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A relatively rare aldehyde dehydrogenase 1 (ALDH1)-positive stem cell-like" subpopulation of tumor cells has the unique ability to initiate and perpetuate tumor growth; moreover� View Publication

Recent research in breast biology has provided support for the cancer stem-cell hypothesis. Two important components of this hypothesis are that tumors originate in mammary stem or progenitor cells as a result of dysregulation of the normally tightly regulated process of self-renewal. As a result,tumors contain and are driven by a cellular subcomponent that retains key stem-cell properties including self-renewal,which drives tumorigenesis and differentiation that contributes to cellular heterogeneity. Advances in stem-cell technology have led to the identification of stem cells in normal and malignant breast tissue. The study of these stem cells has helped to elucidate the origin of the molecular complexity of human breast cancer. The cancer stem-cell hypothesis has important implications for early detection,prevention,and treatment of breast cancer. Both hereditary and sporadic breast cancers may develop through dysregulation of stem-cell self-renewal pathways. These aberrant stem cells may provide targets for the development of cancer prevention strategies. Furthermore,because breast cancer stem cells may be highly resistant to radiation and chemotherapy,the development of more effective therapies for this disease may require the effective targeting of this cell population. View Publication

Systemic hormones are key regulators of postnatal mammary gland development and play an important role in the etiology and treatment of breast cancer. Mammary ductal morphogenesis is controlled by circulating hormones,and these same hormones are also critical mediators of mammary stem cell fate decisions. Recent studies have helped further our understanding of the origin,specification,and fate of mammary stem cells during postnatal development. Here we review recent studies on the involvement of hormone receptors and several transcription factors in mammary stem/progenitor cell differentiation and lineage commitment. View Publication

In recent years,the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated,but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs,and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor,Gleevec (imatinib mesylate; Novartis International,Basel,Switzerland,http://www.novartis.com),to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5,sarcomeric proteins beta-MHC and MLC-2v,and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy,the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating,myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters. View Publication

In models of acute lung injury,CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands,including CXCL1 and CXCL2/3,are chemotactic for PMNs,CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung,aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice,but not in littermate CXCR2-/- mice. Surprisingly,lethally irradiated wild-type mice reconstituted with CXCR2-/- BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium,but CXCR2-/- mice reconstituted with CXCR2-/- BM showed no PMN recruitment. Conversely,CXCR2-/- mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment,inconsistent with a role of CXCR2 on PMNs alone. Cell culture,immunohistochemistry,flow cytometry,and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury. View Publication

An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically,mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models. The results presented here demonstrate the following points: 1) lobular,i.e. secretory,progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly,ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers,although both cell populations are extremely small; 4) as expected,some inocula produce outgrowths with simultaneous development of both lobular and ductal phenotypes--it is not known whether this indicates cooperative interaction between the two epithelial progenitors or signals the presence of a third progenitor type capable of producing both ductular and lobular committed daughters; 5) these findings have important consequences in the design of experiments aimed at testing the effects of known and putative mammary oncogenes and tumor suppressor genes,using techniques which include cellular transformation in vitro followed by in vivo cultivation and evaluation. View Publication

The aim is to investigate the clinical implications of the Oct-4 and Nestin protein in human breast cancers. A total of 346 cases including 26 fresh and 320 paraffin-embedded tumor tissues were selected for characterizing the frequency of CD44(+)CD24(-) tumor cells by flow cytometry and the differential expression of the stem cell-related genes between CD44(+)CD24(-) and non-CD44(+)CD24(-) tumor cells was analyzed by PCR Array and immunofluorescence. In comparison with the non-CD44(+)CD24(-) tumor cells,the CD44(+)CD24(-),particularly for those with high percentage of Oct-4(+) and Nestin(+),tumor cells had higher tumorigenicity by forming mammospheres in vitro. More importantly,42 (13.125%) out of 320 tumor tissues were positive for Oct-4 and Nestin staining. Universal analysis and multivariate analysis revealed that the expression of Oct-4 and Nestin was associated significantly with younger age,pathogenic degrees,lymph node metastasis and triple-negative breast cancer independently (P textless 0.05) as well as shorter survival (P = 0.001). Oct-4 and Nestin were important regulators of the development of breast cancer,and Oct-4 and Nestin may be used as predictors for the prognosis of breast cancers. View Publication

Little is known about the origin of basal-like breast cancers,an aggressive disease that is highly similar to BRCA1-mutant breast cancers. p63 family proteins that are structurally related to the p53 suppressor protein are known to function in stem cell regulation and stratified epithelia development in multiple tissues,and p63 expression may be a marker of basal-like breast cancers. Here we report that ΔNp63 isoforms of p63 are transcriptional targets for positive regulation by BRCA1. Our analyses of breast cancer tissue microarrays and BRCA1-modulated breast cancer cell lines do not support earlier reports that p63 is a marker of basal-like or BRCA1 mutant cancers. Nevertheless,we found that BRCA1 interacts with the specific p63 isoform ΔNp63γ along with transcription factor isoforms AP-2α and AP-2γ. BRCA1 required ΔNp63γ and AP-2γ to localize to an intronic enhancer region within the p63 gene to upregulate transcription of the ΔNp63 isoforms. In mammary stem/progenitor cells,siRNA-mediated knockdown of ΔNp63 expression resulted in genomic instability,increased cell proliferation,loss of DNA damage checkpoint control,and impaired growth control. Together,our findings establish that transcriptional upregulation of ΔNp63 proteins is critical for BRCA1 suppressor function and that defects in BRCA1-ΔNp63 signaling are key events in the pathogenesis of basal-like breast cancer. View Publication

BACKGROUND: Circulating tumor cells (CTCs) in the peripheral blood of breast cancer patients may be an important indicator of metastatic disease and poor prognosis. However,the use of experimental models is required to fully elucidate the functional consequences of CTCs. The purpose of this study was to optimize the sensitivity of multiparameter flow cytometry for detection of human tumor cells in mouse models of breast cancer. METHODS: MDA-MB-468 human breast cancer cells were serially diluted in whole mouse blood. Samples were lysed and incubated with a fluorescein isothiocyanate-conjugated anti-human leukocytic antigen antibody and a phycoerythrin-conjugated anti-mouse pan-leukocyte CD45 antibody. Samples were then immunomagnetically depleted of CD45-positive leukocytes,fixed,permeabilized,and stained with propidium iodide before flow cytometric analysis. RESULTS: Human breast cancer cells could be differentiated from mouse leukocytes based on increased light scatter,cell surface marker expression,and aneuploid DNA content. The method was found to have a lower sensitivity limit of 10(-5) and was effective for detecting human breast cancer cells in vivo in the circulation of experimental mice carrying primary human mammary tumors. CONCLUSIONS: This technique has the potential to be a valuable and sensitive tool for investigating the biological relevance of CTCs in experimental mouse models of breast cancer. View Publication

BACKGROUND: Cancer stem cells (CSCs) are purported to be epithelial tumour cells expressing CD44(+)CD24(lo) that exhibit aldehyde dehydrogenase activity (Aldefluor(+)). We hypothesised that if CSCs are responsible for tumour dissemination,disseminated cells in the bone marrow (BM) would be positive for putative breast CSC markers. Therefore,we assessed the presence of Aldefluor(+) epithelial (CD326(+)CD45(dim)) cells for the presence of the CD44(+)CD24(lo) phenotype in BM of patients with primary breast cancer (PBC). METHODS: BM aspirates were collected at the time of surgery from 66 patients with PBC. Thirty patients received neoadjuvant chemotherapy (NACT) prior to aspiration. BM was analysed for Aldefluor(+) epithelial cells with or without CD44(+)CD24(lo) expression by flow cytometry. BM aspirates from three healthy donors (HD) were subjected to identical processing and analyses and served as controls. RESULTS: Patients with triple-receptor-negative (TN) tumours had a significantly higher median percentage of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population than patients with other immunohistochemical subtypes (P=0.018). Patients with TN tumours or with pN2 or higher pathologic nodal status were more likely to have a proportion of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population above the highest level of HD. Furthermore,patients who received NACT were more likely to have percentages of Aldefluor(+) epithelial cells than the highest level of HD (P=0.004). CONCLUSION: The percentage of CD44(+)CD24(lo) CSC in the BM is higher in PBC patients with high risk tumour features. The selection or enrichment of Aldefluor(+) epithelial cells by NACT may represent an opportunity to target these cells with novel therapies. View Publication