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BACKGROUND: Human breast cancer most frequently originates within a well-defined anatomical structure referred to as the terminal duct lobular unit (TDLU). This structure is endowed with its very own lobular fibroblasts representing one out of two steady-state fibroblast subtypes-the other being interlobular fibroblasts. While cancer-associated fibroblasts (CAFs) are increasingly appreciated as covering a spectrum of perturbed states, we lack a coherent understanding of their relationship-if any-with the steady-state fibroblast subtypes. To address this, we here established two autologous CAF lines representing inflammatory CAFs (iCAFs) and myofibroblast CAFs (myCAFs) and compared them with already established interlobular- and lobular fibroblasts with respect to their origin and impact on tumor formation. METHODS: Primary breast tumor-derived CAFs were transduced to express human telomerase reverse transcriptase (hTERT) and sorted into CD105low and CD105high populations using fluorescence-activated cell sorting (FACS). The two populations were tested for differentiation similarities to iCAF and myCAF states through transcriptome-wide RNA-Sequencing (RNA-Seq) including comparison to an available iCAF-myCAF cell state atlas. Inference of origin in interlobular and lobular fibroblasts relied on RNA-Seq profiles, immunocytochemistry and growth characteristics. Osteogenic differentiation and bone formation assays in culture and in vivo were employed to gauge for origin in bone marrow-derived mesenchymal stem cells (bMSCs). Functional characteristics were assessed with respect to contractility in culture and interaction with tumor cells in mouse xenografts. The cells' gene expression signatures were tested for association with clinical outcome of breast cancer patients using survival data from The Cancer Genome Atlas database. RESULTS: We demonstrate that iCAFs have properties in common with interlobular fibroblasts while myCAFs and lobular fibroblasts are related. None of the CAFs qualify as bMSCs as revealed by lack of critical performance in bone formation assays. Functionally, myCAFs and lobular fibroblasts are almost equally tumor promoting as opposed to iCAFs and interlobular fibroblasts. A myCAF gene signature is found to associate with poor breast cancer-specific survival. CONCLUSIONS: We propose that iCAFs and myCAFs originate in interlobular and lobular fibroblasts, respectively, and more importantly, that the tumor-promoting properties of lobular fibroblasts render the TDLU an epicenter for breast cancer evolution.
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Neoplasias de la Mama , Fibroblastos Asociados al Cáncer , Humanos , Ratones , Animales , Femenino , Neoplasias de la Mama/patología , Osteogénesis , Fibroblastos/metabolismo , Fibroblastos Asociados al Cáncer/patología , Mama/patología , Microambiente TumoralRESUMEN
The glycan moiety Lewis X (LeX) has been implicated in defining progenitor cells as well as playing a role in the progression of solid tumors, including breast cancer. Here, we used the original stage-specific embryonic antigen-1 (SSEA-1) antibody, MC-480, targeting the LeX motif to examine the expression pattern of this marker within the context of a differentiation hierarchy as well as functional properties of breast cancer cells. Immunohistochemical staining revealed the presence of SSEA-1 in a progenitor zone in the normal breast gland. In breast cancer, 81 of 220 carcinomas (37%) were positive for SSEA-1 and a distinct pattern could be correlated to major subtypes. Specifically, estrogen receptor alpha (ERα)-negative tumors showed a higher frequency of SSEA-1 expression compared to ERα-positive tumors, which are generally considered more differentiated (56% vs 29%, p<0.005). Functional assays performed on two representative breast cancer cell lines demonstrated that SSEA-1-expressing cells exhibited cancer stem cell properties as well as having more invasive potential, regardless of ERα status. A potential role of SSEA-1 in metastasis was confirmed by pairwise staining of primary- and corresponding lymph node tumors. Altogether, our data suggest that expression of SSEA-1 in breast cancer contributes to the malignant phenotype.
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Receptor alfa de Estrógeno , Neoplasias , Humanos , Antígeno Lewis X , Neoplasias/patología , Células Madre Neoplásicas/patología , FenotipoRESUMEN
Normal breast luminal epithelial progenitors have been implicated as cell of origin in basal-like breast cancer, but their anatomical localization remains understudied. Here, we combine collection under the microscope of organoids from reduction mammoplasties and single-cell mRNA sequencing (scRNA-seq) of FACS-sorted luminal epithelial cells with multicolor imaging to profile ducts and terminal duct lobular units (TDLUs) and compare them with breast cancer subtypes. Unsupervised clustering reveals eleven distinct clusters and a differentiation trajectory starting with keratin 15+ (K15+) progenitors enriched in ducts. Spatial mapping of luminal progenitors is confirmed at the protein level by staining with critical duct markers. Comparison of the gene expression profiles of normal luminal cells with those of breast cancer subtypes suggests a strong correlation between normal breast ductal progenitors and basal-like breast cancer. We propose that K15+ basal-like breast cancers originate in ductal progenitors, which emphasizes the importance of not only lineages but also cellular position within the ductal-lobular tree.
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BACKGROUND: Dysregulation of brain fluid homeostasis associates with brain pathologies in which fluid accumulation leads to elevated intracranial pressure. Surgical intervention remains standard care, since specific and efficient pharmacological treatment options are limited for pathologies with disturbed brain fluid homeostasis. Such lack of therapeutic targets originates, in part, from the incomplete map of the molecular mechanisms underlying cerebrospinal fluid (CSF) secretion by the choroid plexus. METHODS: The transcriptomic profile of rat choroid plexus was generated by RNA Sequencing (RNAseq) of whole tissue and epithelial cells captured by fluorescence-activated cell sorting (FACS), and compared to proximal tubules. The bioinformatic analysis comprised mapping to reference genome followed by filtering for type, location, and association with alias and protein function. The transporters and associated regulatory modules were arranged in discovery tables according to their transcriptional abundance and tied together in association network analysis. RESULTS: The transcriptomic profile of choroid plexus displays high similarity between sex and species (human, rat, and mouse) and lesser similarity to another high-capacity fluid-transporting epithelium, the proximal tubules. The discovery tables provide lists of transport mechanisms that could participate in CSF secretion and suggest regulatory candidates. CONCLUSIONS: With quantification of the transport protein transcript abundance in choroid plexus and their potentially linked regulatory modules, we envision a molecular tool to devise rational hypotheses regarding future delineation of choroidal transport proteins involved in CSF secretion and their regulation. Our vision is to obtain future pharmaceutical targets towards modulation of CSF production in pathologies involving disturbed brain water dynamics.
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Encéfalo , Plexo Coroideo , Animales , Transporte Biológico/fisiología , Encéfalo/metabolismo , Líquido Cefalorraquídeo/metabolismo , Plexo Coroideo/metabolismo , Células Epiteliales/metabolismo , Epitelio/metabolismo , Ratones , RatasRESUMEN
The myoepithelial (MEP) lineage of human breast comprises bipotent and multipotent progenitors in ducts and terminal duct lobular units (TDLUs). We here assess whether this heterogeneity impacts on oncogenic PIK3CA transformation. Single cell RNA sequencing (scRNA-seq) and multicolor imaging reveal that terminal ducts represent the most enriched source of cells with ductal MEP markers including α-smooth muscle actin (α-SMA), keratin K14, K17 and CD200. Furthermore, we find neighboring CD200high and CD200low progenitors within terminal ducts. When sorted and kept in ground state conditions, their CD200low and CD200high phenotypes are preserved. Upon differentiation, progenitors remain multipotent and bipotent, respectively. Immortalized progenitors are transduced with mutant PIK3CA on an shp53 background. Upon transplantation, CD200low MEP progenitors distinguish from CD200high by the formation of multilayered structures with a hyperplastic inner layer of luminal epithelial cells. We suggest a model with spatially distributed MEP progenitors as founder cells of biphasic breast lesions with implications for early detection and prevention strategies.
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Neoplasias de la Mama , Células Madre Neoplásicas , Oncogenes , Mama , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Fosfatidilinositol 3-Quinasa Clase I/genética , Células Epiteliales/patología , Femenino , HumanosRESUMEN
Full term pregnancy at an early age is the only factor known to consistently protect against breast cancer. Because hormone receptor positive progenitors in the human breast relay endocrine signaling, we here sought to determine whether an experimental mimicry of the third trimester surge of hormones would change their susceptibility to growth stimulation. Hormone receptor positive, reduction mammoplasty-derived human breast epithelial progenitors were exposed to a short-term, pregnancy-level of estradiol, and their subsequent response to estradiol stimulation was analyzed. Exposure to pregnancy-level of estradiol results in subsequent lower sensitivity to estrogen-induced proliferation. Expression array and immunoblotting reveal upregulation of S100A7 and down-regulation of p27, both associated with parity and epithelial differentiation. Notably, we find that the epithelial differentiation is accompanied by upregulation of E-cadherin and down-regulation of vimentin as well as by diminished migration and more mature luminal epithelial differentiation in a mouse transplantation model. Our findings are in support of a de-sensitization mechanism for pregnancy-induced prevention against breast cancer.
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Mama/efectos de los fármacos , Estradiol/farmacología , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Animales , Mama/citología , Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Cadherinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Cultivadas , Estrógenos/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Embarazo , Receptores de Estrógenos/genética , Receptores de Progesterona/genética , Proteína A7 de Unión a Calcio de la Familia S100/genética , Proteína A7 de Unión a Calcio de la Familia S100/metabolismoRESUMEN
CEACAM5 is overexpressed in many primary breast carcinomas. However, the exact role of CEACAM5 in breast cancer tumorigenesis remains unresolved. Here, we examined a repository of 110 cryopreserved primary breast carcinomas by immunohistochemistry to assess the distribution of CEACAM5 in tumor subtypes. The majority of estrogen receptor-positive and HER2-overexpressing tumors were CEACAM5-positive, whereas most of Triple-negative tumors were negative. Assessing sample sets of paired primary breast cancers and corresponding lymph node lesions from a total of 59 patients revealed a high correlation between primary tumor and lymph node with regard to CEACAM5-status. However, a notable subset of sample sets demonstrated intratumoral heterogeneity in the primary tumor, the metastatic lesion or both, suggesting that both CEACAM5-positive and -negative cells can play a role in tumor dissemination. When examining the consequence of expression of CEACAM5 in breast cancer cell lines in culture assays we found that CEACAM5-expressing cells were less invasive. In survival analysis, using cohort studies of breast cancer, expression of CEACAM5 predicted different clinical outcomes depending on molecular subtypes. Altogether, our analysis suggests that CEACAM5 plays a context-dependent role in breast cancer that warrants further investigation.
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The receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony formation and self-maintenance activities while promoting terminal differentiation in vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl + cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when co-opted, maintains acquired stemness in breast cancer cells.
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BACKGROUND: Breast cancer arises within specific regions in the human breast referred to as the terminal duct lobular units (TDLUs). These are relatively dynamic structures characterized by sex hormone driven cyclic epithelial turnover. TDLUs consist of unique parenchymal entities embedded within a fibroblast-rich lobular stroma. Here, we established and characterized a new human breast lobular fibroblast cell line against its interlobular counterpart with a view to assessing the role of region-specific stromal cues in the control of TDLU dynamics. METHODS: Primary lobular and interlobular fibroblasts were transduced to express human telomerase reverse transcriptase (hTERT). Differentiation of the established cell lines along lobular and interlobular pathways was determined by immunocytochemical staining and genome-wide RNA sequencing. Their functional properties were further characterized by analysis of mesenchymal stem cell (MSC) differentiation repertoire in culture and in vivo. The cells' physiological relevance for parenchymal differentiation was examined in heterotypic co-culture with fluorescence-activated cell sorting (FACS)-purified normal breast primary luminal or myoepithelial progenitors. The co-cultures were immunostained for quantitative assessment of epithelial branching morphogenesis, polarization, growth, and luminal epithelial maturation. In extension, myoepithelial progenitors were tested for luminal differentiation capacity in culture and in mouse xenografts. To unravel the significance of transforming growth factor-beta (TGF-ß)-mediated crosstalk in TDLU-like morphogenesis and differentiation, fibroblasts were incubated with the TGF-ß signaling inhibitor, SB431542, prior to heterotypic co-culture with luminal cells. RESULTS: hTERT immortalized fibroblast cell lines retained critical phenotypic traits in culture and linked to primary fibroblasts. Cell culture assays and transplantation to mice showed that the origin of fibroblasts determines TDLU-like and ductal-like differentiation of epithelial progenitors. Whereas lobular fibroblasts supported a high level of branching morphogenesis by luminal cells, interlobular fibroblasts supported ductal-like myoepithelial characteristics. TDLU-like morphogenesis, at least in part, relied on intact TGF-ß signaling. CONCLUSIONS: The significance of the most prominent cell type in normal breast stroma, the fibroblast, in directing epithelial differentiation is largely unknown. Through establishment of lobular and interlobular fibroblast cell lines, we here demonstrate that epithelial progenitors are submitted to stromal cues for site-specific differentiation. Our findings lend credence to considering stromal subtleties of crucial importance in the development of normal breast and, in turn, breast cancer.
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Neoplasias de la Mama/patología , Mama/citología , Diferenciación Celular , Células Epiteliales/citología , Fibroblastos/citología , Células Madre/metabolismo , Células del Estroma/citología , Adulto , Animales , Mama/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular , Técnicas de Cocultivo , Células Epiteliales/metabolismo , Femenino , Fibroblastos/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre/citología , Células del Estroma/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
Cells of the human breast gland express an array of keratins, of which some are used for characterizing both normal and neoplastic breast tissue. However, the expression pattern of certain keratins has yet to be detailed. Here, the expression of a differentiation marker of epidermal epithelium, keratin 10 (K10), was investigated in the human breast gland. While in normal breast tissue generally less than 1% of luminal epithelial cells expressed K10, in women >30 years of age glandular structures with K10-positive (K10pos) cells were found at higher frequency than in younger women. K10pos cells belong to a mature luminal compartment as they were negative for cKIT, positive for Ks20.8, and mostly non-cycling. In breast cancer, around 16% of primary breast carcinomas tested were positive for K10 by immunohistochemistry. Interestingly, K10pos tumor cells generally exhibit features of differentiation similar to their normal counterparts. Although this suggests that K10 is a marker of tumor differentiation, data based on gene expression analysis imply that high levels of K10 dictate a worse outcome for breast cancer patients. These findings can form the basis of future studies that should unravel which role K10 may play as a marker of breast cancer.
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Neoplasias de la Mama/patología , Mama/citología , Mama/patología , Diferenciación Celular , Epidermis/patología , Regulación Neoplásica de la Expresión Génica , Queratina-10/genética , Biomarcadores/metabolismo , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Humanos , Células MCF-7 , PronósticoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Human breast cancer is believed to arise in luminal progenitors within the normal breast. A subset of these are double positive (DP) for basal and luminal keratins and localizes to a putative stem cell zone within ducts. We here present a new protocol based on a combination of CD146 with CD117 and CD326 which provides an up to thirty fold enrichment of the DP cells. We show by expression profiling, colony formation, and morphogenesis that CD146high/CD117high/CD326high DP cells belong to a luminal progenitor compartment. While these DP cells are located quite uniformly in ducts, with age a variant type of DP (vDP) cells, which is mainly CD146-negative, accumulates in lobules. Intriguingly, in specimens with BRCA1 mutations known to predispose for cancer, higher frequencies of lobular vDP cells are observed. We propose that vDP cells are strong candidates for tracing the cellular origin of breast cancer.
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Neoplasias de la Mama/metabolismo , Carcinogénesis , Queratina-14/metabolismo , Queratina-19/metabolismo , Glándulas Mamarias Humanas/metabolismo , Células Madre Neoplásicas/metabolismo , Adolescente , Adulto , Neoplasias de la Mama/patología , Antígeno CD146/metabolismo , Células Cultivadas , Femenino , Voluntarios Sanos , Humanos , Glándulas Mamarias Humanas/patología , Persona de Mediana Edad , Células Madre Neoplásicas/patología , Adulto JovenRESUMEN
Tumorigenesis is increasingly considered to rely on subclones of cells poised to undergo an epithelial to mesenchymal transition (EMT) program. We and others have provided evidence, however, that the tumorigenesis of human breast cancer is not always restricted to typical EMT cells but is also somewhat paradoxically conveyed by subclones of apparently differentiated, non-EMT cells. Here we characterize such non-EMT-like and EMT-like subclones. Through a loss-of-function screen we found that a member of the E3 ubiquitin ligase complexes, FBXO11, specifically fuels tumor formation of a non-EMT-like clone by restraining the p53/p21 pathway. Interestingly, in the related EMT-like clone, FBXO11 operates through the BCL2 pathway with little or no impact on tumorigenesis. These data command caution in attempts to assess tumorigenesis prospectively based on EMT profiling, and they emphasize the importance of next generation subtyping of tumors, that is at the level of clonal composition.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Transición Epitelial-Mesenquimal/genética , Proteínas F-Box/genética , Proteína-Arginina N-Metiltransferasas/genética , Carcinogénesis/genética , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Humanos , Células MCF-7 , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Cancers develop in a complex mutational landscape. Genetic models of tumor formation have been used to explore how combinations of mutations cooperate to promote tumor formation in vivo. Here, we identify lactate dehydrogenase (LDH), a key enzyme in Warburg effect metabolism, as a cooperating factor that is both necessary and sufficient for epidermal growth factor receptor (EGFR)-driven epithelial neoplasia and metastasis in a Drosophila model. LDH is upregulated during the transition from hyperplasia to neoplasia, and neoplasia is prevented by LDH depletion. Elevated LDH is sufficient to drive this transition. Notably, genetic alterations that increase glucose flux, or a high-sugar diet, are also sufficient to promote EGFR-driven neoplasia, and this depends on LDH activity. We provide evidence that increased LDHA expression promotes a transformed phenotype in a human primary breast cell culture model. Furthermore, analysis of publically available cancer data showed evidence of synergy between elevated EGFR and LDHA activity linked to poor clinical outcome in a number of human cancers. Altered metabolism has generally been assumed to be an enabling feature that accelerates cancer cell proliferation. Our findings provide evidence that sugar metabolism may have a more profound role in driving neoplasia than previously appreciated.
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Proteínas de Drosophila/metabolismo , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Hidroliasas/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/fisiopatología , Neoplasias/metabolismo , Neoplasias/fisiopatología , Receptores de Péptidos de Invertebrados/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Drosophila melanogaster , HumanosRESUMEN
CD117 is a putative marker of luminal progenitor cells in the human breast. However, so far mapping the expression pattern of CD117 within the normal gland has not been reported. Here, we examined the anatomical distribution of CD117-expressing cells in lobular and ductal structures by immunohistochemistry. The presence of CD117-positive luminal cells could be divided into three distinct patterns: (1) contiguous, with coherent positive cells and rare negative cells interspaced; (2) patched, with a roughly equal frequency of positive and negative cells distributed focally; or (3) scattered, with few or no positive cells in the structure. Generally, a patched or scattered expression pattern was more frequent in lobules compared with ducts. Furthermore, an age-correlated increase in heterogeneity was observed. When comparing women below and above 21 years of age this heterogeneity was evident for both lobules and ducts. Although CD117-expression was generally segregated from luminal-lineage transcription factor GATA3-positive cells, some did co-express both markers. Finally, co-staining with Ki-67 revealed that a prominent part of cycling cells belonged to the CD117-positive population. Together these data demonstrate the presence of a CD117-expressing progenitor compartment with the capacity to replenish the luminal lineage of the breast gland.
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Mama/química , Mama/ultraestructura , Proteínas Proto-Oncogénicas c-kit/análisis , Adolescente , Adulto , Envejecimiento , Mama/citología , Neoplasias de la Mama/química , Neoplasias de la Mama/etiología , Proliferación Celular , Femenino , Citometría de Flujo , Factor de Transcripción GATA3/análisis , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Persona de Mediana Edad , Adulto JovenRESUMEN
The human breast parenchyma consists of collecting ducts and terminal duct lobular units (TDLUs). The TDLU is the site of origin of most breast cancers. The reason for such focal susceptibility to cancer remains poorly understood. Here, we take advantage of a region-specific heterogeneity in luminal progenitors to interrogate the differentiation repertoire of candidate stem cells in TDLUs. We show that stem-like activity in serial passage culture and in vivo breast morphogenesis relies on the preservation of a myoepithelial phenotype. By enrichment for region-specific progenitors, we identify bipotent and multipotent progenitors in ducts and TDLUs, respectively. We propose that focal breast cancer susceptibility, at least in part, originates from region-specific myoepithelial progenitors.
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Transformación Celular Neoplásica/genética , Células Epiteliales/citología , Glándulas Mamarias Humanas/citología , Células Madre Multipotentes/citología , Células Musculares/citología , Adolescente , Adulto , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Células Epiteliales/metabolismo , Femenino , Expresión Génica , Humanos , Queratina-19/genética , Queratina-19/metabolismo , Glándulas Mamarias Humanas/metabolismo , Persona de Mediana Edad , Células Madre Multipotentes/metabolismo , Células Musculares/metabolismo , Mioepitelioma/diagnóstico , Mioepitelioma/genética , Mioepitelioma/metabolismo , Mioepitelioma/patología , Especificidad de Órganos , Cultivo Primario de Células , PronósticoRESUMEN
Understanding human cancer increasingly relies on insight gained from subtype specific comparisons between malignant and non-malignant cells. The most frequent subtype in breast cancer is the luminal. By far the most frequently used model for luminal breast cancer is the iconic estrogen receptor-positive (ERpos) MCF7 cell line. However, luminal specific comparisons have suffered from the lack of a relevant non-malignant counterpart. Our previous work has shown that transforming growth factor-ß receptor (TGFßR) inhibition suffices to propagate prospectively isolated ERpos human breast luminal cells from reduction mammoplasties (HBEC). Here we demonstrate that transduction of these cells with hTERT/shp16 renders them immortal while remaining true to the luminal lineage including expression of functional ER (iHBECERpos). Under identical culture conditions a major difference between MCF7 and normal-derived cells is the dependence of the latter on TGFßR inhibition for ER expression. In a breast fibroblast co-culture model we further show that whereas MCF7 proliferate concurrently with ER expression, iHBECERpos form correctly polarized acini, and segregate into proliferating and ER expressing cells. We propose that iHBECERpos may serve to shed light on hitherto unappreciated differences in ER regulation and function between normal breast and breast cancer.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Separación Celular/métodos , Glándulas Mamarias Humanas/metabolismo , Receptores de Estrógenos/metabolismo , Biomarcadores de Tumor/genética , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Comunicación Celular , Línea Celular Transformada , Proliferación Celular , Microambiente Celular , Técnicas de Cocultivo , Estradiol/farmacología , Femenino , Fibroblastos/metabolismo , Genotipo , Humanos , Células MCF-7 , Glándulas Mamarias Humanas/efectos de los fármacos , Fenotipo , Receptores de Estrógenos/efectos de los fármacos , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Telomerasa/genética , Telomerasa/metabolismo , Transducción GenéticaRESUMEN
BACKGROUND: The terminal duct lobular unit (TDLU) is the most dynamic structure in the human breast and the putative site of origin of human breast cancer. Although stromal cells contribute to a specialized microenvironment in many organs, this component remains largely understudied in the human breast. We here demonstrate the impact on epithelium of two lineages of breast stromal fibroblasts, one of which accumulates in the TDLU while the other resides outside the TDLU in the interlobular stroma. METHODS: The two lineages are prospectively isolated by fluorescence activated cell sorting (FACS) based on different expression levels of CD105 and CD26. The characteristics of the two fibroblast lineages are assessed by immunocytochemical staining and gene expression analysis. The differentiation capacity of the two fibroblast populations is determined by exposure to specific differentiating conditions followed by analysis of adipogenic and osteogenic differentiation. To test whether the two fibroblast lineages are functionally imprinted by their site of origin, single cell sorted CD271low/MUC1high normal breast luminal epithelial cells are plated on fibroblast feeders for the observation of morphological development. Epithelial structure formation and polarization is shown by immunofluorescence and digitalized quantification of immunoperoxidase-stained cultures. RESULTS: Lobular fibroblasts are CD105high/CD26low while interlobular fibroblasts are CD105low/CD26high. Once isolated the two lineages remain phenotypically stable and functionally distinct in culture. Lobular fibroblasts have properties in common with bone marrow derived mesenchymal stem cells and they specifically convey growth and branching morphogenesis of epithelial progenitors. CONCLUSIONS: Two distinct functionally specialized fibroblast lineages exist in the normal human breast, of which the lobular fibroblasts have properties in common with mesenchymal stem cells and support epithelial growth and morphogenesis. We propose that lobular fibroblasts constitute a specialized microenvironment for human breast luminal epithelial progenitors, i.e. the putative precursors of breast cancer.
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Fibroblastos/citología , Fibroblastos/metabolismo , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/metabolismo , Células del Estroma/citología , Células del Estroma/metabolismo , Biomarcadores , Diferenciación Celular , Linaje de la Célula , Análisis por Conglomerados , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , FenotipoRESUMEN
Investigating the susceptibility of oestrogen receptor-positive (ER(pos)) normal human breast epithelial cells (HBECs) for clinical purposes or basic research awaits a proficient cell-based assay. Here we set out to identify markers for isolating ER(pos) cells and to expand what appear to be post-mitotic primary cells into exponentially growing cultures. We report a robust technique for isolating ER(pos) HBECs from reduction mammoplasties by FACS using two cell surface markers, CD166 and CD117, and an intracellular cytokeratin marker, Ks20.8, for further tracking single cells in culture. We show that ER(pos) HBECs are released from growth restraint by small molecule inhibitors of TGFß signalling, and that growth is augmented further in response to oestrogen. Importantly, ER signalling is functionally active in ER(pos) cells in extended culture. These findings open a new avenue of experimentation with normal ER(pos) HBECs and provide a basis for understanding the evolution of human breast cancer.
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Mama/citología , Células Epiteliales/citología , Estrógenos/metabolismo , Citometría de Flujo/métodos , Receptores de Estrógenos/metabolismo , Mama/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular , Células Cultivadas , Células Epiteliales/metabolismo , Femenino , HumanosRESUMEN
Elucidating the phenotypic evolution of breast cancer through distinct subtypes relies heavily on defining a lineage blueprint of the normal human breast. Here, we show that in normal breast, within the luminal epithelial lineage, a subset of cells characterized by strong staining for endocrine receptors are also characterized by expression of the surface marker CEACAM6. Topographically, this pattern of staining predominates in terminal ductal lobular units, rather than in interlobular ducts. In culture, CEACAM6-expressing cells remain essentially postmitotic under conditions in which the other cells of luminal epithelial lineage are highly proliferative. We examined the pattern of expression among three major breast cancer subtypes: luminal, HER2-enriched, and basal-like. In 104 biopsies, the luminal and HER2-enriched subtypes showed a high proportion of CEACAM6(+) tumors (78% and 83%, respectively); the basal-like subtype showed a low proportion (28%). Further accentuation of this pattern was observed in 13 established breast cancer cell lines. When differentiation was induced by all-trans retinoic acid, CEACAM6 expression strongly correlated with luminal-like differentiation. Furthermore, CEACAM6(+) cancer cells were less proliferative than CEACAM6(-) cells in tumorsphere assays and were less tumorigenic in nude mice. Based on these observations, we propose that luminal and HER2-enriched breast cancers are more closely related than previously thought and may share a common cell of origin.