Healthy Human Breast Research Articles

C.05.03 A case of workplace productivity in patients with depression

In adult women, the water-content represents between 50% and 70% of the mass in normal breast tissues and this percentage is increased within diseased tissues. Water molecules play an essential role in the structural organization of biological tissues such as breast. Then, in this study, we have investigated the influence of the water molecules on the breast tissue organization and their role on the hierarchical tissue rearrangement promoted by tumor growth.SAXS and WAXS techniques were used to analyze healthy, benign and malignant human breast samples in native and lyophilized conditions. The scattering profiles in SAXS and WAXS regime of each tissue type in both conditions were compared in order to identify the structural transformation in these tissues and verify the water influence on the morphological arrangement of normal and pathological human breast tissues.From SAXS, changes at the axial periodicity of collagen fibrils were revealed. Additionally, when the water content has removed a peak at q = 4.17 nm−1 (that was present only in pathological samples) shifted in opposite directions within benign and malignant lesions. From WAXS, water and fatty acids were identified within native samples. However, after freeze-drying, only the fat component was observed in the scattering profiles.Therefore, when the water molecules were removed from the samples, structural changes associated with pathological progression were visible. From this, insights about their influence over the changes promoted by the tumor growth have been proposed. Finally, the findings of this study have the potential to provide valuable information to the development of new target therapy.

Effects of PTEN Loss and Activated KRAS Overexpression on Mechanical Properties of Breast Epithelial Cells.

It has previously been shown that the simultaneous activation of PI3K (phosphatidylinositol 3-kinase) and Ras/MAPK (mitogen-activated protein kinases) pathways facilitate tumor growth despite only inducing cancer cell dormancy individually. Determining the impacts on cellular mechanics each pathway incites alone and in unison is critical to developing non-toxic cancer therapies for triple-negative breast cancers. PTEN (phosphatase and tensin homolog) knockout and activated KRAS (Kristen rat sarcoma viral oncogene homolog) overexpression in healthy MCF-10A human breast epithelial cells activated the PI3K and Ras/MAPK pathways, respectively. Cell stiffness and fluidity were simultaneously measured using atomic force microscopy. Results suggest that PTEN knockout reduced cell stiffness and increased cell fluidity independent of PI3K activation. Effects of activated KRAS overexpression on cell stiffness depends on rigidity of cell culture substrate. Activated KRAS overexpression also counteracts the effects of PTEN knockout.

Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors

Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast tissue. We find that promoter methylation is maintained in this system, even in the absence of the fusion construct, and this prevents cells from engaging senescence arrest. Our data show that the key driver of this phenotype is repression of CDKN2A transcript p16 where myoepithelial cells harbour cancer-like gene expression but do not exhibit anchorage-independent growth. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, which may facilitate tumour initiation.

Anthocyanin‑rich blackcurrant extract inhibits proliferation of the MCF10A healthy human breast epithelial cell line through induction of G0/G1 arrest and apoptosis.

Blackcurrants (Ribesnigrum L., Grossulariaceae) possess a high content of anthocyanin polyphenols, which have been demonstrated to exhibit beneficial effects on health due to their antioxidant and anticarcinogenic prope-rties. The present study investigated novel functions of anthocyanin‑rich blackcurrant extracts (BCEs) in a healthy mammary epithelial cell line, MCF10A. The percentages of viable cells were 85, 75, 53 and 31% following exposure to 50, 100, 200 and 400µg/ml BCE, respectively. The half‑maximal response concentration of BCE was 237.7µg/ml. Microarray and Ingenuity® Pathway Analysis demonstrated that BCE downregulated cell cycle signaling, including upstream genes with mitotic roles such as polo‑like kinase signaling. BCE increased the number of cells in the G0/G1 phase and decreased the number of cells in the S and G2/M phases. Alkaline comet assays demonstrated that 50 and 100µg/ml BCE induced DNA damage in a dose‑dependent manner. Cultures treated with 0, 50, and 100µg/ml BCE contained 4.6, 13.4 and 16.0% apoptotic cells, respectively. As compared with the untreated cultures (1.9%), the number of necrotic cells increased in the 100µg/ml BCE‑treated cultures (from 1.9 to 4.3%) but not in the 50µg/ml BCE‑treated cultures. Reverse transcription‑quantitative polymerase chain reaction analysis demonstrated that BCE reduced mRNA expression of the genomic caretaker lysine‑specific demethylas 5B(KDM5B). The results suggested that blackcurrant anthocyanins may act as cell arrest and death inducers via KDM5B downregulation in healthy breast cells.

Proton and phosphorus magnetic resonance spectroscopy of the healthy human breast at 7T.

In vivo water‐ and fat‐suppressed 1H magnetic resonance spectroscopy (MRS) and 31P magnetic resonance adiabatic multi‐echo spectroscopic imaging were performed at 7 T in duplicate in healthy fibroglandular breast tissue of a group of eight volunteers. The transverse relaxation times of 31P metabolites were determined, and the reproducibility of 1H and 31P MRS was investigated. The transverse relaxation times for phosphoethanolamine (PE) and phosphocholine (PC) were fitted bi‐exponentially, with an added short T 2 component of 20 ms for adenosine monophosphate, resulting in values of 199 ± 8 and 239 ± 14 ms, respectively. The transverse relaxation time for glycerophosphocholine (GPC) was also fitted bi‐exponentially, with an added short T 2 component of 20 ms for glycerophosphatidylethanolamine, which resonates at a similar frequency, resulting in a value of 177 ± 6 ms. Transverse relaxation times for inorganic phosphate, γ‐ATP and glycerophosphatidylcholine mobile phospholipid were fitted mono‐exponentially, resulting in values of 180 ± 4, 19 ± 3 and 20 ± 4 ms, respectively. Coefficients of variation for the duplicate determinations of 1H total choline (tChol) and the 31P metabolites were calculated for the group of volunteers. The reproducibility of inorganic phosphate, the sum of phosphomonoesters and the sum of phosphodiesters with 31P MRS imaging was superior to the reproducibility of 1H MRS for tChol. 1H and 31P data were combined to calculate estimates of the absolute concentrations of PC, GPC and PE in healthy fibroglandular tissue, resulting in upper limits of 0.1, 0.1 and 0.2 mmol/kg of tissue, respectively.

Pattern-based sensing of triple negative breast cancer cells with dual-ligand cofunctionalized gold nanoclusters.

Early detection of breast cancer is a critical component in patient prognosis and establishing effective therapy regimens. Here, we developed an easily accessible yet potentially powerful sensor to detect cancer cell targets by utilizing seven dual-ligand cofunctionalized gold nanoclusters (AuNCs) as both effective cell recognition elements and signal transducers. On the basis of this AuNC multichannel sensor, we have successfully distinguished healthy, cancerous and metastatic human breast cells with excellent reproducibility and high sensitivity. Triple negative breast cancer cells (TNBCs), which exhibit low expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2, were identified. The high accuracy of the blind breast cell sample tests further validates the practical application of the sensor array. In addition, the versatility of the sensor array is further justified by identifying amongst distinct cell types, different cell concentrations and cell mixtures. Notably, the drug-resistant cancer cells can also be efficiently discriminated. Furthermore, the dual-ligand cofunctionalized AuNCs can efficiently differentiate different cells from the peripheral blood of tumor-free and tumor-bearing mice. Taken together, this fluorescent AuNCs based array provides a powerful cell analysis tool with potential applications in biomedical diagnostics.

Biodegradable Glycopolymeric Micelles Obtained by RAFT-controlled Radical Ring-Opening Polymerization

The design and synthesis of an entirely degradable glycopolymer micelle was presented. This design relies on the utilization of RAFT-controlled radical ring-opening polymerization (rROP) technique to afford multiple insertions of cleavable ester linkages onto the backbone of the corona. RAFT polymerization using a macroRAFT agent based on poly(ε-caprolactone) PCL was employed to control the polymerization of well-defined statistical glycopolymers of 1-O-acryloyl-2,3:4,5-di-O-isopropylidene-β-d-fructopyranose (1-O-AiPrFru) and 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) monomer. Three block copolymers were synthesized to generate poly(ε-caprolactone)-b-poly[(1-O-acryloyl-2,3:4,5-di-O-isopropylidene-β-d-fructopyranose)-co-(5,6-benzo-2-methylene-1,3-dioxepane)] (PCL-b-P[(1-O-AiPrFru)-co-(BMDO)]) with varying block lengths. Self-assembly of the deprotected block copolymers generated nonspherical egg shaped micelles, where the shorter chains PCL106-b-P[(1-O-AFru)69-co-(BMDO)9] underwent self-assembly forming micelles with the hydrodynamic diameter (DH) of 106 nm. The biodegradation of these micelles were investigated via enzymatic degradation by Lipase Pseudomonas sp., indicating entirely degradable architectures, which are no longer visible via dynamic light scattering (DLS). SEC further confirmed the appearance of fragmented glycopolymeric units. In vitro cell proliferation assay of the micelles and their degradation products revealed no toxicity against healthy human fibroblast HS27 and breast cancer MDA-MB-231 cell lines. The polymer concentration range tested was up to 0.20 mg·mL–1 with the cell viabilities of ≥95%.

Array-based identification of triple-negative breast cancer cells using fluorescent nanodot-graphene oxide complexes

Early and accurate diagnosis of breast cancer holds great promise to improve treatability and curability. Here, we report the usage of six luminescent nanodot-graphene oxide complexes as novel fluorescent nanoprobes in a sensing array capable of effectively identifying healthy, cancerous, and metastatic human breast cells. The sensory system is based on the utilization of nanoprobe-graphene oxide sensor elements that can be disrupted in the presence of breast cells to give fluorescent readouts. Using this multichannel sensor, we have successfully identified breast cancer cells and distinguished between estrogen receptor positive, human epidermal growth factor receptor-2 positive, and triple negative phenotypes. This approach also allows cell identification at high sensitivity (200 cells) with high reproducibility. The unknown cell sample analysis indicates that the sensor is able to identify 49 out of 50 breast cell samples correctly, with a detection accuracy of 98%. Taken together, this array-based luminescent nanoprobe-graphene oxide sensing platform presents a useful cell screening tool with potential applications in biomedical diagnostics.

Glycerophosphocholine and Glycerophosphoethanolamine Are Not the Main Sources of the In Vivo (31)P MRS Phosphodiester Signals from Healthy Fibroglandular Breast Tissue at 7 T.

The identification of the phosphodiester (PDE) (31)P MR signals in the healthy human breast at ultra-high field. In vivo (31)P MRS measurements at 7 T of the PDE signals in the breast were performed investigating the chemical shifts, the transverse- and the longitudinal relaxation times. Chemical shifts and transverse relaxation times were compared with non-ambiguous PDE signals from the liver. The chemical shifts of the PDE signals are shifted -0.5 ppm with respect to glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE), and the transverse and longitudinal relaxation times for these signals are a factor 3 to 4 shorter than expected for aqueous GPC and GPE. The available experimental evidence suggests that GPC and GPE are not the main source of the PDE signals measured in fibroglandular breast tissue at 7 T. These signals may predominantly originate from mobile phospholipids.

Overcoming limitations in diffusion-weighted MRI of breast by spatio-temporal encoding.

Evaluating the usefulness of diffusion-weighted spatio-temporal encoding (SPEN) methods to provide quantitative apparent diffusion coefficient (ADC)-based characterizations of healthy and malignant human breast tissues, in comparison with results obtained using techniques based on spin-echo echo planar imaging (SE-EPI). Twelve healthy volunteers and six breast cancer patients were scanned at 3T using scanner-supplied diffusion-weighted imaging EPI sequences, as well as two fully refocused SPEN variants programmed in-house. Suitable codes were written to process the data, including calculations of the actual b-values and retrieval of the ADC maps. Systematically better images were afforded by the SPEN scans, with negligible geometrical distortions and markedly weaker ghosting artifacts arising from either fat tissues or from strongly emitting areas such as cysts. SPEN-derived images provided improved characterizations of the fibroglandular tissues and of the lesions' contours. When translated into the calculation of the ADC maps, there were no significant differences between the mean ADCs derived from SPEN and SE-EPI: if reliable images were available, both techniques showed that ADCs decreased by nearly two-fold in the malignant lesion areas. SPEN-based sequences yielded diffusion-weighted breast images with minimal artifacts and distortions, enabling the calculation of improved ADC maps and the identification of decreased ADCs in malignant regions.

Phenotypic plasticity in normal breast derived epithelial cells.

BackgroundNormal, healthy human breast tissue from a variety of volunteer donors has become available for research thanks to the establishment of the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB). Multiple epithelial (K-HME) and stromal cells (K-HMS) were established from the donated tissue. Explant culture was utilized to isolate the cells from pieces of breast tissue. Selective media and trypsinization were employed to select either epithelial cells or stromal cells. The primary, non-transformed epithelial cells, the focus of this study, were characterized by immunohistochemistry, flow cytometry, and in vitro cell culture.ResultsAll of the primary, non-transformed epithelial cells tested have the ability to differentiate in vitro into a variety of cell types when plated in or on biologic matrices. Cells identified include stratified squamous epithelial, osteoclasts, chondrocytes, adipocytes, neural progenitors/neurons, immature muscle and melanocytes. The cells also express markers of embryonic stem cells.ConclusionsThe cell culture conditions employed select an epithelial cell that is pluri/multipotent. The plasticity of the epithelial cells developed mimics that seen in metaplastic carcinoma of the breast (MCB), a subtype of triple negative breast cancer; and may provide clues to the origin of this particularly aggressive type of breast cancer. The KTB is a unique biorepository, and the normal breast epithelial cells isolated from donated tissue have significant potential as new research tools.

Blood Flow Reduction in Breast Tissue due to Mammographic Compression

This study measures hemodynamic properties such as blood flow and hemoglobin concentration and oxygenation in the healthy human breast under a wide range of compressive loads. Because many breast-imaging technologies derive contrast from the deformed breast, these load-dependent vascular responses affect contrast agent-enhanced and hemoglobin-based breast imaging. Diffuse optical and diffuse correlation spectroscopies were used to measure the concentrations of oxygenated and deoxygenated hemoglobin, lipid, water, and microvascular blood flow during axial breast compression in the parallel-plate transmission geometry. Significant reductions (P < .01) in total hemoglobin concentration (∼30%), blood oxygenation (∼20%), and blood flow (∼87%) were observed under applied pressures (forces) of up to 30 kPa (120 N) in 15 subjects. Lipid and water concentrations changed <10%. Imaging protocols based on injected contrast agents should account for variation in tissue blood flow due to mammographic compression. Similarly, imaging techniques that depend on endogenous blood contrasts will be affected by breast compression during imaging.

MO‐D‐141‐08: Multi‐Scale Analysis of Collagen Architecture for Classifying Tumor and Healthy Breast Tissue Images

Purpose: To quantify differences in collagen microarchitecture between healthy human breast tissue and breast cancer images using extended field of view second harmonic generation (EFOV SHG) microscopy coupled with analysis by the curvelet transform. Methods: Two frozen samples of normal associated human breast tissue (NAT) and invasive ductal carcinoma (IDC) were obtained through the University of Wisconsin BioBank. Samples were thawed, fixed, embedded in agar, cut into 300 micron thick sections using a vibratome and imaged using a custom inverted SHG microscope designed to allow for EFOV imaging. Individual SHG Images were captured at 0.79 micron resolution and a FOV of approximately 200 microns. The FOV was extended by stage scanning and image stitching to produce submicron resolution fields of view up to 2 cm2. NAT and IDC tissues were imaged, stitched using the Grid/Collection Stitching ImageJ plugin, and analyzed using features derived from the curvelet transform including variance in orientation and prevalence at various scales. These novel features were tested using support vector machine (SVM) cross validation. Results: IDC tissue contained linearly aligned, crisscrossing collagen while NAT tissue contained wavy, dense collagen. The mean curvelet alignment value for the normal images was approximately 2X higher than that for IDC. In addition, normal images had approximately 20% fewer closely correlated curvelets compared to IDC. Three‐fold cross validation of a linear SVM classifier revealed average sensitivity and specificity of 0.994 and 0.969 respectively. Conclusion: These results demonstrate the excellent potential for collagen architecture quantification based on features derived from the curvelet transform. We have also shown the value of EFOV SHG imaging to capture both large and small scale features necessary for quantifying collagen morphology. Future work will improve EFOV SHG imaging throughput and test classification capabilities of the curvelet based feature set on a large sample population. NIH R01 grants CA114462 and CA136590 to P.J.K. and K.W.E., T32 CA009206 to J.S.B and the Morgridge Institute for Research

Abstract LB-273: The tumor suppressor p16INK4a regulates extensive plasticity in rare somatic cells found in adult human tissue.

Abstract Extensive studies have demonstrated that repression of p16INK4a (CDKN2A) not only silences a powerful tumor suppressor activity, but also is associated with the acquisition of a plastic state, i.e. the ability of a cell to change phenotypes. In epithelial cells, repression of p16INK4a does not only inactivate cell cycle arrest in response to stress but also allows increased expression of chromatin remodeling proteins that are important for epigenetic plasticity underlying differentiation. The up-regulation of such chromatin remodeling proteins sets the expression pattern of pluripotent cells in Drosophila and inhibits differentiation and dictates the decision between progenitor and differentiated states in murine myoblasts. Furthermore, mice engineered for knock-out of BMI-1, a polycomb protein that inhibits p16INK4a transcription, fail to repress p16INK4a activity and fail to generate hematopoietic and neural stem cells. In light of these observations, we reasoned that repression of p16INK4a might also modulate expression of cell surface markers that could be used for the prospective isolation of stem or progenitor cells. We identified cell surface markers associated with repression of p16INK4a and found that they allowed direct isolation of rare cells from healthy human breast tissue that exhibit extensive lineage plasticity. This subpopulation of cells has the ability to transcribe pluripotency markers, Oct3/4, Sox2 and Nanog at levels similar to those measured in human embryonic stem cells and to acquire a plastic state sensitive to environmental programming. In vitro, in vivo and teratoma assays demonstrated that either a directly-sorted (uncultured) or a single cell (clonogenic) cell population from primary human tissue can differentiate into functional derivatives of each germ layer, ectodermal, endodermal and mesodermal. In contrast to other cells that express Oct3/4, Sox2 and Nanog, these human endogenous Plastic Somatic cells (ePS cells) are mortal, express low telomerase activity, expand for an extensive but finite number of population doublings, and maintain a diploid karyotype before arresting in G1. The observation that repressed p16INK4a, a key tumor suppressor gene, is associated with epigenetic and phenotypic plasticity suggests functional links between tumor suppressor genes and plastic states that are yet to be determined and may some day be exploited for cancer intervention. Citation Format: Somdutta Roy, Philippe Gascard, Nancy Dumont, Jianxin Zhao, Deng Pan, Sarah Petrie, Marta Margeta, Thea Dorothy Tlsty. The tumor suppressor p16INK4a regulates extensive plasticity in rare somatic cells found in adult human tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-273. doi:10.1158/1538-7445.AM2013-LB-273

Amide proton transfer imaging of the human breast at 7T: development and reproducibility

Chemical exchange saturation transfer (CEST) can offer information about protons associated with mobile proteins through the amide proton transfer (APT) effect, which has been shown to discriminate tumor from healthy tissue and, more recently, has been suggested as a prognosticator of response to therapy. Despite this promise, APT effects are small (only a few percent of the total signal), and APT imaging is often prone to artifacts resulting from system instability. Here we present a procedure that enables the detection of APT effects in the human breast at 7T while mitigating these issues. Adequate signal-to-noise ratio (SNR) was achieved via an optimized quadrature RF breast coil and 3D acquisitions. To reduce the influence of fat, effective fat suppression schemes were developed that did not degrade SNR. To reduce the levels of ghosting artifacts, dummy scans have been integrated into the scanning protocol. Compared with results obtained at 3T, the standard deviation of the measured APT effect was reduced by a factor of four at 7T, allowing for the detection of APT effects with a standard deviation of 1% in the human breast at 7T. Together, these results demonstrate that the APT effect can be reliably detected in the healthy human breast with a high level of precision at 7T.

Rare somatic cells from human breast tissue exhibit extensive lineage plasticity

We identified cell surface markers associated with repression of p16(INK4a)/cyclin-dependent kinase inhibitor 2A(CDKN2A), a critical determinant in the acquisition of a plastic state. These cell surface markers allowed direct isolation of rare cells from healthy human breast tissue that exhibit extensive lineage plasticity. This subpopulation is poised to transcribe plasticity markers, OCT3/4, SOX2, and NANOG, at levels similar to those measured in human embryonic stem cells and to acquire a plastic state sensitive to environmental programming. In vitro, in vivo, and teratoma assays demonstrated that either a directly sorted (uncultured) or a single-cell (clonogenic) cell population from primary tissue can differentiate into functional derivatives of each germ layer, ectodermal, endodermal, and mesodermal. In contrast to other cells that express OCT3/4, SOX2, and NANOG, these human endogenous plastic somatic cells are mortal, express low telomerase activity, expand for an extensive but finite number of population doublings, and maintain a diploid karyotype before arresting in G1.

Mapping transitions between healthy and pathological lesions in human breast tissues by diffraction enhanced imaging computed tomography (DEI-CT) and small angle x-ray scattering (SAXS)

In this work we have combined the DEI-CT and SAXS technique to study the transition between healthy and pathological breast tissues, which include benign and malignant lesions. The ability of DEI-CT to enhance the contrast between soft tissues was used to localize the tumor region in the sample. Then, the tumor region and its surroundings were scanned by SAXS in order to map the changes promoted by the neoplasias at nano-level.It was clearly observed that pathological tissues present distinguishable SAXS scattering profiles from those of normal tissue. These differences are mainly related to changes in arrangement and diameter of collagen fibrils, evaluated by the higher order of reflection peaks of these fibrils. Differences related to the peak intensities and the total scattered intensity were found by comparing the healthy and pathological regions. The 2nd order of collagen reflection arises only in the healthy region neighboring the benign lesion. A broader peak at q=0.16nm−1 seems to characterize the malignant lesions. Finally, based on this information, the transition between healthy and pathological human breast tissues was mapped which allowed to get insights into the changes promoted by tumors during growth and progression.

Decidua mesenchymal stem cells migrated toward mammary tumors in vitro and in vivo affecting tumor growth and tumor development

Mesenchymal stem cells (MSCs) have affinity to tumor sites where they home, affecting their biology and growth. Previously, we have isolated mesenchymal cells from the decidua of the human placenta named as decidua-derived MSCs (DMSCs). The aims of the present study were to investigate the migration capacity of DMSCs in vitro, and in vivo in a preclinical model of mammary tumors induced by N-nitroso-N-methylurea (NMU). Additionally, we assessed the safety of DMSC administration in vivo and their effect on tumor growth. In vitro studies showed that DMSCs significantly migrate toward both, healthy human breast tissue and breast adenocarcinoma. Nevertheless, the effect on DMSC migration was significantly higher in the presence of tumor tissue. DMSCs also significantly migrated in vitro in the presence of NMU-mammary tumor homogenate when compared with control media alone. In vivo studies showed both migration and engraftment of DMSCs into NMU-induced tumors. Interestingly, DMSCs showed an inhibitory effect on the growth of primary tumors and in the development of new tumors. DMSCs did not affect the growth of secondary tumors, although secondary tumors appeared 2 weeks later, and the number of secondary tumors was lower in the DMSC-treated rats as compared with vehicle-treated rats. To our knowledge, this is the first report showing placental MSCs effect on tumor growth. In conclusion, DMSCs could serve as a therapeutic agent themselves and as a cellular vehicle of anticancer drugs.

Localization of decorin gene expression in normal human breast tissue and in benign and malignant tumors of the human breast

The small extracellular matrix proteoglycan decorin which possesses a potent antitumor activity has been shown to be present in various amounts in the stroma of several tumors including those of the breast. Regarding decorin in breast malignancies the published data are conflicting, i.e., whether breast cancer cells express it or not. Here, we first compared decorin gene expression levels between healthy human breast tissue and selected types of human breast cancer using GeneSapiens databank. Next, we localized decorin mRNA in tissue specimen of normal human breast, intraductal breast papillomas and various histologic types of human breast cancer using in situ hybridization (ISH) with digoxigenin-labeled RNA probes for decorin. We also examined the effect of decorin transduction on the behavior of cultured human breast cancer MCF7 cells. Analysis of GeneSapiens databank revealed that in various human breast cancers decorin expression is significant. However, ISH results clearly demonstrated that human breast cancer cells independently of the type of the cancer do not express decorin mRNA. This was also true for papilloma-forming cells of the human breast. Indeed, decorin gene expression in healthy human breast tissue as well as in benign and malignant tumors of human breast was shown to take place solely in cells of the original stroma. Decorin transduction using decorin adenoviral vector in decorin-negative MCF7 cells resulted in a significant decrease in the proliferation of these cells and changed cell cohesion. Decorin-transduced MCF7 cells also exhibited increased apoptosis. In conclusion, our study shows that in human breast tissue only cells of the original stroma are capable of decorin gene expression. Our study also shows that transduction of decorin in decorin-negative human breast cancer cells markedly modulates the growth pattern of these cells.

Quantitative mapping of total choline in healthy human breast using proton echo planar spectroscopic imaging (PEPSI) at 3 Tesla

To quantitatively measure tCho levels in healthy breasts using Proton-Echo-Planar-Spectroscopic-Imaging (PEPSI). The two-dimensional mapping of tCho at 3 Tesla across an entire breast slice using PEPSI and a hybrid spectral quantification method based on LCModel fitting and integration of tCho using the fitted spectrum were developed. This method was validated in 19 healthy females and compared with single voxel spectroscopy (SVS) and with PRESS prelocalized conventional Magnetic Resonance Spectroscopic Imaging (MRSI) using identical voxel size (8 cc) and similar scan times (∼7 min). A tCho peak with a signal to noise ratio larger than 2 was detected in 10 subjects using both PEPSI and SVS. The average tCho concentration in these subjects was 0.45 ± 0.2 mmol/kg using PEPSI and 0.48 ± 0.3 mmol/kg using SVS. Comparable results were obtained in two subjects using conventional MRSI. High lipid content in the spectra of nine tCho negative subjects was associated with spectral line broadening of more than 26 Hz, which made tCho detection impossible. Conventional MRSI with PRESS prelocalization in glandular tissue in two of these subjects yielded tCho concentrations comparable to PEPSI. The detection sensitivity of PEPSI is comparable to SVS and conventional PRESS-MRSI. PEPSI can be potentially used in the evaluation of tCho in breast cancer. A tCho threshold concentration value of ∼0.7 mmol/kg might be used to differentiate between cancerous and healthy (or benign) breast tissues based on this work and previous studies.