X-Ray Phase-Contrast CT of a Pancreatic Ductal Adenocarcinoma Mouse Model

To explore the potential of grating-based x-ray phase-contrast computed tomography (CT) for preclinical research, a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) was investigated. One ex-vivo mouse specimen was scanned with different grating-based phase-contrast CT imaging setups covering two different settings: i) high-resolution synchrotron radiation (SR) imaging and ii) dose-reduced imaging using either synchrotron radiation or a conventional x-ray tube source. These experimental settings were chosen to assess the potential of phase-contrast imaging for two different types of application: i) high-performance imaging for virtual microscopy applications and ii) biomedical imaging with increased soft-tissue contrast for in-vivo applications. For validation and as a reference, histological slicing and magnetic resonance imaging (MRI) were performed on the same mouse specimen. For each x-ray imaging setup, attenuation and phase-contrast images were compared visually with regard to contrast in general, and specifically concerning the recognizability of lesions and cancerous tissue. To quantitatively assess contrast, the contrast-to-noise ratios (CNR) of selected regions of interest (ROI) in the attenuation images and the phase images were analyzed and compared. It was found that both for virtual microscopy and for in-vivo applications, there is great potential for phase-contrast imaging: in the SR-based benchmarking data, fine details about tissue composition are accessible in the phase images and the visibility of solid tumor tissue under dose-reduced conditions is markedly superior in the phase images. The present study hence demonstrates improved diagnostic value with phase-contrast CT in a mouse model of a complex endogenous cancer, promoting the use and further development of grating-based phase-contrast CT for biomedical imaging applications.     

Inhibition of mTOR Reduces Anal Carcinogenesis in Transgenic Mouse Model

The molecular mechanism of human anal squamous cell carcinoma (ASCC) is unclear, and the accumulating evidence indicate association of ASCC with the activation of the Akt/mTOR pathway. Here we describe a mouse model with spontaneous anal squamous cell cancer, wherein a combined deletion of Tgfbr1 and Pten in stratified squamous epithelia was induced using inducible K14-Cre. Histopathologic analyses confirmed that 33.3% of the mice showed increased susceptibility to ASCC and precancerous lesions. Biomarker analyses demonstrated that the activation of the Akt pathway in ASCC of the Tgfbr1 and Pten double knockout (2cKO) mouse was similar to that observed in human anal cancer. Chemopreventive experiments using mTOR inhibitor-rapamycin treatment significantly delayed the onset of the ASCC tumors and reduced the tumor burden in 2cKO mice by decreasing the phosphorylation of Akt and S6. This is the first conditional knockout mouse model used for investigating the contributions of viral and cellular factors in anal carcinogenesis without carcinogen-mediated induction, and it would provide a platform for assessing new therapeutic modalities for treating and/or preventing this type of cancer.     

Defining Cell Lineages in the Prostate Epithelium

Understanding the stages of cell differentiation in the normal prostate epithelium isessential for the identification of the cell type(s) involved in prostatic carcinogenesis.Prostate glands are composed of three types of epithelial cells (i.e. basal, secretory andneuroendocrine) but the hierarchical relations among these cell types have been longcontroversial. We have recently developed a novel system to define prostate epithelialcell lineages in vivo. We find that, during normal prostate organogenesis, terminallydifferentiated secretory cells derive from p63-positive basal cells, which thusrepresent/include prostate stem cells. Future studies will determine if p63-positive basalcells retain stem cells capabilities in the adult prostate epithelium.     

Iodine Uptake and Prostate Cancer in the TRAMP Mouse Model

Iodine supplementation exerts antitumor effects in several types of cancer. Iodide (I⁻) and iodine (I₂) reduce cell proliferation and induce apoptosis in human prostate cancer cells (LNCaP and DU-145). Both chemical species decrease tumor growth in athymic mice xenografted with DU-145 cells. The aim of this study was to analyze the uptake and effects of iodine in a preclinical model of prostate cancer (transgenic adenocarcinoma of the mouse prostate [TRAMP] mice/SV40-TAG antigens), which develops cancer by 12 wks of age. ¹²⁵I⁻ and ¹²⁵I₂ uptake was analyzed in prostates from wild-type and TRAMP mice of 12 and 24 wks in the presence of perchlorate (inhibitor of the Na⁺/I⁻ symporter [NIS]). NIS expression was quantified by quantitative polymerase chain reaction (qPCR). Mice (6 wks old) were supplemented with 0.125 mg I⁻ plus 0.062 mg I₂/mouse/day for 12 or 24 wks. The weight of the genitourinary tract (GUT), the number of acini with lesions, cell proliferation (levels of proliferating cell nuclear antigen [PCNA] by immunohistochemistry), p53 and p21 expression (by qPCR) and apoptosis (relative amount of nucleosomes by enzyme-linked immunosorbent assay) were evaluated. In both age-groups, normal and tumoral prostates take up both forms of iodine, but only I⁻ uptake was blocked by perchlorate. Iodine supplementation prevented the overexpression of NIS in the TRAMP mice, but had no effect on the GUT weight, cell phenotype, proliferation or apoptosis. In TRAMP mice, iodine increased p53 expression but had no effect on p21 (a p53-dependent gene). Our data corroborate NIS involvement in I⁻ uptake and support the notion that another transporter mediates I₂ uptake. Iodine did not prevent cancer progression. This result could be explained by a strong inactivation of the p53 pathway by TAG antigens.     

The Fine Structure of the Epithelial Cells of the Mouse Prostate : II. Ventral Lobe Epithelium

The fine structure of the epithelial cells of one component of the prostatic complex of the mouse—the ventral lobe—has been investigated by electron microscopy. This organ is composed of small tubules, lined by tall simple cuboidal epithelium, surrounded by smooth muscle and connective tissue. Electron micrographs of the epithelial cells of the ventral lobe show these to be limited by a cell membrane, which appears as a continuous dense line. The nucleus occupies the basal portion of the cell and the nuclear envelope consists of two membranes. The cytoplasmic matrix is of moderately low density. The endoplasmic reticulum consists of elongated, circular, and oval profiles representing the cavities of this system bounded by rough surfaced membranes. The Golgi apparatus appears localized in a region between the apical border and the nucleus, and is composed of the usual elements found in secretory cells (3, 9). At the base of the cells, a basement membrane is visible in close contact with the outer aspect of the cell membrane. A space of varying width, which seems to be occupied by connective tissue, separates the epithelial cells from the surrounding smooth muscle fibers and the blood vessels. Bodies with the appearance of portions of the cytoplasm, mitochondria, or profiles of the endoplasmic reticulum can be seen in the lumina of the acini and on the bases of these pictures and others of the apical region the mechanism of secretion by these cells is discussed. The fine structural organization of these cells is compared with that of another component of the mouse prostate—the coagulating gland.     

Model-Based Tumor Growth Dynamics and Therapy Response in a Mouse Model of De Novo Carcinogenesis

Tumorigenesis is a complex, multistep process that depends on numerous alterations within the cell and contribution from the surrounding stroma. The ability to model macroscopic tumor evolution with high fidelity may contribute to better predictive tools for designing tumor therapy in the clinic. However, attempts to model tumor growth have mainly been developed and validated using data from xenograft mouse models, which fail to capture important aspects of tumorigenesis including tumor-initiating events and interactions with the immune system. In the present study, we investigate tumor growth and therapy dynamics in a mouse model of de novo carcinogenesis that closely recapitulates tumor initiation, progression and maintenance in vivo. We show that the rate of tumor growth and the effects of therapy are highly variable and mouse specific using a Gompertz model to describe tumor growth and a two-compartment pharmacokinetic/ pharmacodynamic model to describe the effects of therapy in mice treated with 5-FU. We show that inter-mouse growth variability is considerably larger than intra-mouse variability and that there is a correlation between tumor growth and drug kill rates. Our results show that in vivo tumor growth and regression in a double transgenic mouse model are highly variable both within and between subjects and that mathematical models can be used to capture the overall characteristics of this variability. In order for these models to become useful tools in the design of optimal therapy strategies and ultimately in clinical practice, a subject-specific modelling strategy is necessary, rather than approaches that are based on the average behavior of a given subject population which could provide erroneous results.     

Loss of Central Auditory Processing in a Mouse Model of Canavan Disease

Canavan Disease (CD) is a leukodystrophy caused by homozygous null mutations in the gene encoding aspartoacylase (ASPA). ASPA-deficiency is characterized by severe psychomotor retardation, and excessive levels of the ASPA substrate N-acetylaspartate (NAA). ASPA is an oligodendrocyte marker and it is believed that CD has a central etiology. However, ASPA is also expressed by Schwann cells and ASPA-deficiency in the periphery might therefore contribute to the complex CD pathology. In this study, we assessed peripheral and central auditory function in the Aspa^lacZ/lacZ rodent model of CD using auditory brainstem response (ABR). Increased ABR thresholds and the virtual loss of waveform peaks 4 and 5 from Aspa^lacZ/lacZ mice, indicated altered central auditory processing in mutant mice compared with Aspa^wt/wt controls and altered central auditory processing. Analysis of ABR latencies recorded from Aspa^lacZ/lacZ mice revealed that the speed of nerve conduction was unchanged in the peripheral part of the auditory pathway, and impaired in the CNS. Histological analyses confirmed that ASPA was expressed in oligodendrocytes and Schwann cells of the auditory system. In keeping with our physiological results, the cellular organization of the cochlea, including the organ of Corti, was preserved and the spiral ganglion nerve fibres were normal in ASPA-deficient mice. In contrast, we detected substantial hypomyelination in the central auditory system of Aspa^lacZ/lacZ mice. In summary, our data suggest that the lack of ASPA in the CNS is responsible for the observed hearing deficits, while ASPA-deficiency in the cochlear nerve fibres is tolerated both morphologically and functionally.     

Hearing Loss in a Mouse Model of 22q11.2 Deletion Syndrome

22q11.2 Deletion Syndrome (22q11DS) arises from an interstitial chromosomal microdeletion encompassing at least 30 genes. This disorder is one of the most significant known cytogenetic risk factors for schizophrenia, and can also cause heart abnormalities, cognitive deficits, hearing difficulties, and a variety of other medical problems. The Df1/+ hemizygous knockout mouse, a model for human 22q11DS, recapitulates many of the deficits observed in the human syndrome including heart defects, impaired memory, and abnormal auditory sensorimotor gating. Here we show that Df1/+ mice, like human 22q11DS patients, have substantial rates of hearing loss arising from chronic middle ear infection. Auditory brainstem response (ABR) measurements revealed significant elevation of click-response thresholds in 48% of Df1/+ mice, often in only one ear. Anatomical and histological analysis of the middle ear demonstrated no gross structural abnormalities, but frequent signs of otitis media (OM, chronic inflammation of the middle ear), including excessive effusion and thickened mucosa. In mice for which both in vivo ABR thresholds and post mortem middle-ear histology were obtained, the severity of signs of OM correlated directly with the level of hearing impairment. These results suggest that abnormal auditory sensorimotor gating previously reported in mouse models of 22q11DS could arise from abnormalities in auditory processing. Furthermore, the findings indicate that Df1/+ mice are an excellent model for increased risk of OM in human 22q11DS patients. Given the frequently monaural nature of OM in Df1/+ mice, these animals could also be a powerful tool for investigating the interplay between genetic and environmental causes of OM.     

PDE2 Is a Novel Target for Attenuating Tumor Formation in a Mouse Model of UVB-Induced Skin Carcinogenesis

Our previous studies demonstrated that the topical application of caffeine is a potent inhibitor of UVB-induced carcinogenesis and selectively increases apoptosis in tumors but not in non-tumor areas of the epidermis in mice that are at a high risk for developing skin cancer. While this effect is mainly through a p53 independent pathway, the mechanism by which caffeine inhibits skin tumor formation has not been fully elucidated. Since caffeine is a non-specific phosphodiesterase inhibitor, we investigated the effects of several PDE inhibitors on the formation of sunburn cells in mouse skin after an acute exposure to ultraviolet light B (UVB). The topical application of a PDE2 inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA hydrochloride), stimulated epidermal apoptosis compared to control (P<0.01) and to a greater extent than caffeine whereas a PDE4 inhibitor attenuated the epidermal apoptosis compared to control (P<0.01). Since PDE2 hydrolyzes cyclic nucleotides, mainly cGMP, the effects of EHNA hydrochloride on epidermal apoptosis following UVB exposure may be mediated, in part, by increased cGMP signaling. Data demonstrated that the topical application of dibutyryl cGMP stimulated epidermal apoptosis (P<0.01) following an acute exposure to UVB. Treating UVB-pretreated mice topically with 3.1 µmole or 0.8 µmole of EHNA hydrochloride attenuated tumor formation to a greater extent than treating with 6.2 µmole caffeine when these compounds were applied once a day, five days a week for 18 weeks. These observations suggest a novel role for PDE2 in UVB-induced tumorigenesis and that PDE2 inhibitors that mediate cGMP signaling may be useful for the prevention and treatment of skin cancer.     

Loss of the V-ATPase B1 Subunit Isoform Expressed in Non-Neuronal Cells of the Mouse Olfactory Epithelium Impairs Olfactory Function

The vacuolar proton-pumping ATPase (V-ATPase) is the main mediator of intracellular organelle acidification and also regulates transmembrane proton (H⁺) secretion, which is necessary for an array of physiological functions fulfilled by organs such as the kidney, male reproductive tract, lung, bone, and ear. In this study we characterize expression of the V-ATPase in the main olfactory epithelium of the mouse, as well as a functional role for the V-ATPase in odor detection. We report that the V-ATPase localizes to the apical membrane microvilli of olfactory sustentacular cells and to the basolateral membrane of microvillar cells. Plasma membrane V-ATPases containing the B1 subunit isoform are not detected in olfactory sensory neurons or in the olfactory bulb. This precise localization of expression affords the opportunity to ascertain the functional relevance of V-ATPase expression upon innate, odor-evoked behaviors in B1-deficient mice. This animal model exhibits diminished innate avoidance behavior (revealed as a decrease in freezing time and an increase in the number of sniffs in the presence of trimethyl-thiazoline) and diminished innate appetitive behavior (a decrease in time spent investigating the urine of the opposite sex). We conclude that V-ATPase-mediated H⁺ secretion in the olfactory epithelium is required for optimal olfactory function.     

条件性敲除APC基因小鼠肠道腺瘤模型的构建

腺瘤性结肠息肉病(adenomatous polyposis coli,APC)基因是家族性腺瘤性息肉病(familial adenomatous polyposis,FAP)的致病基因,APC基因的突变导致小鼠多处产生肿瘤,但肠道条件性敲除APC基因后,小鼠的表型并不清楚。该研究利用Cre-LoxP重组酶系统,在肠道绒毛和隐窝上皮细胞条件性敲除APC基因,并对小鼠表型进行鉴定和分析。将Villin Cre小鼠和APC~(fl/fl)小鼠合笼得到Villin Cre;APC~(fl/+)小鼠;有意思的是后者进一步与APC~(fl/fl)小鼠合笼,却没有得到Villin Cre;APC~(fl/fl)小鼠。进一步解剖Villin Cre;APC~(fl/+)小鼠,发现其自发产生肠道肿瘤,并能携瘤生存,免疫组化显示瘤体组织激活了Wnt信号通路。结果表明成功地构建了小鼠肠道条件性敲除APC基因腺瘤模型,为进一步研究APC基因在肠道发育以及肠道肿瘤的作用提供了优良的工具。     

Examining the Relationship between Pre-Malignant Breast Lesions,Carcinogenesis and Tumor Evolution in the Mammary Epithelium Using an Agent-Based Model

Introduction

Breast cancer, the product of numerous rare mutational events that occur over an extended time period, presents numerous challenges to investigators interested in studying the transformation from normal breast epithelium to malignancy using traditional laboratory methods, particularly with respect to characterizing transitional and pre-malignant states. Dynamic computational modeling can provide insight into these pathophysiological dynamics, and as such we use a previously validated agent-based computational model of the mammary epithelium (the DEABM) to investigate the probabilistic mechanisms by which normal populations of ductal cells could transform into states replicating features of both pre-malignant breast lesions and a diverse set of breast cancer subtypes.

Methods

The DEABM consists of simulated cellular populations governed by algorithms based on accepted and previously published cellular mechanisms. Cells respond to hormones, undergo mitosis, apoptosis and cellular differentiation. Heritable mutations to 12 genes prominently implicated in breast cancer are acquired via a probabilistic mechanism. 3000 simulations of the 40-year period of menstrual cycling were run in wild-type (WT) and BRCA1-mutated groups. Simulations were analyzed by development of hyperplastic states, incidence of malignancy, hormone receptor and HER-2 status, frequency of mutation to particular genes, and whether mutations were early events in carcinogenesis.

Results

Cancer incidence in WT (2.6%) and BRCA1-mutated (45.9%) populations closely matched published epidemiologic rates. Hormone receptor expression profiles in both WT and BRCA groups also closely matched epidemiologic data. Hyperplastic populations carried more mutations than normal populations and mutations were similar to early mutations found in ER+ tumors (telomerase, E-cadherin, TGFB, RUNX3, p < .01). ER- tumors carried significantly more mutations and carried more early mutations in BRCA1, c-MYC and genes associated with epithelial-mesenchymal transition.

Conclusions

The DEABM generates diverse tumors that express tumor markers consistent with epidemiologic data. The DEABM also generates non-invasive, hyperplastic populations, analogous to atypia or ductal carcinoma in situ (DCIS), via mutations to genes known to be present in hyperplastic lesions and as early mutations in breast cancers. The results demonstrate that agent-based models are well-suited to studying tumor evolution through stages of carcinogenesis and have the potential to be used to develop prevention and treatment strategies.     

Germline Genetic Variation Modulates Tumor Progression and Metastasis in a Mouse Model of Neuroendocrine Prostate Carcinoma

Neuroendocrine (NE) differentiation has gained increased attention as a prostate cancer (PC) prognostic marker. The aim of this study is to determine whether host germline genetic variation influences tumor progression and metastasis in C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of aggressive NEPC. TRAMP mice were crossed to the eight progenitor strains of the Collaborative Cross recombinant inbred panel to address this. Tumor growth and metastasis burden were quantified in heterozygous transgene positive F1 male mice at 30 weeks of age. Compared to wild-type C57BL/6J-Tg(TRAMP)824Ng/J males, TRAMP x CAST/EiJ, TRAMP x NOD/ShiLtJ and TRAMP x NZO/HlLtJ F1 males displayed significant increases in tumor growth. Conversely, TRAMP x WSB/EiJ and TRAMP x PWK/PhJ F1 males displayed significant reductions in tumor growth. Interestingly, despite reduced tumor burden, TRAMP x WSB/EiJ males had an increased nodal metastasis burden. Patterns of distant pulmonary metastasis tended to follow the same patterns as that of local dissemination in each of the strains. All tumors and metastases displayed positive staining for NE markers, synaptophysin, and FOXA2. These experiments conclusively demonstrate that the introduction of germline variation by breeding modulates tumor growth, local metastasis burden, and distant metastasis frequency in this model of NEPC. These strains will be useful as model systems to facilitate the identification of germline modifier genes that promote the development of aggressive forms of PC.     

Use of A Mouse Chimaeric Model to Study Cell Migration Patterns In the Small Intestinal Epithelium

The pattern of cells migration in the small intestinal epithelia of a RIII/?ro C57BL/6J mouse aggregation chimaera is demonstrated using Dolichos biflorus agglutinin-peroxidase (DBA) conjugate as a strain-specific marker. Using serial tangential sections of heterogeneously stained villi and plotting the distribution of labelled/unlabelled cells with a drawing tube, and by three-dimensional reconstruction with the aid of computer graphics, we show the migration pathway to be in tight cohorts of similar DBA-peroxidase staining type, which move upwards in straight lines. There is little cell mixing either on the villus or along the crypt-villus junctions. Our observations also show for the first time that a single crypt can feed cells to more than one villus. They also suggest that either cell loss is not confined to the villus tips but can take place from the villus sides, or that there is marked asynchrony of cell production between crypts.