Ectopic Mice Research Articles

Doxycycline-induced exogenous Bmi-1 expression enhances tumor formation in a murine model of oral squamous cell carcinoma

ABSTRACTB Cell-Specific Moloney Murine Leukemia Virus Integration Site 1 (Bmi-1, Bmi1), an epigenetic protein, is necessary for normal stem cell self-renewal in adult animals and for cancer stem cell (CSC) functions in adult animals. To elucidate the functions of Bmi-1 in the oral cavity we created a transgenic mouse line (KrTBmi-1) that expresses ectopic, Flag-tagged Bmi-1 in tongue basal epithelial stem cells only upon doxycycline (DOX) treatment. Genome wide transcriptomics and Ingenuity Pathway Analysis identified several pathways altered by exogenous Bmi-1 expression in the normal tongue epithelium, including EIF2 signaling (P value = 1.58 x 10−49), mTOR signaling (P value = 2.45 x 10−12), oxidative phosphorylation (P = 6.61 x 10−3) and glutathione redox reactions I (P = 1.74 x 10−2). Overall, our data indicate that ectopic Bmi-1 expression has an impact on normal tongue epithelial homeostasis. We then assessed the KrTBmi-1 mice in the 4-nitroquinoline 1-oxide (4-NQO) model of oral carcinogenesis. We found that 80% of mice expressing exogenous Bmi-1 (+DOX, +4-NQO KrTBmi-1; N = 10) developed tumors classified as grade 3 or higher, compared to 60% and 40% of mice expressing just endogenous Bmi-1 (+DOX, +4-NQO Kr and -DOX, +4-NQO KrTBmi-1 groups, respectively; N = 10/group; P value = <0.0001); and 30% of mice expressing ectopic Bmi-1 mice developed 20 or more lesions compared to 10% of mice expressing only endogenous Bmi-1 (P = .009). This demonstrates that exogenous Bmi-1 expression increases the susceptibility of mice to 4-NQO-induced oral carcinogenesis, strengthening the evidence for Bmi-1 as a therapeutic target in human oral squamous cell carcinoma.

Targeting Interleukin-11 Receptor-α Impairs Human Endometrial Cancer Cell Proliferation and Invasion In Vitro and Reduces Tumor Growth and Metastasis In Vivo.

Endometrial cancer contributes to significant morbidity and mortality in women with advanced stage or recurrent disease. IL11 is a cytokine that regulates cell cycle, invasion, and migration, all hallmarks of cancer. IL11 is elevated in endometrial tumors and uterine lavage fluid in women with endometrial cancer, and alters endometrial epithelial cancer cell adhesion and migration in vitro, but its role in endometrial tumorigenesis in vivo is unknown. We injected mice subcutaneously with human-derived Ishikawa or HEC1A endometrial epithelial cancer cells (ectopic), or HEC1A cells into the uterus (orthotopic) to develop endometrial cancer mouse models. Administration of anti-human IL11 receptor (R) α blocking antibody dramatically reduced HEC1A-derived tumor growth in both models and reduced peritoneal metastatic lesion spread in the orthotopic model, compared with IgG. Anti-human IL11Rα retained a well-differentiated, endometrial epithelial phenotype in the HEC1A ectopic mice, suggesting it prevented epithelial-to-mesenchymal transition. Blockade of mouse IL11Rα with anti-mouse IL11Rα antibody did not alter tumor growth, suggesting that cancer epithelial cell IL11 signaling is required for tumor progression. In vitro, anti-human IL11Rα antibody significantly reduced Ishikawa and HEC1A cell proliferation and invasion and promoted apoptosis. Anti-human, but not anti-mouse, IL11Rα antibody reduced STAT3, but not ERK, activation in HEC1A cells in vitro and in endometrial tumors in xenograft mice. We demonstrated that targeted blockade of endometrial cancer epithelial cell IL11 signaling reduced primary tumor growth and impaired metastasis in ectopic and orthotopic endometrial cancer models in vivo Our data suggest that therapeutically targeting IL11Rα could inhibit endometrial cancer growth and dissemination. Mol Cancer Ther; 15(4); 720-30. ©2016 AACR.

Evaluation of a monitored multiplate bioreactor for large-scale expansion of human periosteum derived stem cells for bone tissue engineering applications

In light of the large-scale expansion of human periosteum derived stem cells for the treatment of large bone defects, a multiplate bioreactor system (Pall Integrity Xpansion) in combination with an integrated holographic imaging platform (Ovizio iLine S microscope) was evaluated. The culture process was quantitatively characterized by imaging data, metabolite concentrations and a breakdown of the cell recovery fractions. The resulting cell quality was assessed based on the minimal criteria for mesenchymal stem cells, including viability on cell culture plastic, identity markers and tri-lineage differentiation potential. Additionally, an in vivo bone forming potency assay was used in an ectopic mice model that resulted in compelling bone formation (11.6±3.1% and 12.8±3.3% for the bioreactor and control tissue culture flask condition, respectively). Therefore, it was shown that the bioreactor is able to produce large quantities of cells, while maintaining satisfactory cell quality.

Estrogen Represses Hepatocellular Carcinoma (HCC) Growth via Inhibiting Alternative Activation of Tumor-associated Macrophages (TAMs)

Hepatocarcinoma cancer (HCC) occurs more often in men than in women, and little is known about its underlying molecular mechanisms. We identify that 17β-estradiol (E2) could suppress tumor growth via regulating the polarization of macrophages. Estrogen functions as a suppressor for macrophage alternative activation. These studies introduce a novel mechanism for suppressing male-predominant HCC. Hepatocarcinoma cancer (HCC), one of the most malignant cancers, occurs significantly more often in men than in women; however, little is known about its underlying molecular mechanisms. Here we identified that 17β-estradiol (E2) could suppress tumor growth via regulating the polarization of macrophages. We showed that E2 re-administration reduced tumor growth in orthotopic and ectopic mice HCC models. E2 functioned as a suppressor for macrophage alternative activation and tumor progression by keeping estrogen receptor β (ERβ) away from interacting with ATP5J (also known as ATPase-coupling factor 6), a part of ATPase, thus inhibiting the JAK1-STAT6 signaling pathway. These studies introduce a novel mechanism for suppressing male-predominant HCC.

Spatial and nonspatial Morris maze learning: impaired behavioral flexibility in mice with ectopias located in the prefrontal cortex

About half of BXSB/MpJ- Yaa (BXSB) mice have neocortical ectopias (misplaced clusters of neurons located in layer I of cortex). Previous behavioral studies have suggested that ectopic mice have superior spatial, but equivalent nonspatial, reference memory learning. However, since spatial and nonspatial learning were not assessed in the same apparatus and with the same testing procedure, it is unclear if this conclusion is accurate. We have created a new nonspatial Morris maze for mice that differs from the spatial task only in the type of cues that must be utilized to efficiently locate the platform (intra-maze black/white patterns vs. extra-maze room cues) and does not differ in the level of task complexity or the presence of objects within the maze. Ectopic mice were very good in utilizing extra-maze cues when learning the spatial version and in utilizing intra-maze cues when learning the nonspatial version of the Morris maze, while non-ectopics were not, suggesting that ectopics have superior spatial and nonspatial reference memory. Ectopias in BXSB mice are usually located in prefrontal and/or motor cortex. The prefrontal cortex is involved in behavioral flexibility (e.g. being able to easily switch from using spatial to nonspatial cues). Only ectopic mice with ectopias specifically located in the prefrontal region of cortex demonstrated difficulty switching from using extra-maze to intra-maze cues and vice versa. Thus, the presence of one or more ectopias in the prefrontal region of cortex disrupted one of the normal functions of the prefrontal cortex.

Effects of ectopias and their cortical location on several measures of learning in BXSB mice.

About half of BXSB/MpJ-Yaa mice have ectopias, which are misplaced clusters of neurons located in layer I of cortex. This study replicated several previous findings showing that there are learning differences between mice with ectopias and those without. In addition, we had sufficient numbers of ectopic mice to investigate if ectopics learned differently depending on the cortical location of the ectopia(s). Mice with at least one ectopia located in prefrontal cortex were initially impaired in learning the Morris maze, as well as relearning the Lashley maze when it was inverted, but learned better in the radial-arm maze when compared to ectopic mice with ectopias located in nonprefrontal regions of cortex. Mice with at least one ectopia in motor cortex learned the Lashley maze better than mice with ectopias located outside motor cortex. In sum, the cortical location of the ectopia(s) affected learning performance in certain tasks within the ectopic group, but regardless of the cortical location of the ectopia(s), ectopics still learned differently than nonectopics in several tasks.

Working memory deficits in BXSB mice with neocortical ectopias

Approximately 40–60% of BXSB/MpJ-Yaa mice exhibit neocortical ectopias, which are misplaced clusters of neurons in layer I of cortex. These ectopias are usually located in the prefrontal and/or motor region of cortex in BXSB mice, and are similar in appearance to those found in postmortem analyses of the brains of dyslexic humans. Several within-strain learning differences between mice with ectopias and those without have been reported. In particular, ectopic BXSB mice exhibited superior reference memory learning, but inferior working memory learning in several studies from our laboratory. This study used the Morris water maze delayed matching-to sample task and the water radial-arm maze to asses working memory in female BXSB mice with and without ectopias. In the delayed matching-to sample task, a hidden escape platform remained in a constant position for each four-trial session, but changed position between sessions. Trial 2 was the measure of working memory, i.e., how well did the mouse remember where the platform was located for that session. In the water version of the eight-arm radial maze, hidden escape platforms were located in four of the eight arms, and each platform was removed from the maze once found. This enabled us to assess working and reference memory simultaneously. Ectopic mice demonstrated working memory deficits during the first part of the delayed matching-to sample task compared to nonectopics. Similarly, ectopics made more working memory errors during the latter half of radial-arm maze testing, while not differing from nonectopics in reference memory performance. Additionally, there were significant correlations between measures of working memory in the radial-arm maze and working memory in a delayed matching-to-sample task. These findings are in agreement with other studies demonstrating working memory deficits in ectopic BXSB mice.

Non-spatial water radial-arm maze learning in mice

Recently, we published a method for examining working and reference memory in mice using a spatial version of the water radial-arm maze. Here we describe a non-spatial version of the same maze. BXSB mice were able to learn the maze as shown by the decrease in the number of working and reference memory errors over sessions. This maze was used to examine learning differences between males and females and between mice with misplaced clusters of neurons in layer I of cortex (ectopias) and those without. In a prior study using the spatial version of the water radial-arm maze, male BXSB mice had poorer working memory than females during the acquisition phase. Similarly, in this study male BXSB mice demonstrated impaired working memory during the asymptotic phase of non-spatial radial-arm maze learning. Two prior studies showed that mice with neocortical ectopias demonstrated working memory impairments compared to non-ectopic littermates in the spatial version of the water radial-arm maze. Contrary to this, in the non-spatial radial-arm maze used here, ectopic mice were not impaired in working memory and showed better memory when the working memory ‘load’ was the highest. Overall, both versions of the maze can be useful tools to assess spatial and non-spatial working and reference memory in mice.

Perceptual auditory gap detection deficits in male BXSB mice with cerebrocortical ectopias.

Underlying impairments in rapid auditory processing may contribute to disrupted phonological processing, which in turn characterizes developmental language impairment (LI). Identification of a neurobiological feature of LI that is associated with auditory deficits would further support this model. Accordingly, we found that adult male rats with induced cortical malformations were impaired in rapid auditory processing. Since 40-60% of BXSB mice exhibit spontaneous focal cerebrocortical ectopias (as seen in dyslexics brains), we assessed auditory gap detection in adult male BXSB mice. Ectopic mice were significantly worse than non-ectopics in detecting a 5 ms silent gap, but were not significantly impaired at longer gap durations (10-100 ms). Our results confirm that focal cortical malformations are associated with impairments in rapid auditory processing.

Effects of neocortical ectopias upon the acquisition and retention of a non-spatial reference memory task in BXSB mice

BXSB mice have a 40–60% incidence of small, focal, neocortical dysplasias called ectopias. Ectopics and non-ectopics were compared on horizontal–vertical discrimination learning, a non-spatial cognitive task. No differences were found during acquisition. Ectopic mice, however, were superior when retested 8 weeks later. The results support prior findings that ectopic mice have better long-term spatial memory and extend this conclusion to the non-spatial domain.

Effects of cortical ectopias on spatial delayed-matching-to-sample performance in BXSB mice

BXSB mice have small neocortical anomalies (ectopic collections of neurons in layer I), with an incidence of about 40–60%. Previous studies have found that ectopic mice from this strain are faster than non-ectopcs in learning the Morris water maze (reference memory), but have poorer working memory for spatial learning. The current study continues the investigation of working memory by testing ectopic and non-ectopic BXSB mice on a spatial delayed-matching-to-sample test (S-DMTS; also called spatial learning sets or ‘working memory’water maze). In this test, the mice must find a submerged platform in a pool of water. The platform changes location with every problem, or block of four trials. The subject has ‘matched to sample’ if it locates the platform in less time on the second trial of each problem than it did on the first. Of 33 subjects, 8 had cortical ectopias, one had a small neuron-free gliotic area, and 24 were normal. The normal subjects showed a decrease in time to escape over the first 2 trials of the first 5 problems, while the ectopic subjects did not show a decrease until the third trial, indicating that ectopic mice required more trials to put the platform location into working memory. The site of the ectopias is prefrontal/motor cortex, and we hypothesize that is the cause of the poorer working memory.

Learning and memory in the autoimmune BXSB mouse: effects of neocortical ectopias and environmental enrichment

Approximately 40–60% of BXSB mice have ectopic cell clusters in layer I of neocortex. Prior studies have shown distinct behavioral differences between those with ectopias and their non-ectopic littermates. In this study, female BXSB mice were reared after weaning in either enriched environments or standard cages. Following an initial round of behavioral testing, all mice were housed in standard cages and retested. Enriched cage mice (both ectopic and non-ectopic) showed increased activity, greater speed, and enhanced learning scores across a variety of tests. Additionally, prior test experience itself had significant positive effects on Hebb-Williams maze learning. The presence of ectopias resulted in better Morris maze learning for standard cage reared mice. Further, ectopic mice, regardless of their housing condition, showed better long-term retention in the Morris maze than did their non-ectopic counterparts. These findings show that abnormalities in corticogenesis need not always result in functional deficit.

Learning and memory in the autoimmune BXSB mouse: effects of neocortical ectopias and environmental enrichment

Approximately 40–60% of BXSB mice have ectopic cell clusters in layer I of neocortex. Prior studies have shown distinct behavioral differences between those with ectopias and their non-ectopic littermates. In this study, female BXSB mice were reared after weaning in either enriched environments or standard cages. Following an initial round of behavioral testing, all mice were housed in standard cages and retested. Enriched cage mice (both ectopic and non-ectopic) showed increased activity, greater speed, and enhanced learning scores across a variety of tests. Additionally, prior test experience itself had significant positive effects on Hebb-Williams maze learning. The presence of ectopias resulted in better Morris maze learning for standard cage reared mice. Further, ectopic mice, regardless of their housing condition, showed better long-term retention in the Morris maze than did their non-ectopic counterparts. These findings show that abnormalities in corticogenesis need not always result in functional deficit.

Effects of embryo transfer and cortical ectopias upon the behavior of BXSB-Yaa and BXSB-Yaa + mice

The BXSB-Yaa and BXSB-Yaa + inbred strains of mice differ primarily with respect to the Y chromosome, although there is evidence that they differ on several autosomal genes as well. Each strain has ectopic collections of neurons in neocortical layer I (ectopias), with a higher occurrence in males (58%) than females (42%). Conventionally reared mice from these strains were compared to mice that were transferred, as 8-cell embryos, into the uteri of non-autoimmune recipients, who gave birth to and reared the offspring. The transfer procedure did not change the incidence of ectopias in either sex. There were, however, major differences in behavior. Compared to conventionally reared controls, embryo transfer mice had greater behavioral asymmetry, poorer performance in a black-white discrimination, poorer Morris maze learning, better Lashley maze learning, and better performance in a two-way shuttlebox. Within the transfer groups, females differed as much as males, confirming our prior findings and supporting our thesis that the two strains differ on several autosomal genes in addition to the Y chromosome. These findings show that the intra-uterine environment can powerfully and selectively affect later behavior. When ectopic and non-ectopic mice were compared, BXSB-Yaa mice with neocortical ectopias were better able to learn the Morris spatial maze than non-ectopic controls; this was true whether the mice were conventionally reared or embryo transferred. In contrast, BXSB-Yaa + ectopic mice did not differ from their controls if conventionally reared, but were much worse than controls if embryo transferred.

Neocortical ectopias in BXSB mice: effects upon reference and working memory systems.

BXSB mice have an approximately 40-60% incidence of neocortical ectopias in layer I of the prefrontal/motor cortex. Prior studies have found major behavioral differences between those with ectopias and their non-ectopic littermates. Some of these findings indicate that the two groups differ with respect to spatial reference and working memory. The purpose of this study was to measure reference and working memory in the same animals to test the hypothesis that the ectopics would have better reference memory but less effective working memory. The Lashley III maze has cul-de-sacs which must be eliminated, and T-choices where the animal has to decide whether to go left or right. Ectopic and non-ectopic mice were equally able to learn the maze and did not differ on cul-entry or T-choice errors. Then the maze was inverted and the animals were retested. Turning the maze upside down did not change the relative status of the blind alleys. Therefore, the reference memory knowledge from the prior week's training could be used to avoid entering the culs. However, inverting the maze caused a left-right mirror image reversal of the T-choices. Therefore, prior reference memory information would interfere with learning the new path through the maze, whereas working memory would enable the mouse to eliminate T-choice errors. Ectopic mice made less cul-entry errors and more T-choice errors than their non-ectopic littermates, as predicted.

Environmental enrichment, neocortical ectopias, and behavior in the autoimmune NZB mouse

New Zealand Black (NZB) mice have severe autoimmune disease and approximately 40% have cortical ectopias in layer I of sensorimotor cortex. Because the ectopias are similar to those found in dyslexics, NZB mice have been used as an animal model for developmental learning disorders. In addition, these mice have been used as a model of learning deficits associated with autoimmune disease. To determine whether early intervention would affect learning processes in NZB mice, they were reared after weaning in standard cages or enriched environments. They were given a battery of behavioral tests to measure learning, laterality, and activity, after which they were sacrificed and their brains examined for cortical ectopias. The tests sorted into two behavioral sets. Ectopia-associated behaviors included black-white discrimination learning and the Morris spatial maze. As a group, the mice performed well on these tasks. Ectopic mice had poorer performance than non-ectopics on these measures, and environmental enrichment countered the effects of the ectopias. Autoimmune-associated behavior involved two-way avoidance learning in a shuttlebox. Mice were uniformely poor on this task, ectopias did not affect behavior, and environmental enrichment was without benefit. Evidence from this and other studies shows that poor shuttlebox performance is related to the presence of autoimmune disease. Thus, autoimmune disease and cortical ectopias each appear to affect a separate set of behavioral processes. Environmental enrichment is most effective for behavioral impairments mediated via cortical ectopias, but is much less effective, if at all, if autoimmunity is the primary mediator of the impairments.

Spatial learning, discrimination learning, paw preference and neocortical ectopias in two autoimmune strains of mice

NZB and BXSB mice were given a battery of behavioral tests including paw preference, water escape, Lashley III maze, and discrimination learning. Their brains were then evaluated for cortical ectopias. The incidence of ectopias was 40.5% in NZBs and 48.5% in BXSBs. In the NZB strain left-pawed ectopic mice (both male and female) had the fastest swimming time in the water escape test, while right-pawed ectopics were the slowest. The same findings were obtained for left- and right-pawed ectopic BXSB males, but not for the females. However, on discrimination learning the BXSB males had the exact opposite pattern: right-pawed ectopics were the best learners while left-pawed ectopics were the worst. Male BXSBs and both male and female NZBs were manifesting autoimmune disease at the time of testing, while female BXSBs were not, suggesting that autoimmunity is a necessary background condition for the differential expression of ectopias and paw preference upon learning processes. The finding that the left-pawed ectopic BXSB mice, who were the poorest learners in the non-spatial discrimination learning test, learned best in the spatial water escape test in agreement with the Geschwind hypothesis that pathological events during brain development may, in some instances, produce superiority of function.