Breast Tumor Metastasis and Its Microenvironment: It Takes Both Seed and Soil to Grow a Tumor and Target It for Treatment.

Metastasis remains a major challenge in treating breast cancer. Breast tumors metastasize to organ-specific locations such as the brain, lungs, and bone, but why some organs are favored over others remains unclear. Breast tumors also show heterogeneity, plasticity, and distinct microenvironments. This contributes to treatment failure and relapse. The interaction of breast cancer cells with their metastatic microenvironment has led to the concept that primary breast cancer cells act as seeds, whereas the metastatic tissue microenvironment (TME) is the soil. Improving our understanding of this interaction could lead to better treatment strategies for metastatic breast cancer. Targeted treatments for different subtypes of breast cancers have improved overall patient survival, even with metastasis. However, these targeted treatments are based upon the biology of the primary tumor and often these patients' relapse, after therapy, with metastatic tumors. The advent of immunotherapy allowed the immune system to target metastatic tumors. Unfortunately, immunotherapy has not been as effective in metastatic breast cancer relative to other cancers with metastases, such as melanoma. This review will describe the heterogeneic nature of breast cancer cells and their microenvironments. The distinct properties of metastatic breast cancer cells and their microenvironments that allow interactions, especially in bone and brain metastasis, will also be described. Finally, we will review immunotherapy approaches to treat metastatic breast tumors and discuss future therapeutic approaches to improve treatments for metastatic breast cancer.

Paracrine signalling between keratinocytes and SVF cells results in a new secreted cytokine profile during wound closure.

Stromal vascular fraction (SVF) cells, and the adipose-derived mesenchymal stem cells they contain, have shown enhanced wound healing in vitro and in vivo, yet their clinical application has been limited. In this regard, understanding the mechanisms that govern SVF-enhanced wound healing would improve their application in the clinic. Here, we show that the SVF cells and keratinocytes engage in a paracrine crosstalk during wound closure, which results in a new cytokine profile that is distinct from the cytokines regularly secreted by either cell type on their own. We identify 11 cytokines, 5 of which are not regularly secreted by the SVF cells, whose expressions are significantly increased during wound closure by the keratinocytes. This new cytokine profile could be used to accelerate wound closure and initiate re-epithelialization without the need to obtain the SVF cells from the patient.

A Robust and Standardized Approach to Quantify Wound Closure Using the Scratch Assay.

The scratch assay is an in vitro assay that allows for high-throughput quantification of wound closure by keratinocytes and fibroblasts with relative ease. However, this assay is amenable to experimental variables, which can result in false-positive and false-negative data, making the interpretation of such data difficult. Also, data variability decreases the sensitivity of the scratch assay. Here, we identify important sources of data variation in the scratch assay and provide rational mitigation strategies that enable robust and highly reproducible quantification of scratch width and area, and ultimately the scratch closure rates. By eliminating these sources of variability, the sensitivity of the scratch assay is enhanced, thereby allowing for identification of dependent variables with wide-ranging impacts on wound closure in a robust and standardized manner.

Role of the Microenvironment in Regulating Normal and Cancer Stem Cell Activity: Implications for Breast Cancer Progression and Therapy Response.

The epithelial cells in an adult woman's breast tissue are continuously replaced throughout their reproductive life during pregnancy and estrus cycles. Such extensive epithelial cell turnover is governed by the primitive mammary stem cells (MaSCs) that proliferate and differentiate into bipotential and lineage-restricted progenitors that ultimately generate the mature breast epithelial cells. These cellular processes are orchestrated by tightly-regulated paracrine signals and crosstalk between breast epithelial cells and their tissue microenvironment. However, current evidence suggests that alterations to the communication between MaSCs, epithelial progenitors and their microenvironment plays an important role in breast carcinogenesis. In this article, we review the current knowledge regarding the role of the breast tissue microenvironment in regulating the special functions of normal and cancer stem cells. Understanding the crosstalk between MaSCs and their microenvironment will provide new insights into how an altered breast tissue microenvironment could contribute to breast cancer development, progression and therapy response and the implications of this for the development of novel therapeutic strategies to target cancer stem cells.

Paracrine Crosstalk between Fibroblasts and ER+ Breast Cancer Cells Creates an IL1ß-Enriched Niche that Promotes Tumor Growth.

Breast cancer-induced activated fibroblasts support tumor progression. However, the role of normal fibroblasts in tumor progression remains controversial. In this study, we used modified patient-derived organoid cultures and demonstrate that constitutively secreted cytokines from normal breast fibroblasts initiate a paracrine signaling mechanism with estrogen receptor-positive (ER+) breast cancer cells, which results in the creation of an interleukin (IL)-1ß-enriched microenvironment. We found that this paracrine signaling mechanism is shared between normal and activated fibroblasts. Interestingly, we observed that in reconstructed tumor microenvironment containing autologous ER+ breast cancer cells, activated fibroblasts, and immune cells, tamoxifen is more effective in reducing tumor cell proliferation when this paracrine signaling is blocked. Our findings then suggest that ER+ tumor cells could create a growth-promoting environment without activating stromal fibroblasts and that in breast-conserving surgeries, normal fibroblasts could be a significant modulator of tumor recurrence by enhancing the proliferation of residual breast cancer cells in the tumor-adjacent breast tissue.

Isolation and characterization of a new basal-like luminal progenitor in human breast tissue.

BACKGROUND: Adult stem cells and progenitors are responsible for breast tissue regeneration. Human breast epithelial progenitors are organized in a lineage hierarchy consisting of bipotent progenitors (BPs), myoepithelial- and luminal-restricted progenitors (LRPs) where the LRP differentiation into mature luminal cells requires estrogen receptor (ER) signaling. However, the experimental evidence exploring the relationship between the BPs and LRPs has remained elusive. In this study, we report the presence of a basal-like luminal progenitor (BLP) in human breast epithelial cells. METHODS: Breast reduction samples were used to obtain different subsets of human breast epithelial cell based on cell surface marker expression using flow cytometry. Loss of function and gain of function studies were employed to demonstrate the role of NOTCH3 (NR3)-FRIZZLED7 (FZD7) signaling in luminal cell fate commitment. RESULTS: Our results suggest that, NR3-FZD7 signaling axis was necessary for luminal cell fate commitment. Similar to LRPs, BLPs (NR3highFZD7highCD90+MUC1-ER-) differentiate to generate NR3medFZD7medCD90-MUC1+ER+ luminal cells. Unlike LRPs however, BLP's proliferation and differentiation potentials depend on NR3 and regulated in part by FZD7 signaling. Lastly, we show that BLPs have a higher colony-forming potential than LRPs and that they are continuously generated from the NOTCH3-FZD7low subset of the bipotent progenitors. CONCLUSION: Our data indicate that BPs differentiate to generate basal-like luminal progenitors that in turn differentiate into LRPs. These results provide new insights into the hierarchical organization of human breast epithelial cell and how cooperation between the Notch and Wnt signaling pathways define a new progenitor cell type.

Long Non-Coding RNA H19 Acts as an Estrogen Receptor Modulator that is Required for Endocrine Therapy Resistance in ER+ Breast Cancer Cells.

BACKGROUND/AIMS: Blocking estrogen signaling with endocrine therapies (Tamoxifen or Fulverstrant) is an effective treatment for Estrogen Receptor-α positive (ER+) breast cancer tumours. Unfortunately, development of endocrine therapy resistance (ETR) is a frequent event resulting in disease relapse and decreased overall patient survival. The long noncoding RNA, H19, was previously shown to play a significant role in estrogen-induced proliferation of both normal and malignant ER+ breast epithelial cells. We hypothesized that H19 expression is also important for the proliferation and survival of ETR cells. METHODS: Here we utilized established ETR cell models; the Tamoxifen (Tam)-resistant LCC2 and the Fulvestrant and Tam cross-resistant LCC9 cells. Gain and loss of H19 function were achieved through lentiviral transduction as well as pharmacological inhibitors of the Notch and c-Met receptor signaling pathways. The effects of altered H19 expression on cell viability and ETR were assessed using three-dimensional (3D) organoid cultures and 2D co-cultures with low passage tumour-associated fbroblasts (TAFs). RESULTS: Here we report that treating ETR cells with Tam or Fulvestrant increases H19 expression and that it's decreased expression overcomes resistance to Tam and Fulvestrant in these cells. Interestingly, H19 expression is regulated by Notch and HGF signaling in the ETR cells and pharmacological inhibitors of Notch and c-MET signaling together significantly reverse resistance to Tam and Fulvestrant in an H19-dependent manner in these cells. Lastly, we demonstrate that H19 regulates ERα expression at the transcript and protein levels in the ETR cells and that H19 protects ERα against Fulvestrant-mediated downregulation of ERα protein. We also observed that blocking Notch and the c-MET receptor signaling also overcomes Fulvestrant and Tam resistance in 3D organoid cultures by decreasing ERα and H19 expression in the ETR cells. CONCLUSION: In endocrine therapy resistant breast cancer cells Fulvestrant is ineffective in decreasing ERα levels. Our data suggest that in the ETR cells, H19 expression acts as an ER modulator and that its levels and subsequently ERα levels can be substantially decreased by blocking Notch and c-MET receptor signaling. Consequently, treating ETR cells with these pharmacological inhibitors helps overcome resistance to Fulvestrant and Tamoxifen.

A robust cell culture system for large scale feeder cell-free expansion of human breast epithelial progenitors.

BACKGROUND: Normal human breast epithelial cells are maintained by the proliferation and differentiation of different human breast epithelial progenitors (HBEPs). However, these progenitor subsets can only be obtained at low frequencies, limiting their further characterization. Recently, it was reported that HBEPs can be minimally expanded in Matrigel cocultures with stromal feeder cells. However, variability of generating healthy feeder cells significantly impacts the effective expansion of HBEPs. METHODS: Here, we report a robust feeder cell-free culture system for large-scale expansion of HBEPs in two-dimensional cultures. RESULTS: Using this cell culture system HBEPs can be exponentially expanded as bulk cultures. Moreover, purified HBEP subtypes can also be separately expanded using our cell culture system. The expanded HBEPs retain their undifferentiated phenotype and form distinct epithelial colonies in colony forming cell assays. CONCLUSIONS: The availability of a culture system enabling the large-scale expansion of HBEPs facilitates their application to screening platforms and other cell-based assays.

In vivo evidence supporting a metastasis suppressor role for Stard13 (Dlc2) in ErbB2 (Neu) oncogene induced mouse mammary tumors.

Overexpression of dominant oncogenes and the loss of tumor suppressor genes are basic genetic events in the acquisition of the malignant phenotype. The erb-b2 receptor tyrosine kinase 2 (ERBB-2) proto-oncogene is overexpressed in 20-30% of human breast cancers. The StAR related lipid transfer domain containing 13 gene (STARD13), also known as Deleted in Liver Cancer-2 (DLC-2), maps to chromosome band 13q12.3 and is frequently downregulated in human cancers, including 72% of breast cancers. It encodes a RhoGAP protein with sterile α motif (SAM) and StAR-related lipid transfer (START) domains. The objective of this study was to determine if loss of Stard13 plays a role in mammary tumor progression using transgenic mice expressing the activated ErbB-2 (Neu) oncogene and Cre recombinase (NIC) in mammary epithelium under transcriptional control of the murine mammary tumor virus (MMTV) promoter (MMTV-NIC). These mice were crossed with a conditional Stard13 knockout mouse (floxed exon 3), resulting in simultaneous Neu expression and Stard13 deletion, specifically in the mammary epithelium. We found that loss of Stard13 did not alter tumor growth nor significantly modify overall survival and tumor free survival. However, there was an increase in the total number of lung metastases in the Stard13 heterozygous or homozygous mice compared with the parental MMTV-NIC strain. Altogether our results indicate that Stard13 acts as a metastasis suppressor rather than a tumor suppressor gene, in Neu oncogene induced mammary tumorigenesis.

Breast Cancers Activate Stromal Fibroblast-Induced Suppression of Progenitors in Adjacent Normal Tissue.

Human breast cancer cells are known to activate adjacent "normal-like" cells to enhance their own growth, but the cellular and molecular mechanisms involved are poorly understood. We now show by both phenotypic and functional measurements that normal human mammary progenitor cells are significantly under-represented in the mammary epithelium of patients' tumor-adjacent tissue (TAT). Interestingly, fibroblasts isolated from TAT samples showed a reduced ability to support normal EGF-stimulated mammary progenitor cell proliferation in vitro via their increased secretion of transforming growth factor ß. In contrast, TAT fibroblasts promoted the proliferation of human breast cancer cells when these were co-transplanted in immunodeficient mice. The discovery of a common stromal cell-mediated mechanism that has opposing growth-suppressive and promoting effects on normal and malignant human breast cells and also extends well beyond currently examined surgical margins has important implications for disease recurrence and its prevention.

Population-based analysis of breast cancer treatment by intrinsic sub-type in Manitoba, Canada.

BACKGROUND: Few descriptive epidemiological studies on the incidence, treatment and survival can accurately reflect a whole population. Manitoba, Canada has an accurate cancer registry, a drug information program network and a breast screening program since 1995. This combined with a stable population provides true population data that can accurately describe the region. METHODS: Using a retrospective cohort design all Breast Cancer cases were obtained from 2004-2010 (N=5399) and grouped by Intrinsic sub-type. Identifiable co-morbidities, prescribed endocrine therapy, staging, surgery, treatment and overall and disease-free survival by intrinsic sub-type were evaluated. RESULTS: Prevalence of Luminal A (41.7%), Luminal B HER2- (15.6%), Luminal B HER2+ (8.9%), Basal-like(10.8%), and HER2+ non-luminal (5.1%) were consistent with other descriptive studies in Canada and Spain. Over this time period the number of lumpectomies increased 1.7% per year (P=0.007). There was a steady increase of 3.4% per year in the use of aromatase inhibitors (P=0.005). Pre-menopausal patients had an increased proportion of HER2+ and Basal-like sub-types. The 7year overall/disease-free survival percentages for Luminal A, Luminal B HER2-, Luminal B HER2+, Basal-like, and HER2+ non-luminal were 88.7%/83.6, 78.2%/73.0, 81.5%/73.3%, 67.7%/63.2%, 70.4%/65.6% respectively. CONCLUSIONS: Reasons for variability in the prevalence of intrinsic sub-type by region is not fully understood. Manitoba is unique due the stability of the population, completeness of the registry and length of breast cancer screening program. Few true population-based studies grouped by intrinsic sub-type are available. IMPACT: Descriptive epidemiological studies guide future research by identifying factors that can affect treatment, recurrence, and survival.

Notch-Induced Expression of FZD7 Requires Noncanonical NOTCH3 Signaling in Human Breast Epithelial Cells.

The evolutionarily conserved Notch and Wnt signaling pathways have demonstrated roles in normal mammary gland development and in breast carcinogenesis. We previously reported that in human mammary gland, signaling through NOTCH3 alone regulates the commitment of the undifferentiated bipotential progenitors to the luminal cell fate, indicating that NOTCH3 may regulate the expression of unique genes apart from the other Notch receptors. In this study, we used gain of function and loss of function experiments and found that a Wnt signaling receptor, Frizzled7 (FZD7), is a unique and nonredundant target of NOTCH3 in human breast epithelial cells. Interestingly, neither the constitutively active forms of NOTCH1-2, 4 nor loss of expression of these receptors were able to alter expression of FZD7 in human breast epithelial cells. We further show that FZD7-expressing cells are found more frequently in the luminal progenitor-enriched subpopulation of cells obtained from breast reduction samples compared with the undifferentiated bipotent progenitors. Also, we show that NOTCH3-induced expression of FZD7 occurs in the absence of CSL (CBF1-Suppressor of Hairless-Lag-1). Our data suggest that noncanonical Notch signaling through NOTCH3 could modulate Wnt signaling via FZD7 and in this way, might be involved in luminal cell differentiation.

The Deleted in Liver Cancer 1 (Dlc1) tumor suppressor is haploinsufficient for mammary gland development and epithelial cell polarity.

BACKGROUND: Deleted in Liver Cancer 1 (Dlc1) is a tumor suppressor gene, which maps to human chromosome 8p21-22 and is found frequently deleted in many cancers including breast cancer. The promoter of the remaining allele is often found methylated. The Dlc1 gene encodes a RhoGAP protein that regulates cell proliferation, migration and inhibits cell growth and invasion when restored in Dlc1 deficient tumor cell lines. This study focuses on determining the role of Dlc1 in normal mammary gland development and epithelial cell polarity in a Dlc1 gene trapped (gt) mouse. METHODS: Mammary gland whole mount preparations from 10-week virgin heterozygous Dlc1(gt/+) gene-trapped mice were compared with age-matched wild type (WT) controls. Hematoxylin-Eosin (H&E) and Masson's Trichrome staining of histological sections were carried out. Mammary glands from Dlc1(gt/+) mice and WT controls were enzymatically digested with collagenase and dispase and then cultured overnight to deplete hematopoietic and endothelial cells. The single cell suspensions were then cultured in Matrigel for 12 days. To knockdown Dlc1 expression, primary WT mammary epithelial cells were infected with short hairpin (sh) RNA expressing lentivirus or with a scrambled shRNA control. RESULTS: Dlc1(gt/+) mice showed anomalies in the mammary gland that included increased ductal branching and deformities in terminal end buds and branch points. Compared to the WT controls, Masson's Trichrome staining showed a thickened stromal layer with increased collagen deposition in mammary glands from Dlc1(gt/+) mice. Dlc1(gt/+) primary mammary epithelial cells formed increased solid acinar spheres in contrast with WT and scrambled shRNA control cells, which mostly formed hollow acinar structures when plated in 3D Matrigel cultures. These solid acinar structures were similar to the acinar structures formed when Dlc1 gene expression was knocked down in WT mammary cells by shRNA lentiviral transduction. The solid acinar structures were not due to a defect in apoptosis as determined by a lack of detectible cleaved caspase 3 antibody staining. Primary mammary cells from Dlc1(gt/+) mice showed increased RhoA activity compared with WT cells. CONCLUSIONS: The results illustrate that decreased Dlc1 expression can disrupt the normal cell polarization and mammary ductal branching. Altogether this study suggests that Dlc1 plays a role in maintaining normal mammary epithelial cell polarity and that Dlc1 is haploinsufficient.

Adipose-Derived Stromal Vascular Fraction Differentially Expands Breast Progenitors in Tissue Adjacent to Tumors Compared to Healthy Breast Tissue.

BACKGROUND: Autologous fat grafts supplemented with adipose-derived stromal vascular fraction are used in reconstructive and cosmetic breast procedures. Stromal vascular fraction contains adipose-derived stem cells that are thought to encourage wound healing, tissue regeneration, and graft retention. Although use of stromal vascular fraction has provided exciting perspectives for aesthetic procedures, no studies have yet been conducted to determine whether its cells contribute to breast tissue regeneration. The authors examined the effect of these cells on the expansion of human breast epithelial progenitors. METHODS: From patients undergoing reconstructive breast surgery following mastectomies, abdominal fat, matching tissue adjacent to breast tumors, and the contralateral non-tumor-containing breast tissue were obtained. Ex vivo co-cultures using breast epithelial cells and the stromal vascular fraction cells were used to study the expansion potential of breast progenitors. Breast reduction samples were collected as a source of healthy breast cells. RESULTS: The authors observed that progenitors present in healthy breast tissue or contralateral non-tumor-containing breast tissue showed significant and robust expansion in the presence of stromal vascular fraction (5.2- and 4.8-fold, respectively). Whereas the healthy progenitors expanded up to 3-fold without the stromal vascular fraction cells, the expansion of tissue adjacent to breast tumor progenitors required the presence of stromal vascular fraction cells, leading to a 7-fold expansion, which was significantly higher than the expansion of healthy progenitors with stromal vascular fraction. CONCLUSIONS: The use of stromal vascular fraction might be more beneficial to reconstructive operations following mastectomies compared with cosmetic corrections of the healthy breast. Future studies are required to examine the potential risk factors associated with its use. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Estrogen regulates luminal progenitor cell differentiation through H19 gene expression.

Although the role of estrogen signaling in breast cancer development has been extensively studied, the mechanisms that regulate the indispensable role of estrogen in normal mammary gland development have not been well studied. Because of the unavailability of culture system to maintain estrogen-receptor-positive (ERα(+)) cells in vitro, the molecular mechanisms that regulate estrogen/ERα signaling in the normal human breast are unknown. In the present study, we examined the effects of estrogen signaling on ERα(+) human luminal progenitors using a modified matrigel assay and found that estrogen signaling increased the expansion potential of these progenitors. Furthermore, we found that blocking ERα attenuated luminal progenitor expansion and decreased the luminal colony-forming potential of these progenitors. Additionally, blocking ERα decreased H19 expression in the luminal progenitors and led to the development of smaller luminal colonies. We further showed that knocking down the H19 gene in the luminal progenitors significantly decreased the colony-forming potential of the luminal progenitors, and this phenotype could not be rescued by the addition of estrogen. Lastly, we explored the clinical relevance of the estrogen-H19 signaling axis in breast tumors and found that ERα(+) tumors exhibited a higher expression of H19 as compared with ERα(-) tumors and that H19 expression showed a positive correlation with ERα expression in those tumors. Taken together, the present results indicate that the estrogen-ERα-H19 signaling axis plays a role in regulating the proliferation and differentiation potentials of the normal luminal progenitors and that this signaling network may also be important in the development of ER(+) breast cancer tumors.

Reconstruction of 3-dimensional histology volume and its application to study mouse mammary glands.

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs(1,2,3) is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution. This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

Glutathione-dependent and -independent oxidative stress-control mechanisms distinguish normal human mammary epithelial cell subsets.

Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated "steady-state" normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.

Loss of Igfbp7 causes precocious involution in lactating mouse mammary gland.

BACKGROUND: Insulin like growth factors (IGFs) and their binding proteins (IGFBPs) are secreted peptides that play major roles in regulating the normal development and maturation of mammary gland. While Igfbp7 has been shown to decrease breast tumor growth, its role in regulating the normal mammary gland development has not been studied. To this end, we generated Igfbp7-null mice and examined the development and maturation of mammary glands in the virgin, pregnant and lactating animals. RESULTS: We report here that loss of Igfbp7 significantly retards mammary gland development in the virgin animals. More significantly, the pregnant Igfpb7-null glands contained fewer alveolar structures and that during lactation these glands exhibit the morphological changes that are associated with involution. The transcriptome profile of the Igfbp7-null glands on the lactation day 3 revealed a distinct involution-related gene signature compared to the lactating WT glands. Interestingly, we found that the lactating Igfbp7-null glands exhibit increased expression of Stat3 and enhanced activation of (phosphorylated) Stat3, combined with decreased expression of Stat5 suggesting that the absence of Igfbp7 accelerates the onset of involution. We also found that in absence of Igfpb7, the lactating glands contain increased Igfbp5 protein along with decreased expression of IGF-1 Receptor and Akt activation. Finally, we show that during the normal course of involution, Igfbp7 expression is significantly decreased in the mammary gland. CONCLUSION: Our data suggest that loss of Igfbp7 induces precocious involution possibly through diminished cell survival signals. Our findings identify Igfbp7 as major regulator of involution in the mammary gland.

Membrane potential differences and GABAA receptor expression in hepatic tumor and non-tumor stem cells.

The ability to differentiate tumor initiating stem cells (TISCs) from healthy, normal stem cells (NSCs) could have important diagnostic and therapeutic implications for patients with hepatocellular carcinoma (HCC). The aim of this study was to document and compare cell membrane potentials (PDs) and GABAA receptor subunit expression in hepatic TISCs and NSCs. PD values were determined in CD133(+) Huh-7 TISCs and CD133(+) WBF344 NSCs by single channel microelectrode impalement. GABAA receptor subunit expression was documented using immunohistochemistry (IH) in both cell lines as well as surgically resected HCC and healthy liver tissues. TISCs were significantly depolarized compared with NSCs (-4.0 ± 1.8 versus -11.0 ± 2.4 mV, respectively; p < 0.05). GABAA α6 subunit expression was either absent or markedly attenuated, while γ3 subunit expression was abundant in TISCs and HCC compared with NSCs and healthy liver tissues. Exposure to the GABAA receptor agonist muscimol caused hyperpolarization of TISCs (Δ -4.4 ± 1.1) but depolarization of NSCs (Δ + 5.2 ± 2.3) and attenuation of TISC proliferative activity. We conclude that TISCs and NSCs have significantly different cell membrane potentials and these differences are associated with differences in GABAA receptor subunit expression.