Prospective Isolation and Functional Analysis of Stem and Differentiated Cells from the Mouse Mammary Gland

Prospective isolation and in vitro and in vivo analysis of primary mouse mammary epithelial cells has been used to separate cell subpopulations and identify stem, progenitor and differentiated cell compartments. Progress has been made from cell separation strategies based on a single marker of the luminal epithelial or myoepithelial compartments to use of markers that allow simultaneous isolation of non-epithelial, basal/myoepithelial and luminal epithelial cells. Transplant analysis has shown that mammary stem cells are found in the basal/myoepithelial compartment, whereas in vitro colony progenitors are found in the luminal compartment. A basal population enriched for stem cell activity can be purified from the myoepithelial cells and the most recent data shows that the luminal population can now be prospectively split into estrogen receptor positive and estrogen receptor negative cells. Future work aims to molecularly characterise these populations to identify new drug targets, which can be used to specifically kill breast cancer stem cells.     

Neural EGFL like 2 expressed in myoepithelial cells and suppressed breast cancer cell migration

Breast tissue has a branching structure that contains double-layered cells, consisting primarily of luminal epithelial cells inside and myoepithelial cells outside. Ductal carcinoma in situ (DCIS) still has myoepithelial cells surrounding the cancer cells. However, myoepithelial cells disappear in invasive ductal carcinoma. In this study, we detected expression of neural EGFL like (NELL) 2 and one of its receptors, roundabout guidance receptor (ROBO) 3, in myoepithelial and luminal epithelial cells (respectively) in normal breast tissue. NELL2 also was expressed in myoepithelial cells surrounding the non-cancerous intraductal proliferative lesions and DCIS. However, the expression level and proportion of NELL2-positive cells in DCIS were lower than those in normal and non-cancerous intraductal proliferative lesions. ROBO3 expression was decreased in invasive ductal carcinoma compared to that in normal and non-cancerous intraductal proliferative lesions. An evaluation of NELL2's function in breast cancer cell lines demonstrated that full-length NELL2 suppressed cell adhesion and migration in vitro. In contrast, the N-terminal domain of NELL2 increased cell adhesion in the early phase and migration in vitro in some breast cancer cells. These results suggested that full-length NELL2 protein, when expressed in myoepithelial cells, might serve as an inhibitor of breast cancer cell migration.     

An immunohistochemical study of the breast using antibodies to basal and luminal keratins,alpha-smooth muscle actin,vimentin, collagen IV and laminin

Summary The distribution of simple epithelial (K8/18/ 19) and basal (myoepithelial) (K5/14) keratins, -smooth-muscle actin, vimentin, collagen IV and laminin in normal mammary glands and in benign proliferative lesions was studied using monoclonal antibodies (mAbs). These antibodies (Abs) identified myoepithelial cells and luminal cells specifically. In lesions with adenosis and papillomas, the two-layered formation resembled that of normal glands with a purely myoepithelial-epithelial differentiation. In scleradenotic lesions, the main cell was of myoepithelial immunophenotype with intermixed trabecular-tubular proliferations of simple-type epithelium. The sclerosis seems to be the result of an irregular basal lamina synthesis by the myoepithelial cells. In contrast to these lesions, epitheliosis represents a purely intraluminal cell proliferation of clearly simple epithelial immunophenotype and of cells with a basal keratin phenotype, lacking myoepithelial differentiation antigen actin. The basal keratin type epithelium may represent post-stem or intermediate cells developing into luminal epithelium. Epitheliosis appears to be a purely epithelial hyperplasia with striking similarity to the regeneration of normal breast epithelium. The different proliferative patterns may give an explanation for differences in potential cancer risks of patients with these lesions.Dedicated to Prof. Dr. G. Seifert on the occasion of his 70th birthday     

Analysis of clonal expansions through the normal and premalignant human breast epithelium reveals the presence of luminal stem cells

It is widely accepted that the cell of origin of breast cancer is the adult mammary epithelial stem cell; however, demonstrating the presence and location of tissue stem cells in the human breast has proved difficult. Furthermore, we do not know the clonal architecture of the normal and premalignant mammary epithelium or its cellular hierarchy. Here, we use deficiency in the mitochondrial enzyme cytochrome c oxidase (CCO), typically caused by somatic mutations in the mitochondrial genome, as a means to perform lineage tracing in the human mammary epithelium. PCR sequencing of laser‐capture microdissected cells in combination with immunohistochemistry for markers of lineage differentiation was performed to determine the clonal nature of the mammary epithelium. We have shown that in the normal human breast, clonal expansions (defined here by areas of CCO deficiency) are typically uncommon and of limited size, but can occur at any site within the adult mammary epithelium. The presence of a stem cell population was shown by demonstrating multi‐lineage differentiation within CCO‐deficient areas. Interestingly, we observed infrequent CCO deficiency that was restricted to luminal cells, suggesting that niche succession, and by inference stem cell location, is located within the luminal layer. CCO‐deficient areas appeared large within areas of ductal carcinoma in situ, suggesting that the rate of clonal expansion was altered in the premalignant lesion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Functional consequences of cyclin D1 overexpression in human mammary luminal epithelial cells

The proliferation of eukaryotic cells is primarily regulated by a decision made during the G1 phase of the cell cycle as to remain in the cycle and divide, or to withdraw from the cycle and adopt a different cell fate. During this time, environmental signals, which regulate the synthesis of the G1 cyclins, are coupled to cell division. In this context, mammalian D-type cyclins have been shown to control progression through the G1 phase of the mammalian cell cycle. Specifically, cyclin D1 has been reported frequently to be amplified, over-transcribed and overexpressed in human breast carcinomas. Although the effects of cyclin D1 overexpression have been examined in human breast carcinoma cell lines, the biological consequences of cyclin D1 expression in normal human mammary epithelial cells remain to be elucidated. In this study we have stably over expressed cyclin D1 in human mammary luminal epithelial cells in order to more directly address the role of cyclin D1 in cell cycle control and tumorigenesis of the human breast. Here, we demonstrate that the effect of cyclin D1 overexpression in these cells is to reduce their growth factor dependency, as well as shorten the duration of G1 and correspondingly reduce the mean generation time. Collectively, our data indicate that deregulation of cyclin D1 expression in human mammary epithelial cells can provide a growth advantage and hence contribute to the oncogenic potential of these cells.     

Functional consequences of cyclin D1 overexpression in human mammary luminal epithelial cells

The proliferation of eukaryotic cells is primarily regulated by a decision made during the G1 phase of the cell cycle as to remain in the cycle and divide, or to withdraw from the cycle and adopt a different cell fate. During this time, environmental signals, which regulate the synthesis of the G1 cyclins, are coupled to cell division. In this context, mammalian D-type cyclins have been shown to control progression through the G1 phase of the mammalian cell cycle. Specifically, cyclin D1 has been reported frequently to be amplified, over-tran-scribed and overexpressed in human breast carcinomas. Although the effects of cyclin D1 overexpression have been examined in human breast carcinoma cell lines, the biological consequences of cyclin D1 expression in normal human mammary epithelial cells remain to be elucidated. In this study we have stably over expressed cyclin D1 in human mammary luminal epithelial cells in order to more directly address the role of cyclin D1 in cell cycle control and tumorigenesis of the human breast. Here, we demonstrate that the effect of cyclin D1 overexpression in these cells is to reduce their growth factor dependency, as well as shorten the duration of G1 and correspondingly reduce the mean generation time. Collectively, our data indicate that deregula-tion of cyclin D1 expression in human mammary epithelial cells can provide a growth advantage and hence contribute to the oncogenic potential of these cells. © Rapid Science Ltd.     

RHAMM inhibits cell migration via the AKT/GSK3β/Snail axis in luminal A subtype breast cancer

Breast cancer is one of the most common types of cancer in women. Although the mortality rate of breast cancer has fallen over the past 10 years, effective treatments that reduce the occurrence of breast cancer metastasis remain lacking. In this study, we explored the role of receptor for hyaluronan mediated motility (RHAMM) and the associated signaling pathway in cell migration in luminal A breast cancer. We first examined RHAMM expression levels using human breast tissue microarray and patient breast tissues. We then studied the role of RHAMM in migration in luminal A breast cancer using loss-of-function and gain-of-function strategies in in vitro models and confirmed these findings in an in vivo model. Finally, we investigated signaling molecules that play a role in cell migration using western blot. Our results demonstrated the following: (a) RHAMM shows high expression levels in malignant breast tissue, (b) RHAMM shows low expression levels in luminal A breast cancer compared to other subtypes of breast cancer, (c) RHAMM inhibits cell migration in luminal A breast cancer, and (d) RHAMM inhibits cell migration via the AKT/GSK3β/Snail axis in luminal A breast cancer. This study demonstrates a novel role of RHAMM in cell migration in luminal A breast cancer and suggests that therapeutic strategies involving RHAMM should be considered for various subtypes of breast cancer.     

Stem cells in breast epithelia

The rodent and human nonpregnant mammary glands contain epithelial, intermediate and myoepithelial cells which have all been isolated as cell lines in vitro. Transforming growth factor-α (TGFα) and basic fibroblast growth factor (bFGF) are produced by myoepithelial cells and can stimulate the growth of intermediate stem cells in vitro. Epithelial and intermediate cells behave like stem cells in vitro, since they can differentiate into alveolar-like and myoepithelial cells. The myoepithelial differentiation pathway is associated with the early expression of a calcium-binding regulatory protein called p9Ka and the protease, Cathepsin D. Myoepithelial cells are also present in benign lesions but not in malignant mammary carcinomas of rats or humans, whose resultant cell lines fail to differentiate completely along the myoepithelial cell pathway. Loss of the myoepithelial cell in some invasive carcinomas may be compensated, at least in part, by changes in malignant cells. Over-expression of TGFα and/or erbB receptors may reduce the requirement for TGFα, whilst ectopic production of bFGF and its receptors and p9Ka/Cathespin D may assist in tumorigenesis and in metastasis, respectively. Thus compensation for, or retention of, molecules potentially involved in the differentiation of mammary cells may be a mechanism by which malignancy progresses in some human invasive carcinomas.     

Immunohistochemical expression of monoclonal antibodies (epi-1 and myo-1) derived from human breast cancer against rat mammary tumors.

Immunohistochemical expression of monoclonal antibodies epi-1 and myo-1 derived from human breast cancer cell line (HBC-4W) was examined for DMBA-induced rat mammary tumors. Antibody epi-1 reacted with luminal epithelial cells while antibody myo-1 reacted with myoepithelial cells of the mammary glands in rats, respectively. The reactions with both antibodies were markedly visible, in particular, in the normal mammary gland, tumor-like lesions and benign epithelial mammary tumors in rats, which showed clear two-cell-type structures. Among malignant mammary tumors, adenocarcinoma was strongly positive with antibodies epi-1 and myo-1. However, squamous cell carcinoma and adenoacanthoma mainly reacted with antibody epi-1. On the other hand, the intercellular matrices of pleomorphic cell sarcoma and stromal areas of the normal mammary gland or epithelial tumors were positive with antibody myo-1.     

CD109, a new marker for myoepithelial cells of mammary, salivary, and lacrimal glands and prostate basal cells

The CD109 gene encodes a glycosylphosphatidylinositol (GPI)-anchored cell surface protein. Herein it is shown that CD109 is highly expressed in myoepithelial cells of mammary, salivary, and lacrimal glands; and in prostate basal cells. The anti-CD109 antibody generated by the authors was available for formalin-fixed paraffin section, and it strongly stained myoepithelial cells and basal cells but not ductal, acinar, and secretory cells in these glands. CD109 expression was negative in examined breast ductal carcinomas and prostate adenocarcinomas. These findings indicate that CD109 is a useful marker for the diagnosis of invasive breast and prostate carcinomas.     

Efficacy and mechanism of action of Deguelin in suppressing metastasis of 4T1 cells

Cancer related deaths in breast cancer patients are due to metastasis of the disease. Murine 4T1 cells (Murine mammary cancer cell line developed from 6-thioguanine resistant tumor) provide an excellent research tool for metastasis related studies because these cells are highly aggressive and readily metastasize to the lungs. In this study we determined the effect of Deguelin on in vivo/vitro growth and metastasis of 4T1 cells. Deguelin inhibited the in vitro growth of 4T1 cells in a time and dose dependent manner accompanied with reduced nuclear PCNA immunostaining. In cells treated with Deguelin, reduced expression of nuclear c-Met, and its downstream targets such p-ERK and p-AKT was observed. Deguelin reduced the cell migration in 4T1 cells as determined by scratch wound assay. Combined treatment with Deguelin + ERK or PI3K/AKT inhibitor had no additional effect on cell migration. These results indicated that the action of Deguelin on cell migration may be mediated by AKT and ERK mediated signaling pathways. In vivo, Deguelin treatment significantly inhibited growth of 4T1 cells. Deguelin also reduced the occurrence of metastatic lung lesions by 33 % when cells were injected intravenously into Balb/c female mice. There was no difference in the body weight, nor was there a difference in liver and spleen weights between vehicle treated-control and Deguelin-treated animals, which indicated that Deguelin was nontoxic at the dose used in the present study. These results provide rationale for developing Deguelin as a chemotherapeutic agent for triple negative breast cancer patients.     

AKT1负性调控乳腺癌细胞迁移的机制研究

目的:明确AKT1负性调控乳腺癌细胞迁移的分子机制,为乳腺癌转移的防治提供新思路和新靶点。方法:以乳腺癌MCF-7细胞和MDA-MB-231细胞为研究对象,采用RNA干扰技术敲减AKT1的表达,Western blot检测AKT1总蛋白、β-连环蛋白(β-catenin)总蛋白和核蛋白的表达,免疫荧光检测β-catenin在乳腺癌细胞中的定位,Transwell迁移实验探究β-catenin的核转位是否参与AKT1负性调控乳腺癌细胞迁移的过程。结果:敲减AKT1表达后,β-catenin总蛋白与核蛋白的表达明显上调,乳腺癌MCF-7细胞和MDA-MB-231细胞的迁移能力也明显增强,使用XAV-939抑制β-catenin的核移位后,敲减AKT1表达引起的乳腺癌细胞迁移能力的增强也明显下降。结论:AKT1对乳腺癌细胞迁移的负性调控可能是由β-catenin核转位介导的。     

Genetic alterations in 'normal' luminal and myoepithelial cells of the breast

Chromosomal loci exhibiting loss of heterozygosity (LOH) at high frequency in invasive breast cancer have been investigated in 'normal' breast tissue from patients with carcinoma and from reduction mammoplasty specimens. Duct-lobular units dissected from paraffin-embedded tissues and 485 'normal' luminal and myoepithelial cell clones were studied. Overall, LOH was found in normal cells in 5/10 breast cancer cases and 1/3 reduction mammoplasty specimens. LOH was identified in normal cells adjacent to and distant from the tumour. In one case, all luminal and myoepithelial samples exhibited loss of the same allele on chromosome 13q. One case in which the patient had a germline truncating mutation in the BRCA1 gene exhibited LOH on 17q in 3/33 normal clones. One of these clones showed loss of wild-type allele indicating gene inactivation. This sample also had LOH at markers on chromosomes 11p and 13q. One of 93 clones from three reduction mammoplasties showed allele loss at a locus on chromosome 13q. The identification of LOH in breast lobules suggests that they may be clonal. The demonstration of genetic alteration in luminal and myoepithelial cells provides evidence for the presence of a common stem cell for the two epithelial cell types. LOH has been demonstrated in normal tissues near and away from the carcinoma, suggesting that genetic alterations are likely to be more heterogeneous and widespread than is currently envisaged, and probably occur very early in breast development. Homozygous deletion of BRCA1 per se does not appear to provide clonal advantage.     

Cystic duct dilatations and proliferative epithelial lesions in mouse mammary glands upon keratin 5 promoter-driven overexpression of cyclooxygenase-2

Expression and pharmacological studies support a contribution of cyclooxygenase (COX)-2 to mammary gland tumorigenesis. In a recent transgenic study, mouse mammary tumor virus promoter-driven COX-2 expression in mouse mammary glands was shown to result in alveolar hyperplasia, dysplasia, and carcinomas after multiple rounds of pregnancy and lactation. In the study presented here, the effects of constitutive COX-2 overexpression in keratin 5-positive myoepithelial and luminal cells, driven by the keratin 5 promoter in a hormone-independent manner, was investigated. In nulliparous female mice, aberrant COX-2 overexpression correlated with increased prostaglandin (PG) E(2) levels and caused cystic duct dilatations, adenosis, and fibrosis whereas carcinomas developed rarely. This phenotype depended on COX-2-mediated PGE(2) synthesis and correlated with increased expression of proliferation-associated Ki67 in epithelial cells. No changes in the expression of apoptosis-related Bcl-2, caspase 3, or p53 were observed. Hyperproliferation of the mammary gland epithelial cells was associated with increased aromatase mRNA levels in this tissue. The spontaneous pathologies bear analogies to the human breast with fibrocystic changes. Intriguingly, strong COX-2 expression was observed in fibrocystic changes, as compared to low expression in normal breast epithelium. These results show for the first time that aberrant COX-2 expression contributes to the development of fibrocystic changes (FC), indicating that COX-2 and COX-2-mediated PG synthesis represent potential targets for the therapy of this most frequent benign disorder of the human breast.     

Deregulated activation of matriptase in breast cancer cells

Matriptase is an epithelial-derived, cell surface serine protease. This protease activates hepatocyte growth factor (HGF) and urokinase plasminogen activator (uPA), two proteins thought to be involved in the growth and motility of cancer cells, particularly carcinomas, and in the vascularization of tumors. Thus, matriptase may play an important role in the progression of carcinomas, such as breast cancer. We examined the regulation of activation of matriptase in human breast cancer cells, in comparison to non-transformed mammary epithelial cells 184A1N4 and MCF-10A. Results clearly indicated that unlike non-transformed mammary epithelial cells, breast cancer cells do not respond to the known activators of matriptase, serum and sphingosine 1-phosphate (S1P). Similar levels of activated matriptase were detected in breast cancer cells, grown in the presence or absence of S1P. However, up to five-fold higher levels of activated matriptase were detected in the conditioned media from the cancer cells grown in the absence of serum and S1P, when compared to non-transformed mammary epithelial cells. S1P also induces formation of cortical actin structures in non-transformed cells, but not in breast cancer cells. These results show that in non-transformed cells, S1P induces a rearrangement of the actin cytoskeleton and stimulates proteolytic activity on cell surfaces. In contrast, S1P treatment of breast cancer cells does not activate matriptase, and instead these cells constitutively activate the protease. In addition, breast cancer cells respond differently to S1P in terms of the regulation of actin cytoskeletal structures. Matriptase and its cognate inhibitor, HGF activator inhibitor 1 (HAI-1) colocalize on the cell periphery of breast cancer cells and form stable complexes in the extracellular milieu, suggesting that the inhibitor serves to prevent undesired proteolysis in these cells. Finally, we demonstrate that treatment of T-47D cells with epidermal growth factor (EGF), which promotes cell ruffling, stimulates increased accumulation of activated matriptase at the sites of membrane ruffling, suggesting a possible functional role at these sites. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties

The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC(+)) and epithelial-specific antigen (ESA(+)) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC(-)/ESA(+)). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC(+)/ESA(+) epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC(-)/ESA(+) epithelial cell line was able to generate itself as well as MUC(+)/ESA(+) epithelial cells and Thy-1(+)/alpha-smooth muscle actin(+) (ASMA(+)) myoepithelial cells. The MUC(-)/ESA(+) epithelial cell line further differed from the MUC(+)/ESA(+) epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC(+)/ESA(+) epithelial cell line formed acinus-like spheres. In contrast, the MUC(-)/ESA(+) epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by inoculating the extracellular matrix-embedded cells subcutaneously in nude mice. Thus, MUC(-)/ESA(+) epithelial cells within the luminal epithelial lineage may function as precursor cells of terminal duct lobular units in the human breast.     

Epithelial and mesenchymal subpopulations within normal basal breast cell lines exhibit distinct stem cell/progenitor properties

It has been proposed that epithelial-mesenchymal transition (EMT) in mammary epithelial cells and breast cancer cells generates stem cell features, and that the presence of EMT characteristics in claudin-low breast tumors reveals their origin in basal stem cells. It remains to be determined, however, whether EMT is an inherent property of normal basal stem cells, and if the presence of a mesenchymal-like phenotype is required for the maintenance of all their stem cell properties. We used nontumorigenic basal cell lines as models of normal stem cells/progenitors and demonstrate that these cell lines contain an epithelial subpopulation ("EpCAM+," epithelial cell adhesion molecule positive [EpCAM(pos)]/CD49f(high)) that spontaneously generates mesenchymal-like cells ("Fibros," EpCAM(neg)/CD49f(med/low)) through EMT. Importantly, stem cell/progenitor properties such as regenerative potential, high aldehyde dehydrogenase 1 activity, and formation of three-dimensional acini-like structures predominantly reside within EpCAM+ cells, while Fibros exhibit invasive behavior and mammosphere-forming ability. A gene expression profiling meta-analysis established that EpCAM+ cells show a luminal progenitor-like expression pattern, while Fibros most closely resemble stromal fibroblasts but not stem cells. Moreover, Fibros exhibit partial myoepithelial traits and strong similarities with claudin-low breast cancer cells. Finally, we demonstrate that Slug and Zeb1 EMT-inducers control the progenitor and mesenchymal-like phenotype in EpCAM+ cells and Fibros, respectively, by inhibiting luminal differentiation. In conclusion, nontumorigenic basal cell lines have intrinsic capacity for EMT, but a mesenchymal-like phenotype does not correlate with the acquisition of global stem cell/progenitor features. Based on our findings, we propose that EMT in normal basal cells and claudin-low breast cancers reflects aberrant/incomplete myoepithelial differentiation.     

Establishment of monoclonal antibodies reactive with epithelial and myoepithelial cells in the breast

Monoclonal antibodies which are considered to be able to differentiate epithelial and myoepithelial cells in the breast have been developed. Human mammary carcinoma cell line (HBC-4W) was used for immunization. Monoclonal antibodies-B4B2F10 (epi-1), E9E8B7 (myo-1)-with IgM was examined using tissues from diseased breast by avidin-biotin-peroxidase assay. Epi-1 antibody reacted with epithelial cells while myo-1 antibody reacted with myoepithelial cells in the mammary glands, respectively. The reaction was markedly visible, in particular, in fibroadenoma, mastopathy, and papilloma, which showed clear two-cell-type structures. In the infiltrating ductal carcinoma, epi-1 antibody reacted with carcinoma cells, while myo-1 antibody reacted with stromal cells rather than carcinoma cells suggesting that infiltrating ductal carcinoma was mainly of epithelial origin. In the infiltrating lobular carcinoma, however, myo-1 as well as epi-1 antibodies reacted with carcinoma cells. It is suggested that infiltrating lobular carcinoma was of a mixture of epithelial and myoepithelial cell origin.     

Luminal and cancer cells in the breast show more rapid telomere shortening than myoepithelial cells and fibroblasts

Critically shortened, dysfunctional telomeres may play a role in the genetic instabilities commonly found in cancer. We analyzed 30 surgical specimens of invasive breast carcinoma from women aged 34 to 91 years and estimated telomere lengths as telomere-to-centromere ratio values in the 5 different cell types comprising breast tissue in order to clarify telomere length variations within and between individuals using our tissue quantitative fluorescence in situ hybridization method. We obtained 3 novel findings. (1) In corresponding normal tissues, telomere length decreased in the order myoepithelial cells > normal-appearing fibroblasts > luminal epithelial cells, and telomere lengths were characteristic in these 3 cell types within each individual. (2) As expected, cancer cells had significantly shorter telomeres than myoepithelial cells (P < .0001) and normal-appearing fibroblasts (P = .0161), but there was no significant difference in telomere length between luminal cells and cancer cells (P = .6270). (3) Fibroblasts adjacent to cancer had longer telomeres than normal-appearing fibroblasts distant from cancer (P < .0001). This study, which represents the first reported assessment of telomere length variations in the 5 cell types comprising breast tissue within and between individuals, revealed that normal luminal epithelial cells and cancer cells had the shortest telomeres. Our new findings indicate that telomeres of background luminal cells are as short as those of cancer cells. Tissue quantitative fluorescence in situ hybridization, applicable to analysis of individual cells in tissue sections, is considered to be a powerful technique with considerable promise for studies in oncology.     

Immunohistochemical localisation of keratin and luminal epithelial antigen in myoepithelial and luminal epithelial cells of human mammary and salivary gland tumours

Rabbit antisera to human 40-63 000 MW epidermal keratin, one batch with restricted distribution of reactivity from an initial (aK1) and one with "broad spectrum" distribution of reactivity from a late bleeding (aK), and to "luminal epithelial antigen" (aLEA) were applied to formalin fixed paraffin embedded sections of human normal and neoplastic mammary and salivary glands using an indirect immunoperoxidase method. aK1 reacted with myoepithelial cells, aLEA with luminal epithelial cells and aK with both cell types in normal mammary and salivary gland. In breast carcinomas the majority of intraluminal and infiltrating carcinoma cells reacted with aLEA but not with aK1 which reacted only with surrounding myoepithelial cells. aK reacted with both myoepithelial cells and with intraluminal and infiltrating tumour cells. In the salivary gland adenomas the majority of cells reacted with aK, and those cells arranged in a tubular fashion reacted with aLEA.