Identification of Molecular Distinctions Between Normal Breast-Associated Fibroblasts and Breast Cancer-Associated Fibroblasts

Stromal fibroblasts influence the behavior of breast epithelial cells. Fibroblasts derived from normal breast (NAF) inhibit epithelial growth, whereas fibroblasts from breast carcinomas (CAF) have less growth inhibitory capacity and can promote epithelial growth. We sought to identify molecules that are differentially expressed in NAF versus CAF and potentially responsible for their different growth regulatory abilities. To determine the contribution of soluble molecules to fibroblast–epithelial interactions, NAF were grown in 3D, transwell or direct contact co-cultures with MCF10AT epithelial cells. NAF suppressed proliferation of MCF10AT in both direct contact and transwell co-cultures, but this suppression was significantly greater in direct co-cultures, indicating involvement of both soluble and contact factors. Gene expression profiling of early passage fibroblast cultures identified 420 genes that were differentially expressed in NAF versus CAF. Of the eight genes selected for validation by real-time PCR, FIBULIN 1, was overexpressed in NAF, and DICKKOPF 1, NEUREGULIN 1, PLASMINOGEN ACTIVATOR INHIBITOR 2, and TISSUE PLASMINOGEN ACTIVATOR were overexpressed in CAF. A higher expression of FIBULIN 1 in normal- than cancer-associated fibroblastic stroma was confirmed by immunohistochemistry of breast tissues. Among breast cancers, stromal expression of Fibulin 1 protein was higher in estrogen receptor α-positive cancers and low stromal expression of Fibulin 1 correlated with a higher proliferation of cancer epithelial cells. In conclusion, expression profiling of NAF and CAF cultures identified many genes with potential relevance to fibroblast–epithelial interactions in breast cancer. Furthermore, these early passage fibroblast cultures can be representative of gene expression in stromal fibroblasts in vivo

Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture

BACKGROUND: Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed. METHODS: NAF and CAF were grown with the nontumorigenic MCF10A epithelial cells and their more transformed, tumorigenic derivative, MCF10AT cells, in direct three-dimensional co-cultures on basement membrane material. The proliferation and apoptosis of MCF10A cells and MCF10AT cells were assessed by 5-bromo-2'-deoxyuridine labeling and TUNEL assay, respectively. Additionally, NAF and CAF were compared for expression of insulin-like growth factor II as a potential mediator of their effects on epithelial cell growth, by ELISA and by quantitative, real-time PCR. RESULTS: In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells. However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells. The degree of growth inhibition varied among NAF or CAF from different individuals. In greater numbers, NAF and CAF have less inhibitory effect on epithelial cell growth. The rate of epithelial cell apoptosis was not affected by NAF or CAF. Mean insulin-like growth factor II levels were not significantly different in NAF versus CAF and did not correlate with the fibroblast effect on epithelial cell proliferation. CONCLUSION: Both NAF and CAF have the ability to inhibit the growth of pre-cancerous breast epithelial cells. NAF have greater inhibitory capacity than CAF, suggesting that the ability of fibroblasts to inhibit epithelial cell proliferation is lost during breast carcinogenesis. Furthermore, as the degree of transformation of the epithelial cells increased they became resistant to the growth-inhibitory effects of CAF. Insulin-like growth factor II could not be implicated as a contributor to this differential effect of NAF and CAF on epithelial cell growth

Reduced conditioned fear response in mice that lack Dlx1 and show subtype-specific loss of interneurons

The inhibitory GABAergic system has been implicated in multiple neuropsychiatric diseases such as schizophrenia and autism. The Dlx homeobox transcription factor family is essential for development and function of GABAergic interneurons. Mice lacking the Dlx1 gene have postnatal subtype-specific loss of interneurons and reduced IPSCs in their cortex and hippocampus. To ascertain consequences of these changes in the GABAergic system, we performed a battery of behavioral assays on the Dlx1 mutant mice, including zero maze, open field, locomotor activity, food intake, rotarod, tail suspension, fear conditioning assays (context and trace), prepulse inhibition, and working memory related tasks (spontaneous alteration task and spatial working memory task). Dlx1 mutant mice displayed elevated activity levels in open field, locomotor activity, and tail suspension tests. These mice also showed deficits in contextual and trace fear conditioning, and possibly in prepulse inhibition. Their learning deficits were not global, as the mutant mice did not differ from the wild-type controls in tests of working memory. Our findings demonstrate a critical role for the Dlx1 gene, and likely the subclasses of interneurons that are affected by the lack of this gene, in behavioral inhibition and associative fear learning. These observations support the involvement of particular components of the GABAergic system in specific behavioral phenotypes related to complex neuropsychiatric diseases

Computerized assessment of social approach behavior in mouse

Altered sociability is a core feature of a variety of human neurological disorders, including autism. Social behaviors may be tested in animal models, such as mice, to study the biological bases of sociability and how this is altered in neurodevelopmental disorders. An easily quantifiable social behavior frequently used to assess sociability in the mouse is the tendency to approach and interact with an unfamiliar mouse. Here we present a novel computer-assisted method for scoring social approach behavior in mice using a three-chambered apparatus. We find consistent results between data scored using the computer assisted method and a human observer, making computerized assessment a reliable, low cost, high-throughput method for testing sociability

Computerized Assessment of Social Approach Behavior in Mouse

Altered sociability is a core feature of a variety of human neurological disorders, including autism. Social behaviors may be tested in animal models, such as mice, to study the biological basis of sociability and how this is altered in neurodevelopmental disorders. A quantifiable social behavior frequently used to assess sociability in the mouse is the tendency to approach and interact with an unfamiliar mouse. Here we present a novel computer-assisted method for scoring social approach behavior in mice using a three-chambered apparatus and freely available software. We find consistent results between data scored using the computer-assisted method and a human observer, making computerized assessment a reliable, low cost, high-throughput method for testing sociability.Nancy Lurie Marks Family FoundationSimons FoundationAutism Consortiu

Social Behavioral Deficits Coincide with the Onset of Seizure Susceptibility in Mice Lacking Serotonin Receptor 2c.

The development of social behavior is strongly influenced by the serotonin system. Serotonin 2c receptor (5-HT2cR) is particularly interesting in this context considering that pharmacological modulation of 5-HT2cR activity alters social interaction in adult rodents. However, the role of 5-HT2cR in the development of social behavior is unexplored. Here we address this using Htr2c knockout mice, which lack 5-HT2cR. We found that these animals exhibit social behavior deficits as adults but not as juveniles. Moreover, we found that the age of onset of these deficits displays similar timing as the onset of susceptibility to spontaneous death and audiogenic-seizures, consistent with the hypothesis that imbalanced excitation and inhibition (E/I) may contribute to social behavioral deficits. Given that autism spectrum disorder (ASD) features social behavioral deficits and is often co-morbid with epilepsy, and given that 5-HT2cR physically interacts with Pten, we tested whether a second site mutation in the ASD risk gene Pten can modify these phenotypes. The age of spontaneous death is accelerated in mice double mutant for Pten and Htr2c relative to single mutants. We hypothesized that pharmacological antagonism of 5-HT2cR activity in adult animals, which does not cause seizures, might modify social behavioral deficits in Pten haploinsufficient mice. SB 242084, a 5-HT2cR selective antagonist, can reverse the social behavior deficits observed in Pten haploinsufficient mice. Together, these results elucidate a role of 5-HT2cR in the modulation of social behavior and seizure susceptibility in the context of normal development and Pten haploinsufficiency