathing increased both arterial blood pCO2 and pO2, but decreased blood pH. A fourfold increase in tumour pO2 (measured polarographically) and a twofold increase in image intensity [measured by gradient recalled echo magnetic resonance (MR) imaging sensitive to changes in oxy/deoxyhaemoglobin] were observed. No changes were seen in blood flow measured by laser Doppler flowmetry. Tumour intracellular pH remained neutral, whereas extracellular pH decreased significantly (P < 0.01). Nucleoside triphosphate/inorganic phosphate (NTP/Pi), tissue and plasma glucose increased twofold and lactate decreased in both intra- and extracellular compartments, suggesting a change to a more oxidative metabolism. The improvement in energy status of the tumour was reflected in changes in tissue ions, including Na+, through ionic equilibria. The findings suggest that the metabolic profile of hepatoma 9618a is defined partly by intrinsic tumour properties caused by transformation and partly by tissue hypoxia, but that it can respond to environmental changes induced by carbogen with implications for improvements in therapeutic efficacy.

The objective of this study was first to determine whether three slowly growing early-generation murine transplantable tumours, the T40 fibrosarcoma, T115 mammary carcinoma and T237 lung carcinoma, exhibit patterns of energetics and blood flow during growth that are different from those of the faster growing RIF-I fibrosarcoma. Serial measurements were made with P-31-magnetic resonance spectroscopy (MRS), relating to nutritive blood flow and H-2-magnetic resonance imaging (MRI), which is sensitive to both nutritive and large-vessel (non-nutritive) flow. All four tumour lines showed a decrease in beta NTP/P-i and pH with growth; however, each line showed a different pattern of blood flow that did not correlate with the decrease in energetics. Qualitative histological analysis strongly correlated with the H-2-MRI. Second, their response to 5 mg kg(-1) hydralazine i.v. was monitored by P-31-MRS. A marked decrease in beta NTP/P-i and pH was observed in both the RIF-I fibrosarcoma and the third-generation T115 mammary carcinoma after hydralazine challenge, In contrast, the fourth generation T40 fibrosarcoma and T237 lung carcinoma showed no change in 31P-MRS parameters. However, a fifth-generation T237 cohort, which grew approximately three times faster than fourth-generation T237 cohorts, exhibited a significant deterioration in beta NTP/P-i and pH in response to hydralazine. These data are consistent with a decoupling between large-vessel and nutritive blood flow and indicate that early-generation transplants that have a slow growth rate and vascular tone are more appropriate models of human tumour vasculature than more rapidly growing, repeatedly transplanted tumours.

The direct detection and monitoring of anti-cancer drugs in vivo by magnetic resonance spectroscopy (MRS) may lead to improved anti-cancer strategies. P-31-MRS has been used to detect and quantify ifosfamide (IF) in vivo in GH3 prolactinomas and N-methyl-N-nitrosourea (MNU)-induced mammary tumours in rats. The average concentration of IF in the GH3 protactinoma over the first 2 h following a dose at 250 mg kg(-1) i.v. was calculated to be 0.42 mu mol g(-1) wet weight, with a half-life of elimination (t(1/2)) of 2-4 h. Carbogen (95% oxygen/5% carbon dioxide) breathing increased the amount of IF taken up by the GH3 prolactinoma by 50% (P < 0.01) to 0.68 lambda mol g(-1) wet weight, although t(1/2) elimination rates were unchanged. IF was also detected in the liver in vivo, with a t(1/2) of about 1 h. Carbogen breathing did not affect the maximum peak area (C-max) or the t(1/2) in the liver. Most importantly, the carbogen-induced increase in IF uptake by the tumour caused significant growth delay at all time points in the GH3 tumour growth between day 5 and day 12 (P < 0.01) compared with IF atone. These findings show that carbogen breathing has potential for increasing the efficacy of anti-cancer drugs. isolated GH3 cells were sensitive to the parent drug (IF)in vitro (IC50 = 1.3 +/- 0.2 mM) suggesting that the GH3 cells may be either expressing P450 enzymes or are sensitive to the parent drug per se.

Nuclear magnetic resonance spectroscopy (MRS) offers a non-invasive approach for studying tumour biochemistry and physiology. This review highlights NMR nuclei (P-31, H-1, F-19, C-13, H-2) that have been observed in both pre-clinical and clinical spectroscopic studies of cancer.

Purpose: Gradient-Recalled Echo (GRE) Magnetic Resonance Imaging (MRI), which detects changes in blood vessel deoxyhaemoglobin content, has been investigated as a noninvasive monitor of changes in human tumor oxygenation and blood flow, in response to carbogen (95% O-2, 5% CO2) breathing.Methods and Materials: GRE images (TE = 60 ms, TR = 200 ms, alpha = 40 degrees, 256(2) matrix) were acquired from 31 patients with primary and metastatic disease, prior to and during carbogen breathing. Three patients underwent a follow-up examination after radiotherapy.Results: Seventeen out of 34 tumors showed enhanced image intensity, consistent with an improvement in tumor oxygenation and blood flow, while 11 showed no response; 6 studies were technical failures. In one patient a metastatic node that had eluded orthodox investigation was visualized. A reduction in response was observed in the three patients studied postradiotherapy.Conclusion: This method, which can be performed on a standard clinical MRI instrument, provides a noninvasive measurement of tumor response to oxygenation/blood flow modification. In principle, this should enable the clinician to optimize treatment protocols, such as carbogen breathing, for individual radiotherapy patients. (C) 1997 Elsevier Science Inc.

Gradient-recalled echo magnetic resonance imaging (GRE MRI), which gives information on blood flow and oxygenation changes (Robinson SP, Howe FA, Griffiths JR 1995, int J Radiat Oncol Biol Phys 33: 855), was used to observe the responses of six rodent tumour models to carbogen breathing. In one transplanted rat tumour, the Morris hepatoma 9618a, and a chemically induced rat tumour, the MNU-induced mammary adenocarcinoma, there were marked image intensity increases, similar to those previously observed in the rat GH3 prolactinoma. In contrast, the rat Walker carcinosarcoma showed no response. In two mouse tumours, the RIF-1 fibrosarcoma and the human xenograft HT29, carbogen breathing induced a transient fall in signal intensity that reversed spontaneously within a few minutes. The rat GH3 prolactinoma was xenografted into nude mice, and an increase in image intensity was found in response to carbogen, suggesting that any effects that carbogen may have had on the host were not significant determinants of the tumour response, The increases in GRE image intensity of the MNU, H9618a and GH3 tumours during carbogen breathing are consistent with increases in tumour oxygenation and blood flow, whereas the responses of the RIF-1 and HT29 tumours may be the result of a transient steal effect followed by homeostatic correction.

It is well known that low levels of tissue oxygen (pO2) protect tumour cells from ionising radiation and some chemotherapeutic agents. Thus, numerous studies have been aimed at developing methods to measure tissue oxygenation. An initial discussion of some of the traditional methods for measuring oxygenation is included, followed by a discussion of magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) methods for measuring tumour and normal tissue oxygenation. The latter methods are of interest because of the non-invasive nature of magnetic resonance (MR). Some of the MR methods described herein include: 31P MRS, 1H MRS and MRI, and 19F MRS and MRI. Each method is detailed, including a brief assessment of its ability to measure tumour oxygenation and its potential for clinical application.

Gradient recalled echo (GRE) 1H images can be used to monitor changes in blood oxygenation via the dephasing effects of paramagnetic deoxyhaemoglobin (Hb). We have modulated the blood flow/oxygenation of GH3 rat tumours by i.v. calcitonin gene-related peptide (CGRP) or carbogen (95% O2, 5% CO2) inhalation, and obtained GRE 1H images interleaved with 31P spectra before, during and after the insult. With CGRP the GRE image intensity decreased (6/10) by > 10% with a concomitant 40% decrease (4/4) in beta NTP/P1 and a small decrease in pH. Both the image intensity and 31P spectra returned to near their pre-CGRP levels after 50 min, consistent with a transient episode of hypoxia. Carbogen breathing (5/5) caused > 40% increases in average GRE image intensity, with no significant changes in the 31P spectra (4/4). Three-dimensional GRE images were obtained to confirm that a T2* increase, rather than just an 'in-flow' effect due to increased blood flow, was responsible for the GRE enhancement. Increases in average image intensity > 40% were observed for the three-dimensional GRE images (2/2), indicating a T2* increase. Using Hb as an endogenous contrast agent, the high sensitivity of the GRE technique may provide a method of monitoring heterogeneous tumour perfusion and oxygenation, both in the laboratory and the clinic.

Purpose: The response of tumors to radiotherapy can be enhanced if carbogen (95% O-2, 5% CO2) is breathed. The timing of carbogen administration is critical, and a noninvasive method of monitoring the response of individual tumors would have obvious utility. Functional gradient recalled echo (GRE) magnetic resonance imaging (MRI) techniques are sensitive to changes in the concentrations of deoxyhemoglobin, which, thus, acts as an endogenous contrast agent for oxygenation status and blood flow.Methods and Materials: Subcutaneous GH3 prolactinomas in three rats were imaged at 4.7 Tesla with a GRE H-1 sequence [echo time (TE) = 20 ms, repetition time (TR) = 80 ms, hip angle = 45 degrees, 1 mm slice, 256 phase encode steps, 4 cm field of view, in-plane resolution 0.08 x 0.08 mm, acquisition time = 4 min]. The rats breathed air or carbogen for four periods of 20 min; three control rats breathed only air.Results: Carbogen breathing caused increases of up to 100% in the GRE image intensity of the tumors. Reversion to air breathing caused the image intensity to fall; essentially the same response was observed with the second cycle of carbogen and air breathing. Control rat tumors showed no significant change.Conclusions: The response of tumors to carbogen can be monitored noninvasively by GRE MRI. In principle, this could be due to an increase in oxygen content of the blood, a decrease in tumor cell oxygen consumption, or an increase in tumor blood flow. The very large changes in signal intensity suggest that a blood flow increase is the most probable explanation. If this technique can be successfully applied in man, it should be possible to optimize carbogen treatment for individual radiotherapy patients, and perhaps also to enhance tumor uptake of chemotherapeutic agents.

Pattern recognition has been applied to the analysis of in vivo P-31 NMR spectra. Using four different classes of tumour and three types of normal tissue, cluster analysis and artificial neural networks were successful in separating and classifying the majority of samples analysed. Although the phosphomonoester and P(i) regions appeared to be the most important spectral features, data representing the entire P-31 spectrum were required for best separation of the tumour and tissue classes.

1H nuclear magnetic resonance at 360 MHz shows that SK&F 96365 (1-(beta-[3-(p-methoxyphenyl)-propyloxy]-p-methoxyphenethyl)-1H- imidazole hydrochloride), an antagonist of mammalian receptor-operated calcium channels, interacts with the calcium-binding regulatory protein calmodulin (CaM). This may be inferred by a number of chemical shift changes in the spectrum of the calcium-saturated protein induced by addition of the compound. Moreover, two well-resolved singlets corresponding to the 2-proton of the SK&F 96365 imidazolium moiety are observed in the spectrum over a wide range of protein:compound ratios. Separation of rac SK&F 96365 into its two enantiomers by high-performance liquid chromatography on a cellulose tris (4-methylbenzoate) column enabled us to show that the doubling of this NMR signal in the presence of CaM is due to a propensity of the protein to distinguish between the two optical isomers of the compound.