miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors.

Expression of BRCA1 is commonly decreased in sporadic breast tumors, and this correlates with poor prognosis of breast cancer patients. Here we show that BRCA1 transcripts are selectively enriched in the Argonaute/miR-182 complex and miR-182 downregulates BRCA1 expression. Antagonizing miR-182 enhances BRCA1 protein levels and protects them from IR-induced cell death, while overexpressing miR-182 reduces BRCA1 protein, impairs homologous recombination-mediated repair, and render cells hypersensitive to IR. The impaired DNA repair phenotype induced by miR-182 overexpression can be fully rescued by overexpressing miR-182-insensitive BRCA1. Consistent with a BRCA1-deficiency phenotype, miR-182-overexpressing breast tumor cells are hypersensitive to inhibitors of poly (ADP-ribose) polymerase 1 (PARP1). Conversely, antagonizing miR-182 enhances BRCA1 levels and induces resistance to PARP1 inhibitor. Finally, a clinical-grade PARP1 inhibitor impacts outgrowth of miR-182-expressing tumors in animal models. Together these results suggest that miR-182-mediated downregulation of BRCA1 impedes DNA repair and may impact breast cancer therapy.

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.

Cd11b(+) myeloid cells support hepatic metastasis through down-regulation of angiopoietin-like 7 in cancer cells.

UNLABELLED: Myeloid cells are known to mediate metastatic progression. Here, we attempted to elucidate the mechanisms underlying these effects by identifying gene expression alterations in cancer cells forming hepatic metastases after myeloid cell depletion. Hepatic metastases are heavily infiltrated by CD11b(+) myeloid cells. We established hepatic metastases in transgenic CD11b-diphtheria toxin receptor mice by intrasplenic injection of MC38 colon and Lewis lung carcinoma cells before depleting myeloid cells with diphtheria toxin. Myeloid cell depletion inhibited metastatic growth with a marked diminishment of tumor vasculature. Expression of ANGPTL7 (angiopoietin-like 7), a protein not previously linked to metastasis, was highly up-regulated in cancer cells after myeloid cell depletion. This effect was duplicated in tissue culture, where coculture of cancer cells with tumor-conditioned myeloid cells from liver metastases or myeloid cell conditioned media down-regulated ANGPTL7 expression. Analogous to myeloid cell depletion, overexpression of ANGPTL7 in cancer cells significantly reduced hepatic metastasis formation and angiogenesis. We found that ANGPTL7 itself has strong antiangiogenic effects in vitro. Furthermore, analysis of The Cancer Genome Atlas colorectal and breast cancer data sets revealed striking ANGPTL7 underexpression in cancerous compared to normal tissues. Also, ANGPTL7 was down-regulated in metastatic liver colonies of colorectal cancer patients compared to their adjacent liver tissue. CONCLUSION: Myeloid cells promote liver metastasis by down-regulating ANGPTL7 expression in cancer cells; our findings implicate ANGPTL7 as a mediator of metastatic progression and a potential target for interference with liver metastases.

Ultrasonography-guided intracardiac injection: an improvement for quantitative brain colonization assays.

Brain metastasis is a frequent occurrence in patients with cancer, with devastating consequences. The current animal models for brain metastasis are highly variable, leading to a need for improved in vivo models that recapitulate the clinical disease. Herein, we describe an experimental brain metastasis model that uses ultrasonographic guidance to perform intracardiac injections. This method is easy to perform, giving consistent and quantitative results. Demonstrating the utility of this method, we have assessed a variety of metastatic cell lines for their ability to develop into brain metastases. Those cell lines that were competent at brain colonization could be detected in the brain vasculature 4 hours after intracardiac injection, and a few adherent cells persisted until colonization occurred. In contrast, those cell lines that were deficient in brain colonization were infrequently found 4 hours after introduction into the arterial circulation and were not detected at later time points. All of these cells were capable of brain colonization after intraparenchymal injection. We propose that adherence to the brain vasculature may be the key limiting step that determines the ability of a cancer cell to form brain metastases successfully. Identifying brain endothelium-specific adhesion molecules may enable development of screening modalities to detect brain-colonizing cancer cells and therapies to prevent these metastatic cells from seeding the brain.

Recruitment of a myeloid cell subset (CD11b/Gr1 mid) via CCL2/CCR2 promotes the development of colorectal cancer liver metastasis.

UNLABELLED: Liver metastasis from colorectal cancer is a leading cause of cancer mortality. Myeloid cells play pivotal roles in the metastatic process, but their prometastatic functions in liver metastasis remain incompletely understood. To investigate their role, we simulated liver metastasis in C57BL/6 mice through intrasplenic inoculation of MC38 colon carcinoma cells. Among the heterogeneous myeloid infiltrate, we identified a distinct population of CD11b/Gr1(mid) cells different from other myeloid populations previously associated with liver metastasis. These cells increased in number dramatically during establishment of liver metastases and were recruited from bone marrow by tumor-derived CCL2. Liver metastasis of Lewis lung carcinoma cells followed this pattern but this mechanism is not universal as liver colonization by B16F1 melanoma cells did not recruit similar subsets. Inhibition of CCL2 signaling and absence of its cognate receptor CCR2 reduced CD11b/Gr1(mid) recruitment and decreased tumor burden. Depletion of the CD11b/Gr1(mid) subset in a transgenic CD11b-diphtheria toxin receptor mouse model markedly reduced tumor cell proliferation. There was no evidence for involvement of an adaptive immune response in the prometastatic effects of CD11b/Gr1(mid) cells. Additionally, an analogous myeloid subset was found in liver metastases of some colorectal cancer patients. CONCLUSION: Collectively, our findings highlight the importance of myeloid cells--in this case a selective CD11b/Gr1(mid) subset--in sustaining development of colorectal cancer liver metastasis and identify a potential target for antimetastatic therapy.

Subcutaneous tumor volume measurement in the awake, manually restrained mouse using MRI.

PURPOSE: To describe a combination of techniques using the excellent volumetric capacities of magnetic resonance imaging (MRI) while avoiding anesthesia and maintaining high-throughput capability for tumor volume measurement in the awake mouse. This approach presents an alternative to calipers which, although cheap, fast, and easy to use, introduce many biases for tumor volume estimation. MATERIALS AND METHODS: The murine CaNT subcutaneous xenograft model was used. A quiet and modestly T2-weighted spin-echo scan was acquired at 4.7T (TE = 15 msec, TR = 1100 msec, 0.5 mm isotropic resolution) while the awake mouse was held by hand in the magnet. This method was compared to standard MR in the anesthetized mouse and caliper measurements. RESULTS: The combination of techniques used allows rapid, accurate, and reproducible measurement of subcutaneous tumor volumes in awake mice. It is less sensitive to both intra- and interoperator-derived biases and avoids confounds from the compliance of the fat and skin around the tumor, as well as from the tumor itself. Moreover, the data remain available for retrieval and scrutiny and reanalysis. CONCLUSION: Rapid, accurate, and precise tumor volumetry can be performed in the awake mouse by handheld positioned MR.

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.

Benzodiazepine binding site occupancy by the novel GABAA receptor subtype-selective drug 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) in rats, primates, and humans.

The GABA(A) receptor alpha2/alpha3 subtype-selective compound 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023; also known as MK-0777) is a triazolopyridazine that has similar, subnanomolar affinity for the benzodiazepine binding site of alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors and has partial agonist efficacy at the alpha2 and alpha3 but not the alpha1 or alpha5 subtypes. The purpose of the present study was to define the relationship between plasma TPA023 concentrations and benzodiazepine binding site occupancy across species measured using various methods. Thus, occupancy was measured using either in vivo [(3)H]flumazenil binding or [(11)C]flumazenil small-animal positron emission tomography (microPET) in rats, [(123)I]iomazenil gamma-scintigraphy in rhesus monkeys, and [(11)C]flumazenil PET in baboons and humans. For each study, plasma-occupancy curves were derived, and the plasma concentration of TPA023 required to produce 50% occupancy (EC(50)) was calculated. The EC(50) values for rats, rhesus monkeys, and baboons were all similar and ranged from 19 to 30 ng/ml, although in humans, the EC(50) was slightly lower at 9 ng/ml. In humans, a single 2-mg dose of TPA023 produced in the region of 50 to 60% occupancy in the absence of overt sedative-like effects. Considering that nonselective full agonists are associated with sedation at occupancies of less than 30%, these data emphasize the relatively nonsedating nature of TPA023.

Late changes in blood-brain barrier permeability in a rat tMCAO model of stroke detected by gadolinium-enhanced MRI.

OBJECTIVES: After cerebral ischaemia the blood-brain barrier (BBB) may be compromised and this has been observed in both clinical and preclinical studies. The timing of BBB disruption after ischaemia has long been considered to be biphasic, however some groups contest this view. Therefore, the purpose of this study was to characterize the BBB permeability timecourse in a rat model at both acute and chronic time points. METHODS: Unilateral transient middle cerebral artery occlusion (tMCAO) was performed in 15 male Sprague Dawley rats. Change in T1-weighted MR signal before and after an injection of gadolinium-based contrast agent was calculated voxelwise to derive a BBB permeability index (BBBPI) at both early (6 h, 12 h, and 24 h) and late (7 and 14 days) time points. RESULTS: As expected, BBBPI in the non-lesioned ROI was not significantly different from pre-occlusion baseline at any time point. However, BBBPI in the ipsilateral (lesioned) ROI was statistically different to baseline at day 7 (p < 0.001) and day 14 (p < 0.01) post-tMCAO. There was a small, but not-significant increase in BBBPI in the earlier phase (at 6 hours). DISCUSSION: Our results indicate a significant late opening of the BBB. This is important as the majority of previous studies have only characterised an early acute BBB permeability in ischemia. However, the later period of increased permeability may indicate an optimal time for drug delivery across the BBB, when it is especially suited to drugs targeting delayed processes.

Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

Activin receptor-like kinase 1 (ALK1)-mediated endothelial cell signalling in response to bone morphogenetic protein 9 (BMP9) and BMP10 is of significant importance in cardiovascular disease and cancer. However, detailed molecular mechanisms of ALK1-mediated signalling remain unclear. Here, we report crystal structures of the BMP10:ALK1 complex at 2.3 Å and the prodomain-bound BMP9:ALK1 complex at 3.3 Å. Structural analyses reveal a tripartite recognition mechanism that defines BMP9 and BMP10 specificity for ALK1, and predict that crossveinless 2 is not an inhibitor of BMP9, which is confirmed by experimental evidence. Introduction of BMP10-specific residues into BMP9 yields BMP10-like ligands with diminished signalling activity in C2C12 cells, validating the tripartite mechanism. The loss of osteogenic signalling in C2C12 does not translate into non-osteogenic activity in vivo and BMP10 also induces bone-formation. Collectively, these data provide insight into ALK1-mediated BMP9 and BMP10 signalling, facilitating therapeutic targeting of this important pathway.

Early-stage 11C-Flumazenil PET predicts day-14 selective neuronal loss in a rodent model of transient focal cerebral ischemia.

Predicting tissue outcome early after stroke is an important goal. MRI >3 h accurately predicts infarction but is insensitive to selective neuronal loss (SNL). Previous studies suggest that chronic-stage 11C-flumazenil PET (FMZ-PET) is a validated marker of SNL in rats, while early-stage FMZ-PET may predict infarction. Whether early FMZ-PET also predicts SNL is unknown. Following 45-min distal MCA occlusion, adult rats underwent FMZ-PET at 1 h and 48 h post-reperfusion to map distribution volume (VT), which reflects GABA-A receptor binding. NeuN immunohistochemistry was performed at Day 14. In each rat, VT and %NeuN loss were determined in 44 ROIs spanning the hemisphere. NeuN revealed isolated SNL and cortical infarction in five and one rats, respectively. In the SNL subgroup, VT-1 h was mildly reduced and only weakly predicted SNL, while VT-48 h was significantly increased and predicted SNL both individually (p < 0.01, Kendall) and across the group (p < 0.001), i.e. the higher the VT, the stronger the SNL. Similar correlations were found in the rat with infarction. Our findings suggest GABA-A receptors are still present on injured neurons at the 48 h timepoint, and the increased 48 h VT observed here is consistent with earlier rat studies showing early GABA-A receptor upregulation. That FMZ binding at 48 h was predictive of SNL may have clinical implications.

Neither in vivo MRI nor behavioural assessment indicate therapeutic efficacy for a novel 5HT(1A) agonist in rat models of ischaemic stroke.

BACKGROUND: 5HT1A agonists have previously been shown to promote recovery in animal models of stroke using ex vivo outcome measures which have raised the hopes for a potential clinical implementation. The purpose of this study was to evaluate the potential neuroprotective properties of a novel 5HT1A agonist DU123015 in 2 different models of transient focal ischaemic stroke of varying severities using both in vivo neuroimaging and behavioural techniques as primary outcome measures. For these studies, the NMDA receptor antagonist MK-801 was also utilized as a positive control to further assess the effectiveness of the stroke models and techniques used. RESULTS: In contrast to MK-801, no significant therapeutic effect of DU123015 on lesion volume in either the distal MCAo or intraluminal thread model of stroke was found. MK-801 significantly reduced lesion volume in both models; the mild distal MCAo condition (60 min ischaemia) and the intraluminal thread model, although it had no significant impact upon the lesion size in the severe distal MCAo condition (120 min ischaemia). These therapeutic effects on lesion size were mirrored on a behavioural test for sensory neglect and neurological deficit score in the intraluminal thread model. CONCLUSION: This study highlights the need for a thorough experimental design to test novel neuroprotective compounds in experimental stroke investigations incorporating: a positive reference compound, different models of focal ischaemia, varying the duration of ischaemia, and objective in vivo assessments within a single study. This procedure will help us to minimise the translation of less efficacious compounds.

Prospective gating control for highly efficient cardio-respiratory synchronised short and constant TR MRI in the mouse.

PURPOSE: Cardiac and respiratory motion derived image artefacts are reduced when data are acquired with cardiac and respiratory synchronisation. Where steady state imaging techniques are required in small animals, synchronisation is most commonly performed using retrospective gating techniques but these invoke an inherent time penalty. This paper reports the development of prospective gating techniques for cardiac and respiratory motion desensitised MRI with significantly reduced minimum scan time compared to retrospective gating. METHODS: Prospective gating incorporating the automatic reacquisition of data corrupted by motion at the entry to each breath was implemented in short TR 3D spoiled gradient echo imaging. Motion sensitivity was examined over the whole mouse body for scans performed without gating, with respiratory gating, and with cardio-respiratory gating. The gating methods were performed with and without automatic reacquisition of motion corrupted data immediately after completion of the same breath. Prospective cardio-respiratory gating, with acquisition of 64 k-space lines per cardiac R-wave, was used to enable whole body DCE-MRI in the mouse. RESULTS: Prospective cardio-respiratory gating enabled high fidelity steady state imaging of physiologically mobile organs such as the heart and lung. The automatic reacquisition of data corrupted by motion at the entry to each breath minimised respiratory motion artefact and enabled a highly efficient data capture that was adaptive to changes in the inter-breath interval. Prospective cardio-respiratory gating control enabled DCE-MRI to be performed over the whole mouse body with the acquisition of successive image volumes every 12-15 s at 422 µm isotropic resolution. CONCLUSIONS: Highly efficient cardio-respiratory motion desensitised steady state MRI can be performed in small animals with prospective synchronisation, centre-out phase-encode ordering, and the automatic reacquisition of data corrupted by motion at the entry to each breath. The method presented is robust against spontaneous changes in the breathing rate. Steady state imaging with prospective cardio-respiratory gating is much more efficient than with retrospective gating, and enables the examination of rapidly changing systems such as those found when using DCE-MRI.

Imaging of brain hypoxia in permanent and temporary middle cerebral artery occlusion in the rat using 18F-fluoromisonidazole and positron emission tomography: a pilot study.

In acute stroke, the target of therapy is the severely hypoxic but salvageable tissue. Previous human studies using 18F-fluoromisonidazole and positron emission tomography (18F-FMISO PET) have shown high tracer retention indicative of tissue hypoxia, which had normalized at repeat scan >48 h later. In the only validation study of 18F-FMISO, using ex vivo autoradiography in thread middle cerebral artery occluded (MCAo) rats, there was unexpected high uptake as late as 22 h after reperfusion, raising questions about the use of 18F-FMISO as a hypoxia tracer. Here we report a pilot study of 18F-FMISO PET in experimental stroke. Spontaneous hypertensive rats were subjected to distal clip MCAo. Three-hour dynamic PET was performed in 7 rats: 3 normals, 1 with permanent MCAo (two sessions: 30 mins and 48 h after clip), and 3 with temporary MCAo (45 mins, n=1; 120 mins, n=2; scanning started 30 mins after clip removal). Experiments were terminated by perfusion-fixation for standard histopathology. Late tracer retention was assessed by both compartmental modelling and simple side-to-side ratios. In the initial PET session of the permanent MCAo rat, striking trapping of 18F-FMISO was observed in the affected cortex, which had normalized 48 h later; histopathology revealed pannecrosis. In contrast, there was no demonstrable tracer retention in either temporary MCAo models, and histopathology showed ischemic changes only. These results document elevated 18F-FMISO uptake in the stroke area only in the early phase of MCAo, but not after early reperfusion nor when tissue necrosis has developed. These findings strongly support the validity of 18F-FMISO as a marker of viable hypoxic tissue/penumbra after stroke.

The MHP36 line of murine neural stem cells expresses functional CXCR1 chemokine receptors that initiate chemotaxis in vitro.

Chemokines help to establish cerebral inflammation after ischemia, which comprises a major component of secondary brain injury. The CXCR4 chemokine receptor system induces neural stem cell migration, and hence has been implicated in brain repair. We show that CXCR1 and interleukin-8 also stimulate chemotaxis in murine neural stem cells from the MHP36 cell line. The presence of CXCR1 was confirmed by reverse transcriptase PCR and immunohistochemistry. Interleukin-8 evoked intracellular calcium currents, upregulated doublecortin (a protein expressed by migrating neuroblasts), and elicited positive chemotaxis in vitro. Therefore, effectors of the early innate immune response may also influence brain repair mechanisms.

Metabolic acidosis and other determinants of hemoglobin-oxygen dissociation in severe childhood Plasmodium falciparum malaria.

Metabolic acidosis is a common complication of severe malaria caused by Plasmodium falciparum. The factors contributing to the acidosis were assessed in 62 children with severe falciparum malaria (cases) and in 29 control children who had recently recovered from mild or moderate malaria. The acidosis was largely caused by the accumulation of both lactic and 3-hydroxybutyric acids. The determinants of oxygen release to the tissues were also examined; although there was no difference between cases and controls in respect of 2,3-bisphosphoglycerate and mean corpuscular hemoglobin concentration, there was a marked increase in P(50) in the cases, caused by pyrexia, low pH, and base deficit. There was substantial relative or actual hypoglycemia in many cases. The relationship of these observations to therapeutic strategy is discussed.

Multi-modal characterisation of the neocortical clip model of focal cerebral ischaemia by MRI, behaviour and immunohistochemistry.

The neocortical clip model of focal cerebral ischaemia has previously been used with success in neuroprotection studies. To further improve its translational qualities, we have characterised this model using a combination of serial Magnetic Resonance Imaging (MRI), neurological assessment, the bilateral asymmetry test (BAT) and immunohistochemistry. The right MCA was occluded in spontaneously hypertensive rats for 0, 60 and 120 min. MRI was performed pre-surgery, 1, 3 and 7 days post-surgery. Behavioural assessment was performed 2 days before and 3 and 7 days post-surgery whilst neurological deficits were monitored daily. Neuroimaging results showed that 0 min of MCA occlusion did not produce a lesion, whereas occlusion for 60 min produced a lesion that remained stable over time. Occlusion for 120 min caused a more severe lesion 1 day post-surgery, but decreased by 7 days. Behaviour, neurological scores and histological lesion volumes correlated strongly with MRI lesion volume. Immunohistochemistry revealed neuronal loss, astrogliosis and macrophage infiltration in lesioned cortices. The neocortical clip model produced ischaemic lesions that are restricted to cortical territories of the MCA. The duration of occlusion dictates lesion severity which may prove useful for probing therapeutic interventions at different stages of stroke progression. The correlation of MRI with two different behavioural measures and post-mortem histology strengthens the basis for MRI providing an in vivo surrogate marker for structural and behavioural deficits caused by a cortical stroke.

High-Resolution Magnetic Resonance Imaging of the Regenerating Adult Zebrafish Heart.

The adult zebrafish is a well-established model for studying heart regeneration, but due to its tissue opaqueness, repair has been primarily assessed using destructive histology, precluding repeated investigations of the same animal. We present a high-resolution, non-invasive in vivo magnetic resonance imaging (MRI) method incorporating a miniature respiratory and anaesthetic perfusion set-up for live adult zebrafish, allowing for visualization of scar formation and heart regeneration in the same animal over time at an isotropic 31 µm voxel resolution. To test the method, we compared well and poorly healing cardiac ventricles using a transgenic fish model that exhibits heat-shock (HS) inducible impaired heart regeneration. HS-treated groups revealed persistent scar tissue for 10 weeks, while control groups were healed after 4 weeks. Application of the advanced MRI technique allowed clear discrimination of levels of repair following cryo- and resection injury for several months. It further provides a novel tool for in vivo time-lapse imaging of adult fish for non-cardiac studies, as the method can be readily applied to image wound healing in other injured or diseased tissues, or to monitor tissue changes over time, thus expanding the range of questions that can be addressed in adult zebrafish and other small aquatic species.

Automated MicroSPECT/MicroCT Image Analysis of the Mouse Thyroid Gland.

BACKGROUND: The ability of thyroid follicular cells to take up iodine enables the use of radioactive iodine (RAI) for imaging and targeted killing of RAI-avid thyroid cancer following thyroidectomy. To facilitate identifying novel strategies to improve 131I therapeutic efficacy for patients with RAI refractory disease, it is desired to optimize image acquisition and analysis for preclinical mouse models of thyroid cancer. METHODS: A customized mouse cradle was designed and used for microSPECT/CT image acquisition at 1 hour (t1) and 24 hours (t24) post injection of 123I, which mainly reflect RAI influx/efflux equilibrium and RAI retention in the thyroid, respectively. FVB/N mice with normal thyroid glands and TgBRAFV600E mice with thyroid tumors were imaged. In-house CTViewer software was developed to streamline image analysis with new capabilities, along with display of 3D voxel-based 123I gamma photon intensity in MATLAB. RESULTS: The customized mouse cradle facilitates consistent tissue configuration among image acquisitions such that rigid body registration can be applied to align serial images of the same mouse via the in-house CTViewer software. CTViewer is designed specifically to streamline SPECT/CT image analysis with functions tailored to quantify thyroid radioiodine uptake. Automatic segmentation of thyroid volumes of interest (VOI) from adjacent salivary glands in t1 images is enabled by superimposing the thyroid VOI from the t24 image onto the corresponding aligned t1 image. The extent of heterogeneity in 123I accumulation within thyroid VOIs can be visualized by 3D display of voxel-based 123I gamma photon intensity. CONCLUSIONS: MicroSPECT/CT image acquisition and analysis for thyroidal RAI uptake is greatly improved by the cradle and the CTViewer software, respectively. Furthermore, the approach of superimposing thyroid VOIs from t24 images to select thyroid VOIs on corresponding aligned t1 images can be applied to studies in which the target tissue has differential radiotracer retention from surrounding tissues.

MRI-guided radiotherapy of the SK-N-SH neuroblastoma xenograft model using a small animal radiation research platform.

OBJECTIVE: Neuroblastoma has one of the lowest survival rates of all childhood cancers, despite the use of intensive treatment regimens. Preclinical models of neuroblastoma are essential for testing new multimodality protocols, including those that involve radiotherapy (RT). The aim of this study was to develop a robust method for RT planning and tumour response monitoring based on combined MRI and cone-beam CT (CBCT) imaging and to apply it to a widely studied mouse xenograft model of neuroblastoma, SK-N-SH. METHODS: As part of a tumour growth inhibition study, SK-N-SH xenografts were generated in BALB/c nu/nu mice. Mice (n = 8) were placed in a printed MR- and CT-compatible plastic cradle, imaged using a 4.7-T MRI scanner and then transferred to a small animal radiation research platform (SARRP) irradiator with on-board CBCT. MRI/CBCT co-registration was performed to enable RT planning using the soft-tissue contrast afforded by MRI prior to delivery of RT (5 Gy). Tumour response was assessed by serial MRI and calliper measurements. RESULTS: SK-N-SH xenografts formed soft, deformable tumours that could not be differentiated from surrounding normal tissues using CBCT. MR images, which allowed clear delineation of tumours, were successfully co-registered with CBCT images, allowing conformal RT to be delivered. MRI measurements of tumour volume 4 days after RT correlated strongly with length of survival time. CONCLUSION: MRI allowed precision RT of SK-N-SH tumours and provided an accurate means of measuring tumour response. Advances in knowledge: MRI-based RT planning of murine tumours is feasible using an SARRP irradiator.