Jane Halliday

A quantitative comparison of the influence of individual versus population-derived vascular input functions on dynamic contrast enhanced-MRI in small animals

For quantitative analysis of dynamic contrast enhanced magnetic resonance imaging data, the time course of the concentration of the contrast agent in the blood plasma, or vascular input function (VIF), is required. We compared pharmacokinetic parameters derived using individual and population‐based VIFs in mice for two different contrast agents, gadopentetate dimeglumine and P846. Eleven mice with subcutaneous 4T1 breast cancer xenografts were imaged at 7 T. A precontrast T1 map was acquired along with dynamic T1‐weighted gradient echo images before, during, and after a bolus injection of contrast agent delivered via a syringe pump. Each animals individual VIF and derived population‐averaged VIF were used to extract parameters from the signal‐time curves of tumor tissue at both the region of interest and voxel level. The results indicate that for both contrast agents, Ktrans values estimated using population‐averaged VIF have a high correlation (concordance correlation coefficient > 0.85) with Ktrans values estimated using individual VIF on both a region of interest and voxel level. This work supports the validity of using of a population‐based VIF with a stringent injection protocol in preclinical dynamic contrast enhanced magnetic resonance imaging studies. Magn Reson Med, 2011.

Exploring R-2* and R-1 as imaging biomarkers of tumor oxygenation

To investigate the combined use of hyperoxia‐induced ΔR2* and ΔR1 as a noninvasive imaging biomarker of tumor hypoxia.

MRI measurements of vessel calibre in tumour xenografts: Comparison with vascular corrosion casting

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.

Investigating Temporal Fluctuations in Tumor Vasculature With Combined Carbogen and Ultrasmall Superparamagnetic Iron Oxide Particle (CUSPIO) Imaging

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 R2* response were identified, and compared with histological analysis of the vasculature. In particular, a strong association between a negative ΔR2*carbogen followed by positive ΔR2*USPIO with the uptake of the perfusion marker Hoechst 33342 was determined. Regions of tumor tissue where there was a significant ΔR2*carbogen but no significant ΔR2*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 ΔR2*carbogen and ΔR2*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. Magn Reson Med 66:227–234, 2011.

False-negative MRI biomarkers of tumour response to targeted cancer therapeutics.

Background: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.Methods:First, in investigating imaging biomarkers of invasion, the response of orthotopic murine PC3 prostate xenografts to the Src inhibitor saracatinib was assessed using susceptibility contrast MRI. Second, the longitudinal response of chemically induced rat mammary adenocarcinomas to the VEGFR2 inhibitor vandetanib was monitored by intrinsic susceptibility MRI, to identify the time window of transient vascular normalisation.Results:No significant differences in fractional blood volume (%), vessel calibre (μm), native T1 (ms) or apparent water diffusion coefficient were determined, despite reduced expression of activated Fak and paxillin in the saracatinib cohort. Treatment with vandetanib elicited a 60% antitumour response (P<0.01), 80% inhibition in vessel density (P<0.05) and reduction in hypoxia (P<0.05). There was, however, no significant change in tumour baseline R2* (s−1) or carbogen-induced ΔR2* with treatment.Conclusion:Reporting negative imaging biomarker responses is important, to avoid the risk of clinical trials using the same biomarkers being undertaken with a false expectation of success, and the abandonment of promising new therapeutics based on a false-negative imaging biomarker response being mistaken for a true-negative.

Evaluation of novel combined carbogen USPIO (CUSPIO) imaging biomarkers in assessing the antiangiogenic effects of cediranib (AZD2171) in rat C6 gliomas.

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 ΔR2*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.

Manganese enhancement in non-CNS organs.

Manganese‐enhanced magnetic resonance imaging (MEMRI) is a novel imaging technique capable of monitoring calcium influx, in vivo. Manganese (Mn2+) ions, similar to calcium ions (Ca2+), are taken up by activated cells where their paramagnetic properties afford signal enhancement in T1‐weighted MRI methodologies. In this study we have assessed Mn2+ distribution in mice using magnetization‐prepared rapid gradient echo (MP‐RAGE) based MRI, by measuring changes in T1–effective relaxation times (T1‐eff), effective R1‐relaxation rates (R1‐eff) and signal intensity (SI) profiles over time. The manganese concentration in the tissue was also determined using inductively coupled plasma atomic emission spectrometry (ICP‐AES). Our results show a strong positive correlation between infused dose of MnCl2 and the tissue manganese concentration. Furthermore, we demonstrate a linear relationship between R1‐eff and tissue manganese concentration and tissue‐specific Mn2+ distribution in murine tissues following dose‐dependent Mn2+ administration. This data provides an optimized MnCl2 dose regimen for an MP‐RAGE based sequence protocol for specific target organs and presents a potential 3D MRI technique for in vivo imaging of Ca2+ entry during Ca2+‐dependent processes in a wide range of tissues. Copyright

Response of HT29 colorectal xenograft model to cediranib assessed with 18 F-fluoromisonidazole positron emission tomography, dynamic contrast-enhanced and diffusion-weighted MRI.

Cediranib is a small‐molecule pan‐vascular endothelial growth factor receptor inhibitor. The tumor response to short‐term cediranib treatment was studied using dynamic contrast‐enhanced and diffusion‐weighted MRI at 7 T, as well as 18F‐fluoromisonidazole positron emission tomography and histological markers. Rats bearing subcutaneous HT29 human colorectal tumors were imaged at baseline; they then received three doses of cediranib (3 mg/kg per dose daily) or vehicle (dosed daily), with follow‐up imaging performed 2 h after the final cediranib or vehicle dose. Tumors were excised and evaluated for the perfusion marker Hoechst 33342, the endothelial cell marker CD31, smooth muscle actin, intercapillary distance and tumor necrosis. Dynamic contrast‐enhanced MRI‐derived parameters decreased significantly in cediranib‐treated tumors relative to pretreatment values [the muscle‐normalized initial area under the gadolinium concentration curve decreased by 48% (p = 0.002), the enhancing fraction by 43% (p = 0.003) and Ktrans by 57% (p = 0.003)], but remained unchanged in controls. No change between the pre‐ and post‐treatment tumor apparent diffusion coefficients in either the cediranib‐ or vehicle‐treated group was observed over the course of this study. The 18F‐fluoromisonidazole mean standardized uptake value decreased by 33% (p = 0.008) in the cediranib group, but showed no significant change in the control group. Histological analysis showed that the number of CD31‐positive vessels (59 per mm2), the fraction of smooth muscle actin‐positive vessels (80–87%) and the intercapillary distance (0.17 mm) were similar in cediranib‐ and vehicle‐treated groups. The fraction of perfused blood vessels in cediranib‐treated tumors (81 ± 7%) was lower than that in vehicle controls (91 ± 3%, p = 0.02). The necrotic fraction was slightly higher in cediranib‐treated rats (34 ± 12%) than in controls (26 ± 10%, p = 0.23). These findings suggest that short‐term treatment with cediranib causes a decrease in tumor perfusion/permeability across the tumor cross‐section, but changes in vascular morphology, vessel density or tumor cellularity are not manifested at this early time point. Copyright

Response of HT29 Colorectal Xenograft Model to Cediranib Assessed with 18F-FMISO PET, Dynamic Contrast-Enhanced and Diffusion-Weighted MRI

Cediranib is a small‐molecule pan‐vascular endothelial growth factor receptor inhibitor. The tumor response to short‐term cediranib treatment was studied using dynamic contrast‐enhanced and diffusion‐weighted MRI at 7 T, as well as 18F‐fluoromisonidazole positron emission tomography and histological markers. Rats bearing subcutaneous HT29 human colorectal tumors were imaged at baseline; they then received three doses of cediranib (3 mg/kg per dose daily) or vehicle (dosed daily), with follow‐up imaging performed 2 h after the final cediranib or vehicle dose. Tumors were excised and evaluated for the perfusion marker Hoechst 33342, the endothelial cell marker CD31, smooth muscle actin, intercapillary distance and tumor necrosis. Dynamic contrast‐enhanced MRI‐derived parameters decreased significantly in cediranib‐treated tumors relative to pretreatment values [the muscle‐normalized initial area under the gadolinium concentration curve decreased by 48% (p = 0.002), the enhancing fraction by 43% (p = 0.003) and Ktrans by 57% (p = 0.003)], but remained unchanged in controls. No change between the pre‐ and post‐treatment tumor apparent diffusion coefficients in either the cediranib‐ or vehicle‐treated group was observed over the course of this study. The 18F‐fluoromisonidazole mean standardized uptake value decreased by 33% (p = 0.008) in the cediranib group, but showed no significant change in the control group. Histological analysis showed that the number of CD31‐positive vessels (59 per mm2), the fraction of smooth muscle actin‐positive vessels (80–87%) and the intercapillary distance (0.17 mm) were similar in cediranib‐ and vehicle‐treated groups. The fraction of perfused blood vessels in cediranib‐treated tumors (81 ± 7%) was lower than that in vehicle controls (91 ± 3%, p = 0.02). The necrotic fraction was slightly higher in cediranib‐treated rats (34 ± 12%) than in controls (26 ± 10%, p = 0.23). These findings suggest that short‐term treatment with cediranib causes a decrease in tumor perfusion/permeability across the tumor cross‐section, but changes in vascular morphology, vessel density or tumor cellularity are not manifested at this early time point. Copyright

Investigating the Vascular Phenotype of Subcutaneously and Orthotopically Propagated PC3 Prostate Cancer Xenografts Using Combined Carbogen Ultrasmall Superparamagnetic Iron Oxide MRI.

Abstract The aim of this study was to use the combined carbogen-ultrasmall superparamagnetic iron oxide (CUSPIO) magnetic resonance imaging (MRI) method, which uses spatial correlations in independent susceptibility imaging biomarkers, to investigate and compare the impact of tumor size and anatomical site on vascular structure and function in vivo. Mice bearing either subcutaneous or orthotopic PC3 LN3 prostate tumors were imaged at 7 T, using a multi-gradient echo sequence to quantify R2*, before and during carbogen (95% O2/5% CO2) breathing, and subsequently following intravenous administration of USPIO particles. Carbogen and USPIO-induced changes in R2* were used to inform on hemodynamic vasculature and fractional blood volume (%), respectively. The CUSPIO imaging data were also segmented to identify and assess five categories of R2* response. Small and large subcutaneous and orthotopic tumor cohorts all exhibited significantly (P < 0.05) different median baseline R2*, &Dgr;R2*carbogen, and fractional blood volume. CUSPIO imaging showed that small subcutaneous tumors predominantly exhibited a negative &Dgr;R2*carbogen followed by a positive &Dgr;R2*USPIO, consistent with a well perfused tumor vasculature. Large subcutaneous tumors exhibited a small positive &Dgr;R2*carbogen and relatively low fractional blood volume, suggesting less functional vasculature. Orthotopic tumors revealed a large, positive &Dgr;R2*carbogen, consistent with vascular steal, and which may indicate that vascular function is more dependent on site of implantation than tumor size. Regions exhibiting significant &Dgr;R2*carbogen, but no significant &Dgr;R2*USPIO, suggesting transient vascular shutdown over the experimental timecourse, were apparent in all 3 cohorts. CUSPIO imaging can inform on efficient drug delivery via functional vasculature in vivo, and on appropriate tumor model selection for pre-clinical therapy trials