Characterization of Human Mammary Epithelial Stem Cells
Characterization of Human Mammary Epithelial Stem Cells

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Publisher:

Published: 2009

Total Pages: 18

ISBN-13:

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The mammary epithelium of normal adult female mice contains stem cells with extensive in vivo regenerative and self-renewal potential. Indirect evidence has suggested that analogous cells exist in the mammary glands of adult women, and are candidate targets for the first transforming mutations that lead to the evolution of breast cancer stem cells. The objective of this grant was to determine whether these hypothesized normal human mammary stem cells might be detected and quantified by a robust and specific assay that could be used to enable the purification and phenotypic properties of these cells, and to derive information about their frequency and how they are regulated. During the 3 years of this grant, I established conditions that allow human mammary gland structures to be reproducibly generated in subrenal xenografts in highly immune deficient mice, starting with small innocula of dissociated human mammary cells. The regenerated glands are similar in morphology and cellular organization to normal human mammary glands, bounded by a basement membrane with an outer layer of myo-epithelial cells and an inner layer of polarized luminal cells that can be induced to produce milk. I also established that the presence of regenerated structures can be determined by detecting the in vitro clonogenic progenitors they contain and this endpoint can serve as an objective indicator of the presence of a primitive stem-like cell in the initial cells transplanted. This retrospective functional assay allows limiting dilution analysis of positive xenograft yields to derive mammary stem cell frequencies in differently manipulated populations. Using this approach I found the frequency of stem cells in normal human mammary tissue to be ~1 per 5000 cells and their phenotype to be CD49f+ EpCAM-/low CD31- CD45-

Regulating Human Mammary Epithelial Stem Cells Transformation
Regulating Human Mammary Epithelial Stem Cells Transformation

Author: Flora Clément

Publisher:

Published: 2017

Total Pages: 0

ISBN-13:

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It has been shown for a number of cancers that a cell population characterized by stem cell (SC) properties and therapeutic resistance is likely responsible for relapse several years after treatment. Current therapies kill most of the tumor cells, but fail to eradicate the so-called cancer stem cells (CSC). Therefore a complete cure of the disease will require the eradication of the tumor-sustaining CSC. We propose to study these CSC in the context of breast cancer as the existence of CSC as already been highlighted in this epithelia.CD10 is a membrane enzyme able to cleave several peptide of the microenvironment (such as oxytocin, bombesin, enkephalin.. ) that can also interact with intracellular signalling pathway through its direct interaction with PTEN. Our results, and those of the literature, indicate that CD10 enzyme controls the fate of SC and is deregulated in normal breast and cancerous tissues. We showed that CD10 membrane expression allows the maintenance of immature cells partly through its enzymatic function that inhibits mammary stem cells differentiation. As CD10 has been described in breast cancer initiation, progression and resistance, we then decided to test the role of CD10 in tumor context. Our strategy consists in flow cytometry cell sorting for CD10+/CD10- cells to compare the functional properties of both sub-population. Only CD10+ cells are able to regenerate both CD10+ and CD10- subpopulations, and CD10+ cells exhibit higher expression of immature genes. Interestingly, modulating CD10 using stable expression of CD10 in our models and Sh strategies do not mimick the normal functions of CD10, indicating that CD10 could be more a marker of a certain population with immature properties prone to transformation rather than a driver. To better characterize the role of CD10 in luminal breast transformation, we developed a new human mammary model, initiated from immature cells to obtain transformed luminal epithelial cells and their resistant counterpart. We observed a higher level of CD10 expression during mammary epithelial cell transformation process. We then performed a microarray on CD10+ and CD10- subpopulations. Preliminary analysis seems to confirm that CD10 is a potential marker for a stem cell population prone to transformation rather than a direct driver of the cell transformation.

Characterization of Breast Cancer Stem Cells
Characterization of Breast Cancer Stem Cells

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Published: 2004

Total Pages: 0

ISBN-13:

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Cellular markers which identify putative breast epithelial stem cells have been report Attention has focused on the possibility that certain clinical aspects of breast cancer could result from transformed stem cells residing in the malignant tumor. Breast cancer may therefore originate from neoplastic transformation of normal breast epithelial stem cells. These transformed stem cells may exist in the tumor as rare cells with properties which drive multiple aspects of tumorigenesis. The predictions of this model included slower cell cycle progression greater resistance to DNA damage, increased in vitro invasion, and in vivo tumorigenesis. The application tested the hypothesis that transformed stem cells drive multiple aspects of breast tumorigenesis by functionally characterizing their biological properties. The hypothesis was tested by sorting of putative breast cancer stem cell populations followed by cell cycle analysis, in vitro and in vivo proliferation. and invasion assays, and sensitivity to chemotherapy and radiation induced DNA damage. Putative cancer stem cells generally exhibited slower cell cycle progression, greater resistance to DNA damage, and increased tumor formation. However, the degree of stem-like properties of these cells varied widely depending on the line from which they were isolated.

Mammary Stem Cells
Mammary Stem Cells

Author: Maria dM. Vivanco

Publisher: Humana

Published: 2022-02-18

Total Pages: 354

ISBN-13: 9781071621929

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This second edition provides an overview of recent developments and approaches used by researchers to investigate the properties and functions of mammary epithelial and stem cells, which will contribute to understand the heterogeneity of the mammary gland and of breast cancer. Chapters detail processes used to characterize stem cells, single cell RNA sequencing, computational methods, sophisticated imaging techniques, and a variety of model systems, among others. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Mammary Stem Cells: Methods and Protocols, Second Edition aims to make available protocols used to navigate the intricate behavior of mammary stem cells and to gain further knowledge to take us closer to the design of innovative strategies to prevent and treat breast cancer.

Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the P53 Or BRCAl Genes
Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the P53 Or BRCAl Genes

Author: Raymond White

Publisher:

Published: 1997

Total Pages: 18

ISBN-13:

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The overall goal of this project is to identify and characterize the consequences of human mammary epithelial cells (HMEC) that become deficient in normal p53 and BRCA1 gene functions. Our work during the third year of the funding period has produced good progress establishing new systems to create and characterize HMEC with an altered tumor suppressor gene activity. In collaboration with Dr. Bissell's laboratory (UC Berkeley), we have established three-dimensional culture system in our laboratory. This system will allow us to see differences of the growth properties, expression and distribution of certain cell lineage markers, morphology and behavior in between normal cells and partially transformed cells. Our recent acquisition of a Microarray Spotting and Scanning instrument enabled us the use of this robust and sensitive microarray technology to establish genetic expression profiles from any cell source of interest. We have also applied the differential display protocol to identify genes with altered expression in a model system of normal and neoplastic epithelial cells. We firmly believe that these new systems will provide us critical information to our understanding of breast carcinogenesis.

Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the P53 Or BRCA1 Genes
Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the P53 Or BRCA1 Genes

Author:

Publisher:

Published: 1999

Total Pages: 0

ISBN-13:

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The overall goal of this project is to identify and characterize the consequences of human mammary epithelial cells (HMEC) that become deficient in normal p53 or BRCA-l gene functions. We have created retroviral vectors which allow us conditionally express the E6/E7 gene of human papillomavirus type 16 (HPVl6), dominant-negative p53 gene, or anti-sense BRCA-l gene. The consequences of transduction of these viral constructs into primary human mammary epithelial cells will be discovered through controlled in vitro comparisons between genetically altered derivatives and their isogenic parent cells. As we proposed in our last report, we are now focused our efforts on microarray-based comparisons to identify breast cancer related genes. During the past year we have successfully implemented the Microarray Spotting and Scanning techniques. This includes development of robust fluorescent labeling and hybridization protocols as well as the preparation and testing of over 23,000 minimally redundant cDNA target samples for deposition on the microarray slides. We have compared expression profiles from several distinct breast cell lines we had created. We believe that this system will provide us critical information to our understanding of early breast carcinogenesis.

Characterization of an In Vitro Human Breast Epithelial Organoid System
Characterization of an In Vitro Human Breast Epithelial Organoid System

Author:

Publisher:

Published: 2001

Total Pages: 114

ISBN-13:

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The objectives of this study are: (1) to identify factors that regulate the growth and differentiation of organoids formed by two types of normal human breast epithelial cells (HBEC) in Matrigel; (2) to characterize the expression and function of estrogen receptors (ER) in normal and in vitro neoplastically transformed HBEC; and (3) to determine if a HBEC type with stem cell characteristics (Type I) is more susceptible to telomerase activation and immortalization. The major results are: (1) Type I HBEC in conjunction with Type II cells are capable of forming ductal and end bud or lobule 1-like structures in Matrigel which preserve the undifferentiated state of HBEC for a long time, evidence that Type I HBEC are stem cells; (2) Type I normal HBEC and their neoplastically transformed clones express a variant ER-A in vitro on plastic while expressing a wild type ER-A in tumors developed in nude mice or grown in vitro in Matrigel (3) The -46kd variant ER-A is the result of splicing deletion of exon 1 in ER-A transcript; Type I HBEC use promoter A while immortal and tumorigenic Type I cells use both promoter A and C for transcription; and (4) high susceptibility of Type I HBEC to telomerase activation and immortalization, a basis on which Type I EBEC (stem cells) are major targets for carcinogenesis.