Faculty Bibliography

PURPOSE: In this study, we evaluated a genetic approach for in vivo multimodal molecular imaging of vasculature in a mouse model of melanoma. PROCEDURES: We used a novel transgenic mouse, Ts-Biotag, that genetically biotinylates vascular endothelial cells. After inoculating these mice with B16 melanoma cells, we selectively targeted endothelial cells with (strept)avidinated contrast agents to achieve multimodal contrast enhancement of Tie2-expressing blood vessels during tumor progression. RESULTS: This genetic targeting system provided selective labeling of tumor vasculature and showed in vivo binding of avidinated probes with high specificity and sensitivity using microscopy, near infrared, ultrasound, and magnetic resonance imaging. We further demonstrated the feasibility of conducting longitudinal three-dimensional (3D) targeted imaging studies to dynamically assess changes in vascular Tie2 from early to advanced tumor stages. CONCLUSIONS: Our results validated the Ts-Biotag mouse as a multimodal targeted imaging system with the potential to provide spatio-temporal information about dynamic changes in vasculature during tumor progression.

In vivo imaging modalities provide powerful tools for the noninvasive longitudinal characterization of preclinical cancer models. Medulloblastoma (MB) is the most common malignant brain tumor in children, and the subject of intense research, much of which involves mouse models. Manganese-enhanced magnetic resonance imaging (MEMRI) produces unparalleled images of the cerebellum, the site of most MBs [1,2]. For this reason, longitudinal MEMRI of preclinical medulloblastoma models enables analysis of the region of origin, monitoring of tumor progression, and treatment response evaluation. In this study, we present the initial MEMRI characterization of a novel mouse medulloblastoma model with an activating mutation in the Smo gene, which exhibit different growth characteristics than those observed in previous studies of Ptch1 knockout mice [1]. SmoM2 mice were engineered by crossing Atoh1-CreER [3] male mice with homozygous R26-floxedSTOP-SmoM2 females [4]. The SmoM2 mutation was induced by subcutaneous injection of low dose (1mug/g) Tamoxifen (TMX) at postnatal day P2. Biweekly imaging sessions using 7-Tesla MRI (Bruker) began at postnatal day P21. MnCl2 (50-60 mg/kg) was injected intraperitoneally 24 hours before imaging. Scan protocol: 1 min low-resolution pilot, 20 min 150mum resolution T1-weighted GE sequence (TE/TR = 4/30 ms; FA = 20degree; FOV = 19.2 mm x 19.2 mm x 12 mm; Matrix = 128 x 128 x 80). Images were analyzed in 3-space using Amira and Fiji. Morphological characterization was corroborated with histology as shown in Fig1. Longitudinal MEMRI results are summarized in Fig2. Based on our preliminary results, all SmoM2 mice had preneoplastic lesions, while approximately half developed into full tumor morphology (n=21). Of the mice with tumors, approximately 72% developed bilateral tumors and the remaining developed tumors in either the right or left hemisphere. Approximately 50% of animals with bilateral tumors exhibited regression in one lateral tumor and progression in the other, or progression in both tumors (n=8). General disease progression is as follows: at approximately postnatal week W3, small lesions are apparent in the majority of interlobule spaces including the mid vermis; at ~W7, regions of proliferative lesion thickening are apparent and smaller lesions regress; at ~W13 significant tumor encroachment into the forebrain as well as expansion of the third and fourth ventricles are apparent. Tumors were observed to originate in the posterior hemispheres, shift and compress the normal appearing cerebellum as they progress, and finally encroach into the forebrain. Estimated tumor volume doubling time is approximately 4.5 days at early timepoints (W11.5). Noticeable symptoms - including delayed tail-pull reflex, ataxia, and hydrocephalus - in SmoM2 mice were apparent as early as W10. In addition to qualitative understanding of tumor progression, we have manually segmented and quantified tumor volume at these key timepoints in an effort to produce a unified growth model. Current efforts in automated segmentation and hierarchical clustering-based classification of tumors will guide upcoming preclinical trials of anticancer therapeutics

Determining the cellular basis of brain growth is an important problem in developmental neurobiology. In the mammalian brain, the cerebellum is particularly amenable to studies of growth because it contains only a few cell types, including the granule cells, which are the most numerous neuronal subtype. Furthermore, in the mouse cerebellum granule cells are generated from granule cell precursors (gcps) in the external granule layer (EGL), from 1 day before birth until about 2 weeks of age. The complexity of the underlying cellular processes (multiple cell behaviors, three spatial dimensions, time-dependent changes) requires a quantitative framework to be fully understood. In this paper, a differential equation-based model is presented, which can be used to estimate temporal changes in granule cell numbers in the EGL. The model includes the proliferation of gcps and their differentiation into granule cells, as well as the process by which granule cells leave the EGL. Parameters describing these biological processes were derived from fitting the model to histological data. This mathematical model should be useful for understanding altered gcp and granule cell behaviors in mouse mutants with abnormal cerebellar development and cerebellar cancers.

The mouse is the mammalian model of choice for investigating cardiovascular biology, given our ability to manipulate it by genetic, pharmacologic, mechanical, and environmental means. Imaging is an important approach to phenotyping both function and structure of cardiac and vascular components. This review details commonly used imaging approaches, with a focus on echocardiography and magnetic resonance imaging and brief overviews of other imaging modalities. We also briefly outline emerging imaging approaches but caution that reliability and validity data may be lacking. (c) 2016 by John Wiley & Sons, Inc.

PURPOSE: Manganese (Mn) is an effective contrast agent and biologically active metal, which has been widely used for Mn-enhanced MRI (MEMRI). The purpose of this study was to develop and test a Mn binding protein for use as a genetic reporter for MEMRI. METHODS: The bacterial Mn-binding protein, MntR was identified as a candidate reporter protein. MntR was engineered for expression in mammalian cells, and targeted to different subcellular organelles, including the Golgi Apparatus where cellular Mn is enriched. Transfected HEK293 cells and B16 melanoma cells were tested in vitro and in vivo, using immunocytochemistry, MR imaging and relaxometry. RESULTS: Subcellular targeting of MntR to the cytosol, endoplasmic reticulum and Golgi apparatus was verified with immunocytochemistry. After targeting to the Golgi, MntR expression produced robust R1 changes and T1 contrast in cells, in vitro and in vivo. Co-expression with the divalent metal transporter DMT1, a previously described Mn-based reporter, further enhanced contrast in B16 cells in culture, but in the in vivo B16 tumor model tested was not significantly better than MntR alone. CONCLUSION: This second-generation reporter system both expands the capabilities of genetically encoded reporters for imaging with MEMRI and provides important insights into the mechanisms of Mn biology which create endogenous MEMRI contrast. Magn Reson Med, 2014. (c) 2014 Wiley Periodicals, Inc.

The widespread use of the mouse as a model system to study brain development has created the need for noninvasive neuroimaging methods that can be applied to early postnatal mice. The goal of this study was to optimize in vivo three- (3D) and four-dimensional (4D) manganese (Mn)-enhanced MRI (MEMRI) approaches for acquiring and analyzing data from the developing mouse brain. The combination of custom, stage-dependent holders and self-gated (motion-correcting) 3D MRI sequences enabled the acquisition of high-resolution (100-mum isotropic), motion artifact-free brain images with a high level of contrast due to Mn-enhancement of numerous brain regions and nuclei. We acquired high-quality longitudinal brain images from two groups of FVB/N strain mice, six mice per group, each mouse imaged on alternate odd or even days (6 3D MEMRI images at each day) covering the developmental stages between postnatal days 1 to 11. The effects of Mn-exposure, anesthesia and MRI were assessed, showing small but significant transient effects on body weight and brain volume, which recovered with time and did not result in significant morphological differences when compared to controls. Metrics derived from deformation-based morphometry (DBM) were used for quantitative analysis of changes in volume, position and signal intensity of a number of brain regions. The cerebellum, a brain region undergoing significant changes in size and patterning at early postnatal stages, was analyzed in detail to demonstrate the spatiotemporal characterization made possible by this new atlas of mouse brain development. These results show that MEMRI is a powerful tool for quantitative analysis of mouse brain development, with great potential for in vivo phenotype analysis in mouse models of neurodevelopmental diseases.

A prominent feature of the developing mammalian brain is the widespread migration of neural progenitor (NP) cells during embryogenesis. A striking example is provided by NP cells born in the ventral forebrain of mid-gestation stage mice, which subsequently migrate long distances to their final positions in the cortex and olfactory bulb. Previous studies have used two-dimensional histological methods, making it difficult to analyze three-dimensional (3D) migration patterns. Unlike histology, magnetic resonance microimaging (micro-MRI) is a non-destructive, quantitative and inherently 3D imaging method for analyzing mouse embryos. To allow mapping of migrating NP cells with micro-MRI, cells were labeled in situ in the medial (MGE) and lateral (LGE) ganglionic eminences, using targeted in utero ultrasound-guided injection of micron-sized particles of iron-oxide (MPIO). Ex vivo micro-MRI and histology were then performed 5-6days after injection, demonstrating that the MPIO had magnetically labeled the migrating NP populations, which enabled 3D visualization and automated segmentation of the labeled cells. This approach was used to analyze the distinct patterns of migration from the MGE and LGE, and to construct rostral-caudal migration maps from each progenitor region. Furthermore, abnormal migratory phenotypes were observed in Nkx2.1-/- embryos, most notably a significant increase in cortical neurons derived from the Nkx2.1-/- LGE. Taken together, these results demonstrate that MPIO labeling and micro-MRI provide an efficient and powerful approach for analyzing 3D cell migration patterns in the normal and mutant mouse embryonic brain.

Volumetric analysis of brain ventricles (BVs) is important to the study of normal and abnormal development of the central nervous system of mouse embryos. High-frequency ultrasound (HFU) is frequently used to image embryos because it is real-time, non-invasive, and provides fine-resolution images. However, manual segmentation of BVs from 3D HFU volumes remains challenging and time consuming. Therefore, automatic segmentation, staging, and mutant detection algorithms are needed for studies with large embryo counts. An accurate and automatic method to segment BVs from high-frequency ultrasound images has been stated in a prior work. This paper presents novel algorithms for deriving the Y-skeleton of a BV region and decomposing the BV region into five components (fourth ventricle, aqueduct, third ventricle and two lateral ventricles). Embryo staging and mutant detection are accomplished by analyzing the volume profile along the BV skeleton and the volumes of the five BV components

Mouse models have increased our understanding of the pathogenesis of medulloblastoma (MB), the most common malignant pediatric brain tumor that often forms in the cerebellum. A major goal of ongoing research is to better understand the early stages of tumorigenesis and to establish the genetic and environmental changes that underlie MB initiation and growth. However, studies of MB progression in mouse models are difficult due to the heterogeneity of tumor onset times and growth patterns and the lack of clinical symptoms at early stages. Magnetic resonance imaging (MRI) is critical for noninvasive, longitudinal, three-dimensional (3D) brain tumor imaging in the clinic but is limited in resolution and sensitivity for imaging early MBs in mice. In this study, high-resolution (100 mum in 2 hours) and high-throughput (150 mum in 15 minutes) manganese-enhanced MRI (MEMRI) protocols were optimized for early detection and monitoring of MBs in a Patched-1 (Ptch1) conditional knockout (CKO) model. The high tissue contrast obtained with MEMRI revealed detailed cerebellar morphology and enabled detection of MBs over a wide range of stages including pretumoral lesions as early as 2 to 3 weeks postnatal with volumes close to 0.1 mm(3). Furthermore, longitudinal MEMRI allowed noninvasive monitoring of tumors and demonstrated that lesions within and between individuals have different tumorigenic potentials. 3D volumetric studies allowed quantitative analysis of MB tumor morphology and growth rates in individual Ptch1-CKO mice. These results show that MEMRI provides a powerful method for early in vivo detection and longitudinal imaging of MB progression in the mouse brain.