The future of mammary stem cell biology: the power of in vivo transplants

The recent review by Smith and Medina [1] of in vivo transplantation models and their role in investigating mammary stem cell (MaSC) biology provides comprehensive coverage of the history and complexity of the ‘gold standard ’ MaSC assay in mice. This includes a description of the pioneering studies that showed that mammary epithelial outgrowths can be generated in cleared mammary fat pads transplanted with explants or admixtures of mammary cells [2]. However, this approach clearly does not lend itself to prospective analysis of isolated subpopulations in order to identify which cells possess in vivo regenerative activity. More recently, success in obtaining complex mammary gland structures from transplanted suspensions of single cells has now made this possible [3-7]. Moreover, the regenerated structures have been shown to contain daughter cells with the same in viv

CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells

INTRODUCTION: Breast cancer is thought to arise in mammary epithelial stem cells. There is, therefore, a large amount of interest in identifying these cells. The breast is a complex tissue consisting of two epithelial layers (an outer myoepithelial/basal layer and an inner luminal epithelial layer) as well as a large non-epithelial component (fibroblasts, endothelial cells, lymphocytes, adipocytes, neurons and myocytes). The definitive identification of a mammary epithelial stem cell population is critically dependent on its purity. To date, this has been hampered by the lack of suitable markers to separate out the two epithelial layers, and to remove contaminating non-epithelial cells. METHODS: Mouse mammary glands were dissociated and stained with CD24. Cells were sorted into separate populations based on CD24 expression and assessed for luminal epithelial and myoepithelial/basal markers by direct fluorescent microscopy and real time PCR. The stem/progenitor potential of these cell populations was assessed in vivo by cleared mammary fat pad transplantation. RESULTS: Three populations of CD24 expressing cells were identified: CD24(Negative), CD24(Low )and CD24(High). Staining of these cells with cytokeratin markers revealed that these populations correspond to non-epithelial, myoepithelial/basal and luminal epithelial cells, respectively. Cell identities were confirmed by quantitative PCR. Cleared mammary fat pad transplantation of these cell populations revealed that extensive mammary fat pad repopulation capacity segregates with the CD24(Low )cells, whilst CD24(High )cells have limited repopulation capacity. CONCLUSION: Differential staining of mammary epithelial cells for CD24 can be used to simultaneously isolate pure populations of non-epithelial, myoepithelial/basal and luminal epithelial cells. Furthermore, mammary fat pad repopulation capacity is enriched in the CD24(Low )population. As separation is achieved using a single marker, it will be possible to incorporate additional markers to further subdivide these populations. This will considerably facilitate the further analysis of mammary epithelial subpopulations, whilst ensuring high purity, which is key for understanding mammary epithelial stem cells in normal tissue biology and carcinogenesis

Interaction between drug and placebo effects: a cross-over balanced placebo design trial

<p>Abstract</p> <p>Background</p> <p>The total effect of a medication is the sum of its drug effect, placebo effect (meaning response), and their possible interaction. Current interpretation of clinical trials' results assumes no interaction. Demonstrating such an interaction has been difficult due to lack of an appropriate study design.</p> <p>Methods</p> <p>180 adults were randomized to caffeine (300 mg) or placebo groups. Each group received the assigned intervention described by the investigators as caffeine or placebo, in a randomized crossover design. 4-hour-area-under-the-curve of energy, sleepiness, nausea (on 100 mm visual analog scales), and systolic blood pressure levels as well as caffeine pharmacokinetics (in 22 volunteers nested in the caffeine group) were determined. Caffeine drug, placebo, placebo-plus-interaction, and total effects were estimated by comparing outcomes after, receiving caffeine described as placebo to receiving placebo described as placebo, receiving placebo described as caffeine or placebo, receiving caffeine described as caffeine or placebo, and receiving caffeine described as caffeine to receiving placebo described as placebo, respectively.</p> <p>Results</p> <p>The placebo effect on area-under-the-curve of energy (mean difference) and sleepiness (geometric mean ratio) was larger than placebo-plus-interaction effect (16.6 [95% CI, 4.1 to 29.0] vs. 8.4 [-4.2 to 21.0] mm*hr and 0.58 [0.39 to 0.86] vs. 0.69 [0.49 to 0.97], respectively), similar in size to drug effect (20.8 [3.8 to 37.8] mm*hr and 0.49 [0.30 to 0.91], respectively), and its combination with the later was larger than total caffeine effect (29.5 [11.9 to 47.1] mm*hr and 0.37 [0.22 to 0.64]). Placebo-plus-interaction effect increased caffeine terminal half-life by 0.40 [0.12 to 0.68] hr (P = 0.007).</p> <p>Conclusions</p> <p>Drug and placebo effects of a medication may be less than additive, which influences the interpretation of clinical trials. The placebo effect may increase active drug terminal half-life, a novel mechanism of placebo action.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov identification number - NCT00426010.</p

A phase I trial of the selective oral cyclin-dependent kinase inhibitor seliciclib (CYC202; R-Roscovitine), administered twice daily for 7 days every 21 days

Seliciclib (CYC202; R-roscovitine) is the first selective, orally bioavailable inhibitor of cyclin-dependent kinases 1, 2, 7 and 9 to enter clinical trial. Preclinical studies showed antitumour activity in a broad range of human tumour xenografts. A phase I trial was performed with a 7-day b.i.d. p.o. schedule. Twenty-one patients (median age 62 years, range: 39–73 years) were treated with doses of 100, 200 and 800 b.i.d. Dose-limiting toxicities were seen at 800 mg b.i.d.; grade 3 fatigue, grade 3 skin rash, grade 3 hyponatraemia and grade 4 hypokalaemia. Other toxicities included reversible raised creatinine (grade 2), reversible grade 3 abnormal liver function and grade 2 emesis. An 800 mg portion was investigated further in 12 patients, three of whom had MAG3 renograms. One patient with a rapid increase in creatinine on day 3 had a reversible fall in renal perfusion, with full recovery by day 14, and no changes suggestive of renal tubular damage. Further dose escalation was precluded by hypokalaemia. Seliciclib reached peak plasma concentrations between 1 and 4 h and elimination half-life was 2–5 h. Inhibition of retinoblastoma protein phosphorylation was not demonstrated in peripheral blood mononuclear cells. No objective tumour responses were noted, but disease stabilisation was recorded in eight patients; this lasted for a total of six courses (18 weeks) in a patient with ovarian cancer

Breast cancer stem cells: tools and models to rely on

There is increasing evidence for the "cancer stem cell (CSC) hypothesis", which holds that cancers are driven by a cellular component that has stem cell properties, including self-renewal, tumorigenicity and multi-lineage differentiation capacity. Researchers and oncologists see in this model an explanation as to why cancer may be so difficult to cure, as well as a promising ground for novel therapeutic strategies. Given the specific stem cell features of self-renewal and differentiation, which drive tumorigenesis and contribute to cellular heterogeneity, each marker and assay designed to isolate and characterize CSCs has to be functionally validated. In this review, we survey tools and markers available or promising to identify breast CSCs. We review the main models used to study breast CSCs and how they challenge the CSC hypothesis

Epigenetic Regulation of Cell Type–Specific Expression Patterns in the Human Mammary Epithelium

Differentiation is an epigenetic program that involves the gradual loss of pluripotency and acquisition of cell type–specific features. Understanding these processes requires genome-wide analysis of epigenetic and gene expression profiles, which have been challenging in primary tissue samples due to limited numbers of cells available. Here we describe the application of high-throughput sequencing technology for profiling histone and DNA methylation, as well as gene expression patterns of normal human mammary progenitor-enriched and luminal lineage-committed cells. We observed significant differences in histone H3 lysine 27 tri-methylation (H3K27me3) enrichment and DNA methylation of genes expressed in a cell type–specific manner, suggesting their regulation by epigenetic mechanisms and a dynamic interplay between the two processes that together define developmental potential. The technologies we developed and the epigenetically regulated genes we identified will accelerate the characterization of primary cell epigenomes and the dissection of human mammary epithelial lineage-commitment and luminal differentiation

Basal cytokeratins and their relationship to the cellular origin and functional classification of breast cancer

Recent publications have classified breast cancers on the basis of expression of cytokeratin-5 and -17 at the RNA and protein levels, and demonstrated the importance of these markers in defining sporadic tumours with bad prognosis and an association with BRCA1-related breast cancers. These important observations using different technology platforms produce a new functional classification of breast carcinoma. However, it is important in developing hypotheses about the pathogenesis of this tumour type to review the nomenclature that is being used to emphasize potential confusion between terminology that defines clinical subgroups and markers of cell lineage. This article reviews the lineages in the normal breast in relation to what have become known as the 'basal-like' carcinomas

MMTV-Wnt1 and -ΔN89β-Catenin Induce Canonical Signaling in Distinct Progenitors and Differentially Activate Hedgehog Signaling within Mammary Tumors

Canonical Wnt/β-catenin signaling regulates stem/progenitor cells and, when perturbed, induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTV-Wnt1 and MMTV-ΔN89β-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-ΔN89β-catenin transgenes induce tumors with different phenotypes. Using axin2/conductin reporter genes we show that MMTV-Wnt1 and MMTV-ΔN89β-catenin activate canonical Wnt signaling within distinct cell-types. ΔN89β-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18+ER−PR−CD24highCD49flow profile and progenitor properties. In contrast, MMTV-Wnt1 induced canonical signaling in K14+ basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types. MMTV-Wnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that large melanocytic nevi are a hitherto unreported hallmark of early hyperplastic Wnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14+/p63+ subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-ΔN89β-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand

Pregnancy in the mature adult mouse does not alter the proportion of mammary epithelial stem/progenitor cells

Introduction In humans, an early full-term pregnancy reduces lifetime breast cancer risk by up to 50% whereas a later pregnancy (>35 years old) can increase lifetime risk. Several mechanisms have been suggested, including changes in levels of circulating hormones, changes in the way the breast responds to these hormones, changes in gene expression programmes which may alter susceptibility to transformation and changes to mammary stem cell numbers or behaviour. Previous studies have shown that the mammary tissue isolated from both virgin and parous mice has the ability to repopulate a cleared mammary fat pad in transplant experiments. Limited dilution transplant assays have demonstrated that early pregnancy (at 5 weeks of age) reduces stem/progenitor cell numbers in the mouse mammary epithelium by twofold. However, the effects on stem/progenitor cell numbers in the mammary epithelium of a pregnancy in older animals have not yet been tested. Methods Mice were put through a full-term pregnancy at 9 weeks of age, when the mammary epithelium is mature. The total mammary epithelium was purified from parous 7-week post-lactation and age-matched virgin mice and analysed by flow cytometry and limiting dilution cleared fat pad transplants. Results There were no significant differences in the proportions of different mammary epithelial cell populations or numbers of CD24+/Low Sca-1- CD49fHigh cells (stem cell enriched basal mammary epithelial compartment). There was no significant difference in stem/progenitor cell frequency based on limiting dilution transplants between the parous and age-matched virgin epithelium. Conclusions Although differences between parous and virgin mammary epithelium at later time points post lactation or following multiple pregnancies cannot be ruled out, there are no differences in stem/progenitor cell numbers between mammary epithelium isolated from parous animals which were mated at 9 weeks old and virgin animals. However, a recent report has suggested that animals that were mated at 5 weeks old have a twofold reduction in stem/progenitor cell numbers. This is of interest given the association between early, but not late, pregnancy and breast cancer risk reduction in humans. However, a mechanistic connection between stem cell numbers and breast cancer risk remains to be established

Phenotypic and Functional Characterization of Human Mammary Stem/Progenitor Cells in Long Term Culture

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. Methodology: Single cell suspensions derived from human breast `organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. Principal Findings: We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. Conclusions: Thus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance