Endothelial cell death after ionizing radiation does not impair vascular structure in mouse tumor models.

The effect of radiation therapy on tumor vasculature has long been a subject of debate. Increased oxygenation and perfusion have been documented during radiation therapy. Conversely, apoptosis of endothelial cells in irradiated tumors has been proposed as a major contributor to tumor control. To examine these contradictions, we use multiphoton microscopy in two murine tumor models: MC38, a highly vascularized, and B16F10, a moderately vascularized model, grown in transgenic mice with tdTomato-labeled endothelium before and after a single (15 Gy) or fractionated (5 × 3 Gy) dose of radiation. Unexpectedly, even these high doses lead to little structural change of the perfused vasculature. Conversely, non-perfused vessels and blind ends are substantially impaired after radiation accompanied by apoptosis and reduced proliferation of their endothelium. RNAseq analysis of tumor endothelial cells confirms the modification of gene expression in apoptotic and cell cycle regulation pathways after irradiation. Therefore, we conclude that apoptosis of tumor endothelial cells after radiation does not impair vascular structure.

Determination of oxygen relaxivity in oxygen nanobubbles at 3 and 7 Tesla.

OBJECTIVE: Oxygen-loaded nanobubbles have shown potential for reducing tumour hypoxia and improving treatment outcomes, however, it remains difficult to noninvasively measure the changes in partial pressure of oxygen (PO2) in vivo. The linear relationship between PO2 and longitudinal relaxation rate (R1) has been used to noninvasively infer PO2 in vitreous and cerebrospinal fluid, and therefore, this experiment aimed to investigate whether R1 is a suitable measurement to study oxygen delivery from such oxygen carriers. METHODS: T1 mapping was used to measure R1 in phantoms containing nanobubbles with varied PO2 to measure the relaxivity of oxygen (r1Ox) in the phantoms at 7 and 3 T. These measurements were used to estimate the limit of detection (LOD) in two experimental settings: preclinical 7 T and clinical 3 T MRI. RESULTS: The r1Ox in the nanobubble solution was 0.00057 and 0.000235 s-1/mmHg, corresponding to a LOD of 111 and 103 mmHg with 95% confidence at 7 and 3 T, respectively. CONCLUSION: This suggests that T1 mapping could provide a noninvasive method of measuring a > 100 mmHg oxygen delivery from therapeutic nanobubbles.

Olaparib increases the therapeutic index of hemithoracic irradiation compared with hemithoracic irradiation alone in a mouse lung cancer model.

BACKGROUND: The radiosensitising effect of the poly(ADP-ribose) polymerase inhibitor olaparib on tumours has been reported. However, its effect on normal tissues in combination with radiation has not been well studied. Herein, we investigated the therapeutic index of olaparib combined with hemithoracic radiation in a urethane-induced mouse lung cancer model. METHODS: To assess tolerability, A/J mice were treated with olaparib plus whole thorax radiation (13 Gy), body weight changes were monitored and normal tissue effects were assessed by histology. In anti-tumour (intervention) studies, A/J mice were injected with urethane to induce lung tumours, and were then treated with olaparib alone, left thorax radiation alone or the combination of olaparib plus left thorax radiation at 8 weeks (early intervention) or 18 weeks (late intervention) after urethane injection. Anti-tumour efficacy and normal tissue effects were assessed by visual inspection, magnetic resonance imaging and histology. RESULTS: Enhanced body weight loss and oesophageal toxicity were observed when olaparib was combined with whole thorax but not hemithorax radiation. In both the early and late intervention studies, olaparib increased the anti-tumour effects of hemithoracic irradiation without increasing lung toxicity. CONCLUSIONS: The addition of olaparib increased the therapeutic index of hemithoracic radiation in a mouse model of lung cancer.

Tumour irradiation combined with vascular-targeted photodynamic therapy enhances antitumour effects in pre-clinical prostate cancer.

BACKGROUND: There is a need to improve the treatment of prostate cancer (PCa) and reduce treatment side effects. Vascular-targeted photodynamic therapy (VTP) is a focal therapy for low-risk low-volume localised PCa, which rapidly disrupts targeted tumour vessels. There is interest in expanding the use of VTP to higher-risk disease. Tumour vasculature is characterised by vessel immaturity, increased permeability, aberrant branching and inefficient flow. FRT alters the tumour microenvironment and promotes transient 'vascular normalisation'. We hypothesised that multimodality therapy combining fractionated radiotherapy (FRT) and VTP could improve PCa tumour control compared against monotherapy with FRT or VTP. METHODS: We investigated whether sequential delivery of FRT followed by VTP 7 days later improves flank TRAMP-C1 PCa tumour allograft control compared to monotherapy with FRT or VTP. RESULTS: FRT induced 'vascular normalisation' changes in PCa flank tumour allografts, improving vascular function as demonstrated using dynamic contrast-enhanced magnetic resonance imaging. FRT followed by VTP significantly delayed tumour growth in flank PCa allograft pre-clinical models, compared with monotherapy with FRT or VTP, and improved overall survival. CONCLUSION: Combining FRT and VTP may be a promising multimodal approach in PCa therapy. This provides proof-of-concept for this multimodality treatment to inform early phase clinical trials.

Choice of reference measurements affects quantification of long diffusion time behaviour using stimulated echoes.

PURPOSE: To demonstrate how reference data affect the quantification of the apparent diffusion coefficient (ADC) in long diffusion time measurements with diffusion-weighted stimulated echo acquisition mode (DW-STEAM) measurements, and to present a modification to avoid contribution from crusher gradients in DW-STEAM. METHODS: For DW-STEAM, reference measurements at long diffusion times have significant b0 value, because b = 0 cannot be achieved in practice as a result of the need for signal spoiling. Two strategies for acquiring reference data over a range of diffusion times were considered: constant diffusion weighting (fixed-b0 ) and constant gradient area (fixed-q0 ). Fixed-b0 and fixed-q0 were compared using signal calculations for systems with one and two diffusion coefficients, and experimentally using data from postmortem human corpus callosum samples. RESULTS: Calculations of biexponential diffusion decay show that the ADC is underestimated for reference images with b > 0, which can induce an apparent time-dependence for fixed-q0 . Restricted systems were also found to be affected. Experimentally, the exaggeration of the diffusion time-dependent effect under fixed-q0 versus fixed-b0 was in a range predicted theoretically, accounting for 62% (longitudinal) and 35% (radial) of the time dependence observed in white matter. CONCLUSIONS: Variation in the b-value of reference measurements in DW-STEAM can induce artificial diffusion time dependence in ADC, even in the absence of restriction. Magn Reson Med 79:952-959, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

The pH low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions.

Current strategies for early detection of breast and other cancers are limited in part because some lesions identified as potentially malignant do not develop into aggressive tumors. Acid pH has been suggested as a key characteristic of aggressive tumors that might distinguish aggressive lesions from more indolent pathology. We therefore investigated the novel class of molecules, pH low insertion peptides (pHLIPs), as markers of low pH in tumor allografts and of malignant lesions in a mouse model of spontaneous breast cancer, BALB/neu-T. pHLIP Variant 3 (Var3) conjugated with fluorescent Alexa546 was shown to insert into tumor spheroids in a sequence-specific manner. Its signal reflected pH in murine tumors. It was induced by carbonic anhydrase IX (CAIX) overexpression and inhibited by acetazolamide (AZA) administration. By using (31)P magnetic resonance spectroscopy (MRS), we demonstrated that pHLIP Var3 was retained in tumors of pH equal to or less than 6.7 but not in tissues of higher pH. In BALB/neu-T mice at different stages of the disease, the fluorescent signal from pHLIP Var3 marked cancerous lesions with a very low false-positive rate. However, only ∼60% of the smallest lesions retained a pHLIP Var3 signal, suggesting heterogeneity in pH. Taken together, these results show that pHLIP can identify regions of lower pH, allowing for its development as a theranostic tool for clinical applications.

Robust and high resolution hyperpolarized metabolic imaging of the rat heart at 7 T with 3D spectral-spatial EPI.

PURPOSE: Hyperpolarized metabolic imaging has the potential to revolutionize the diagnosis and management of diseases where metabolism is dysregulated, such as heart disease. We investigated the feasibility of imaging rodent myocardial metabolism at high resolution at 7 T. METHODS: We present here a fly-back spectral-spatial radiofrequency pulse that sidestepped maximum gradient strength requirements and enabled high resolution metabolic imaging of the rodent myocardium. A 3D echo-planar imaging readout followed, with centric ordered z-phase encoding. The cardiac gated sequence was used to image metabolism in rodents whose metabolic state had been manipulated by being fasted, fed, or fed and given the pyruvate dehydrogenase kinase inhibitor dichloroacetate. RESULTS: We imaged hyperpolarized metabolites with a spatial resolution of 2×2×3.8 mm(3) and a temporal resolution of 1.8 s in the rat heart at 7 T. Significant differences in myocardial pyruvate dehydrogenase flux were observed between the three groups of animals, concomitant with the known biochemistry. CONCLUSION: The proposed sequence was able to image in vivo metabolism with excellent spatial resolution in the rat heart. The field of view enabled the simultaneous multi-organ acquisition of metabolic information from the rat, which is of great utility for preclinical research in cardiovascular disease. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance.

A System-Agnostic, Adaptable and Extensible Animal Support Cradle System for Cardio-Respiratory-Synchronised, and Other, Multi-Modal Imaging of Small Animals.

Standardisation of animal handling procedures for a wide range of preclinical imaging scanners will improve imaging performance and reproducibility of scientific data. Whilst there has been significant effort in defining how well scanners should operate and how in vivo experimentation should be practised, there is little detail on how to achieve optimal scanner performance with best practices in animal welfare. Here, we describe a system-agnostic, adaptable and extensible animal support cradle system for cardio-respiratory-synchronised, and other, multi-modal imaging of small animals. The animal support cradle can be adapted on a per application basis and features integrated tubing for anaesthetic and tracer delivery, an electrically driven rectal temperature maintenance system and respiratory and cardiac monitoring. Through a combination of careful material and device selection, we have described an approach that allows animals to be transferred whilst under general anaesthesia between any of the tomographic scanners we currently or have previously operated. The set-up is minimally invasive, cheap and easy to implement and for multi-modal, multi-vendor imaging of small animals.

A simple, open and extensible gating Control unit for cardiac and respiratory synchronisation control in small animal MRI and demonstration of its robust performance in steady-state maintained CINE-MRI.

Prospective cardiac gating during MRI is hampered by electromagnetic induction from the rapidly switched imaging gradients into the ECG detection circuit. This is particularly challenging in small animal MRI, as higher heart rates combined with a smaller myocardial mass render routine ECG detection challenging. We have developed an open-hardware system that enables continuously running MRI scans to be performed in conjunction with cardio-respiratory gating such that the relaxation-weighted steady state magnetisation is maintained throughout the scan. This requires that the R-wave must be detected reliably even in the presence of rapidly switching gradients, and that data previously acquired that were corrupted by respiratory motion re-acquired. The accurately maintained steady-state magnetisation leads to an improvement in image quality and removes alterations in intensity that may otherwise occur throughout the cardiac cycle and impact upon automated image analysis. We describe the hardware required to enable this and demonstrate its application and robust performance using prospectively cardio-respiratory gated CINE imaging that is operated at a single, constant TR. Schematics, technical drawings, component listing and assembly instructions are made publicly available.

Combining sonodynamic therapy with chemoradiation for the treatment of pancreatic cancer.

Treatment options for patients with pancreatic cancer are limited and survival prospects have barely changed over the past 4 decades. Chemoradiation treatment (CRT) has been used as neoadjuvant therapy in patients with borderline resectable disease to reduce tumour burden and increase the proportion of patients eligible for surgery. Antimetabolite drugs such as gemcitabine and 5-fluorouracil are known to sensitise pancreatic tumours to radiation treatment. Likewise, photodynamic therapy (PDT) has also been shown to enhance the effect of radiation therapy. However, PDT is limited to treating superficial lesions due to the attenuation of light by tissue. The ability of the related technique, sonodynamic therapy (SDT), to enhance CRT was investigated in two murine models of pancreatic cancer (PSN-1 and BxPC-3) in this study. SDT uses low intensity ultrasound to activate an otherwise non-toxic sensitiser, generating toxic levels of reactive oxygen species (ROS) locally. It is applicable to greater target depths than PDT due to the ability of ultrasound to propagate further than light in tissue. Both CRT and the combination of CRT plus SDT delayed tumour growth in the two tumour models. In the PSN-1 model, but not the BxPC-3 model, the combination treatment caused an increase in survival relative to CRT alone (p = 0.038). The improvement in survival conferred by the addition of SDT in this model may be related to differences in tumour architecture between the two models. MRI and US images showed that PSN-1 tumours were less well perfused and vascularised than BxPC-3 tumours. This poor vascularisation may explain why PSN-1 tumours were more susceptible to the effects of vascular damage exerted by SDT treatment.

Improved detection of molecularly targeted iron oxide particles in mouse brain using B0 field stabilised high resolution MRI.

PURPOSE: High resolution multi-gradient echo (MGE) scanning is typically used for detection of molecularly targeted iron oxide particles. The images of individual echoes are often combined to generate a composite image with improved SNR from the early echoes and boosted contrast from later echoes. In 3D implementations prolonged scanning at high gradient duty cycles induces a B0 shift that predominantly affects image alignment in the slow phase encoding dimension of 3D MGE images. The effect corrupts the composite echo image and limits the image resolution that is realised. A real-time adaptive B0 stabilisation during respiration gated 3D MGE scanning is shown to reduce image misalignment and improve detection of molecularly targeted iron oxide particles in composite images of the mouse brain. METHODS: An optional B0 measurement block consisting of a 16 µs hard pulse with FA 1°, an acquisition delay of 3.2 ms, followed by gradient spoiling in all three axes was added to a respiration gated 3D MGE scan. During the acquisition delay of each B0 measurement block the NMR signal was routed to a custom built B0 stabilisation unit which mixed the signal to an audio frequency nominally centred around 1000 Hz to enable an Arduino based single channel receiver to measure frequency shifts. The frequency shift was used to effect correction to the main magnetic field via the B0 coil. The efficacy of B0 stabilisation and respiration gating was validated in vivo and used to improve detection of molecularly targeted microparticles of iron oxide (MPIO) in a mouse model of acute neuroinflammation. RESULTS: Without B0 stabilisation 3D MGE image data exhibit varying mixtures of translation, scaling and blurring, which compromise the fidelity of the composite image. The real-time adaptive B0 stabilisation minimises corruption of the composite image as the images from the different echoes are properly aligned. The improved detection of molecularly targeted MPIO easily compensates for the scan time penalty of 14% incurred by the B0 stabilisation method employed. Respiration gating of the B0 measurement and the MRI scan was required to preserve high resolution detail, especially towards the back of the brain. CONCLUSIONS: High resolution imaging for the detection of molecularly targeted iron oxide particles in the mouse brain requires good stabilisation of the main B0 field, and can benefit from a respiration gated image acquisition strategy.

Manganese-free chow, a refined non-invasive solution to reduce gastrointestinal signal for T1-weighted magnetic resonance imaging of the mouse abdomen.

Commercial mouse chow is designed to provide a complete, nutrient-rich diet, and it can contain upwards of 100 mg/kg manganese, an essential mineral. Manganese acts as a relaxation time-shortening contrast agent for both T1 and T2, and where standard chow is hydrated in the gastrointestinal tract, bright signals are produced when using T1-weighted imaging (T1WI). As a result of peristalsis, gastrointestinal hyperintensities result in temporally unstable signals, leading to image ghosting and decreased resolution from that prescribed. To avoid the problem, various methods of gastrointestinal tract modulation, including the use of intestinal cleansing with laxatives and dietary modulation, have been reported. Here, dietary modulation has been extended to the use of a biologically innocuous, long-term change of diet. In this study, we report on the use of a commercially available manganese-free chow to improve the image quality of the gastrointestinal tract. This manganese-free chow, apart from the omitted manganese which is available in tap water, is a complete diet and readily available. We investigated the time-dependent, diet-related gastrointestinal intensities on short-TR T1WI magnetic resonance imaging; monitored body mass, food and water consumption and standard blood biochemistry analysis following diet change; and determined manganese concentration in blood plasma following a five-day change to manganese-free chow. We show that the manganese-free chow presents a refinement to other gastrointestinal tract modulation, as it avoids the need for invasive procedures for gut voiding and can be provided ad libitum so that animals can be maintained with no need for prescribed diet change before imaging.

Enhanced antitumor immunity through sequential targeting of PI3Kδ and LAG3.

BACKGROUND: Despite striking successes, immunotherapies aimed at increasing cancer-specific T cell responses are unsuccessful in most patients with cancer. Inactivating regulatory T cells (Treg) by inhibiting the PI3Kδ signaling enzyme has shown promise in preclinical models of tumor immunity and is currently being tested in early phase clinical trials in solid tumors. METHODS: Mice bearing 4T1 mammary tumors were orally administered a PI3Kδ inhibitor (PI-3065) daily and tumor growth, survival and T cell infiltrate were analyzed in the tumor microenvironment. A second treatment schedule comprised PI3Kδ inhibitor with anti-LAG3 antibodies administered sequentially 10 days later. RESULTS: As observed in human immunotherapy trials with other agents, immunomodulation by PI3Kδ-blockade led to 4T1 tumor regressor and non-regressor mice. Tumor infiltrating T cells in regressors were metabolically fitter than those in non-regressors, with significant enrichments of antigen-specific CD8+ T cells, T cell factor 1 (TCF1)+ T cells and CD69- T cells, compatible with induction of a sustained tumor-specific T cell response. Treg numbers were significantly reduced in both regressor and non-regressor tumors compared with untreated tumors. The remaining Treg in non-regressor tumors were however significantly enriched with cells expressing the coinhibitory receptor LAG3, compared with Treg in regressor and untreated tumors. This striking difference prompted us to sequentially block PI3Kδ and LAG3. This combination enabled successful therapy of all mice, demonstrating the functional importance of LAG3 in non-regression of tumors on PI3Kδ inhibition therapy. Follow-up studies, performed using additional cancer cell lines, namely MC38 and CT26, indicated that a partial initial response to PI3Kδ inhibition is an essential prerequisite to a sequential therapeutic benefit of anti-LAG3 antibodies. CONCLUSIONS: These data indicate that LAG3 is a key bottleneck to successful PI3Kδ-targeted immunotherapy and provide a rationale for combining PI3Kδ/LAG3 blockade in future clinical studies.

Magnetic resonance imaging of oxygen microbubbles.

Oxygen loaded microbubbles are being investigated as a means of reducing tumour hypoxia in order to improve response to cancer therapy. To optimise this approach, it is desirable to be able to measure changes in tissue oxygenation in real-time during treatment. In this study, the feasibility of using magnetic resonance imaging (MRI) for this purpose was investigated. Longitudinal relaxation time (T1) measurements were made in simple hydrogel phantoms containing two different concentrations of oxygen microbubbles. T1 was found to be unaffected by the presence of oxygen microbubbles at either concentration. Upon application of ultrasound to destroy the microbubbles, however, a statistically significant reduction in T1 was seen for the higher microbubble concentration. Further work is needed to assess the influence of physiological conditions upon the measurements, but these preliminary results suggest that MRI could provide a method for quantifying the changes in tissue oxygenation produced by microbubbles during therapy.

Cardio-Respiratory synchronized bSSFP MRI for high throughput in vivo lung tumour quantification.

The identification and measurement of tumours is a key requirement in the study of tumour development in mouse models of human cancer. Disease burden in autochthonous tumours, such as those arising in the lung, can be seen with non-invasive imaging, but cannot be accurately measured using standard tools such as callipers. Lung imaging is further complicated in the mouse due to instabilities arising from the rapid but cyclic cardio-respiratory motions, and the desire to use free-breathing animals. Female A/JOlaHsd mice were either injected (i.p.) with PBS 0.1ml/10g body weight (n = 6), or 10% urethane/PBS 0.1ml/10g body weight (n = 12) to induce autochthonous lung tumours. Cardio-respiratory synchronised bSSFP MRI, at 200 µm isotropic resolution was performed at 8, 13 and 18 weeks post induction. Images from the same mouse at different time points were aligned using threshold-based segmented masks of the lungs (ITK-SNAP and MATLAB) and tumour volumes were determined via threshold-based segmentation (ITK-SNAP).Scan times were routinely below 10 minutes and tumours were readily identifiable. Image registration allowed serial measurement of tumour volumes as small as 0.056 mm3. Repetitive imaging did not lead to mouse welfare issues. We have developed a motion desensitised scan that enables high sensitivity MRI to be performed with high throughput capability of greater than 4 mice/hour. Image segmentation and registration allows serial measurement of individual, small tumours. This allows fast and highly efficient volumetric lung tumour monitoring in cohorts of 30 mice per imaging time point. As a result, adaptive trial study designs can be achieved, optimizing experimental and welfare outcomes.

Reduced respiratory motion artefact in constant TR multi-slice MRI of the mouse.

PURPOSE: Multi-slice scanning in the abdomen and thorax of small animals is compromised by the effects of respiration unless imaging and respiration are synchronised. To avoid the signal modulations that result from respiration motion and a variable TR, blocks of fully relaxed slices are typically acquired during inter-breath periods, at the cost of scan efficiency. This paper reports a conceptually simple yet effective prospective gating acquisition mode for multi-slice scanning in free breathing small animals at any fixed TR of choice with reduced sensitivity to respiratory motion. METHODS: Multi-slice scan modes have been implemented in which each slice has its own specific projection or phase encode loop index counter. When a breath is registered RF pulses continue to be applied but data are not acquired, and the corresponding counters remain fixed so that the data are acquired one TR later, providing it coincides with an inter-breath period. The approach is refined to reacquire the slice data that are acquired immediately before each breath is detected. Only the data with reduced motion artefact are used in image reconstruction. The efficacy of the method is demonstrated in the RARE scan mode which is well known to be particularly useful for tumour visualization. RESULTS: Validation in mice with RARE demonstrates improved stability with respect to ungated scanning where signal averaging is often used to reduce artefacts. SNR enhancement maps demonstrate the improved efficiency of the proposed method that is equivalent to at least a 2.5 fold reduction in scan time with respect to ungated signal averaging. A steady-state magnetisation transfer contrast prepared gradient echo implementation is observed to highlight tumour structure. Supplementary simulations demonstrate that only small variations in respiration rate are required to enable efficient sampling with the proposed method. CONCLUSIONS: The proposed prospective gating acquisition scheme enables efficient multi-slice scanning in small animals at the optimum TR with reduced sensitivity to respiratory motion. The method is compatible with a wide range of complementary methods including non-Cartesian scan modes, partially parallel imaging, and compressed sensing. In particular, the proposed scheme reduces the need for continual close monitoring to effect operator intervention in response to respiratory rate changes, which is both difficult to maintain and precludes high throughput.

Tumor pH and Protein Concentration Contribute to the Signal of Amide Proton Transfer Magnetic Resonance Imaging.

Abnormal pH is a common feature of malignant tumors and has been associated clinically with suboptimal outcomes. Amide proton transfer magnetic resonance imaging (APT MRI) holds promise as a means to noninvasively measure tumor pH, yet multiple factors collectively make quantification of tumor pH from APT MRI data challenging. The purpose of this study was to improve our understanding of the biophysical sources of altered APT MRI signals in tumors. Combining in vivo APT MRI measurements with ex vivo histological measurements of protein concentration in a rat model of brain metastasis, we determined that the proportion of APT MRI signal originating from changes in protein concentration was approximately 66%, with the remaining 34% originating from changes in tumor pH. In a mouse model of hypopharyngeal squamous cell carcinoma (FaDu), APT MRI showed that a reduction in tumor hypoxia was associated with a shift in tumor pH. The results of this study extend our understanding of APT MRI data and may enable the use of APT MRI to infer the pH of individual patients' tumors as either a biomarker for therapy stratification or as a measure of therapeutic response in clinical settings. SIGNIFICANCE: These findings advance our understanding of amide proton transfer magnetic resonance imaging (APT MRI) of tumors and may improve the interpretation of APT MRI in clinical settings.

A Carbon-Fiber Sheet Resistor for MR-, CT-, SPECT-, and PET-Compatible Temperature Maintenance in Small Animals.

A magnetic resonance (MR)-, computed tomography (CT)-, single-photon emission computed tomography (SPECT)-, and positron emission tomography (PET)-compatible carbon-fiber sheet resistor for temperature maintenance in small animals where space limitations prevent the use of circulating fluids was developed. A 250 Ω carbon-fiber sheet resistor was mounted to the underside of an imaging cradle. Alternating current, operating at 99 kHz, and with a power of 1-2 W, was applied to the resistor providing a cradle base temperature of ∼37°C. Temperature control was implemented with a proportional-integral-derivative controller, and temperature maintenance was demonstrated in 4 mice positioned in both MR and PET/SPECT/CT scanners. MR and CT compatibility were also shown, and multimodal MR-CT-PET-SPECT imaging of the mouse abdomen was performed in vivo. Core temperature was maintained at 35.5°C ± 0.2°C. No line-shape, frequency, or image distortions attributable to the current flow through the heater were observed on MR. Upon CT imaging, no heater-related artifacts were observed when carbon-fiber was used. Multimodal imaging was performed and images could be easily coregistered, displayed, analyzed, and presented. Carbon fiber sheet resistors powered with high-frequency alternating current allow homeothermic maintenance that is compatible with multimodal imaging. The heater is small, and it is easy to produce and integrate into multimodal imaging cradles.

Ultrasound-mediated cavitation enhances the delivery of an EGFR-targeting liposomal formulation designed for chemo-radionuclide therapy.

Nanomedicines allow active targeting of cancer for diagnostic and therapeutic applications through incorporation of multiple functional components. Frequently, however, clinical translation is hindered by poor intratumoural delivery and distribution. The application of physical stimuli to promote tumour uptake is a viable route to overcome this limitation. In this study, ultrasound-mediated cavitation of microbubbles was investigated as a mean of enhancing the delivery of a liposome designed for chemo-radionuclide therapy targeted to EGFR overexpressing cancer. Method: Liposomes (111In-EGF-LP-Dox) were prepared by encapsulation of doxorubicin (Dox) and surface functionalisation with Indium-111 tagged epidermal growth factor. Human breast cancer cell lines with high and low EGFR expression (MDA-MB-468 and MCF7 respectively) were used to study selectivity of liposomal uptake, subcellular localisation of drug payload, cytotoxicity and DNA damage. Liposome extravasation following ultrasound-induced cavitation of microbubbles (SonoVue®) was studied using a tissue-mimicking phantom. In vivo stability, pharmacokinetic profile and biodistribution were evaluated following intravenous administration of 111In-labelled, EGF-functionalised liposomes to mice bearing subcutaneous MDA-MB-468 xenografts. Finally, the influence of ultrasound-mediated cavitation on the delivery of liposomes into tumours was studied. Results: Liposomes were loaded efficiently with Dox, surface decorated with 111In-EGF and showed selective uptake in MDA-MB-468 cells compared to MCF7. Following binding to EGFR, Dox was released into the intracellular space and 111In-EGF shuttled to the cell nucleus. DNA damage and cell kill were higher in MDA-MB-468 than MCF7 cells. Moreover, Dox and 111In were shown to have an additive cytotoxic effect in MDA-MB-468 cells. US-mediated cavitation increased the extravasation of liposomes in an in vitro gel phantom model. In vivo, the application of ultrasound with microbubbles increased tumour uptake by 66% (p<0.05) despite poor vascularisation of MDA-MB-468 xenografts (as shown by DCE-MRI). Conclusion:111In-EGF-LP-Dox designed for concurrent chemo-radionuclide therapy showed specificity for and cytotoxicity towards EGFR-overexpressing cancer cells. Delivery to tumours was enhanced by the use of ultrasound-mediated cavitation indicating that this approach has the potential to deliver cytotoxic levels of therapeutic radionuclide to solid tumours.

Imaging of Claudin-4 in Pancreatic Ductal Adenocarcinoma Using a Radiolabelled Anti-Claudin-4 Monoclonal Antibody.

PURPOSE: Despite its widespread use, the positron emission tomography (PET) radiotracer 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has been shown in clinical settings to be ineffective for improving early diagnosis of pancreatic ductal adenocarcinoma (PDAC). A promising biomarker for PDAC detection is the tight junction protein claudin-4. The purpose of this study was to evaluate a new single-photon emission computed tomography (SPECT) imaging agent, [111In]anti-claudin-4 mAb, with regard to its ability to allow visualisation of claudin-4 in a xenograft and a genetically engineered mouse model of PDAC. PROCEDURES: The ability of [111In]anti-claudin-4 mAb to selectively target claudin-4 was assessed using two human xenograft tumour models with differential claudin-4 status in mice. [111In]anti-claudin-4 mAb was also used to detect PDAC development in genetically engineered KPC mice. The PDAC status of these mice was confirmed with [18F]FDG-PET, magnetic resonance imaging (MRI), histology, and immunofluorescence microscopy. RESULTS: High uptake of [111In]anti-claudin-4 mAb was observed in PDAC xenografts in mice, reaching 16.9 ± 4.5 % of injected dose per gram (% ID/g) at 72 h post-injection. This uptake was mediated specifically by the expression of claudin-4. Uptake of [111In]anti-claudin-4 mAb also enabled clear visualisation of spontaneous PDAC formation in KPC mice. CONCLUSIONS: [111In]anti-claudin-4 mAb allows non-invasive detection of claudin-4 upregulation during development of PDAC and could potentially be used to aid in the early detection and characterisation of this malignancy.