Evidence of steady-state fibroblast subtypes in the normal human breast as cells-of-origin for perturbed-state fibroblasts in breast cancer.

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.

Ductal keratin 15+ luminal progenitors in normal breast exhibit a basal-like breast cancer transcriptomic signature.

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.

Myoepithelial progenitors as founder cells of hyperplastic human breast lesions upon PIK3CA transformation.

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.

Desensitization of human breast progenitors by a transient exposure to pregnancy levels of estrogen.

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.

Fibroblasts direct differentiation of human breast epithelial progenitors.

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.

Publisher Correction: A CD146 FACS Protocol Enriches for Luminal Keratin 14/19 Double Positive Human Breast Progenitors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A CD146 FACS Protocol Enriches for Luminal Keratin 14/19 Double Positive Human Breast Progenitors.

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.

Aggressiveness of non-EMT breast cancer cells relies on FBXO11 activity.

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.

Proof of region-specific multipotent progenitors in human breast epithelia.

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.

Establishment of a normal-derived estrogen receptor-positive cell line comparable to the prevailing human breast cancer subtype.

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.

Evidence of two distinct functionally specialized fibroblast lineages in breast stroma.

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.

Propagation of oestrogen receptor-positive and oestrogen-responsive normal human breast cells in culture.

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.

Tumor initiating but differentiated luminal-like breast cancer cells are highly invasive in the absence of basal-like activity.

The majority of human breast cancers exhibit luminal epithelial differentiation. However, most aggressive behavior, including invasion and purported cancer stem cell activity, are considered characteristics of basal-like cells. We asked the following questions: Must luminal-like breast cancer cells become basal-like to initiate tumors or to invade? Could luminally differentiated cells within a basally initiated hierarchy also be tumorigenic? To answer these questions, we used rare and mutually exclusive lineage markers to isolate subsets of luminal-like and basal-like cells from human breast tumors. We enriched for populations with or without prominent basal-like traits from individual tumors or single cell cloning from cell lines and recovered cells with a luminal-like phenotype. Tumor cells with basal-like traits mimicked phenotypic and functional behavior associated with stem cells assessed by gene expression, mammosphere formation and lineage markers. Luminal-like cells without basal-like traits, surprisingly, were fully capable of initiating invasive tumors in NOD SCID gamma (NSG) mice. In fact, these phenotypically pure luminal-like cells generated larger and more invasive tumors than their basal-like counterparts. The tumorigenicity and invasive potential of the luminal-like cancer cells relied strongly on the expression of the gene GCNT1, which encodes a key glycosyltransferase controlling O-glycan branching. These findings demonstrate that basal-like cells, as defined currently, are not a requirement for breast tumor aggressiveness, and that within a single tumor there are multiple "stem-like" cells with tumorigenic potential casting some doubt on the hypothesis of hierarchical or differentiative loss of tumorigenicity.

The Na+/H+ exchanger NHE1, but not the Na+, HCO3(-) cotransporter NBCn1, regulates motility of MCF7 breast cancer cells expressing constitutively active ErbB2.

We and others have shown central roles of the Na(+)/H(+) exchanger NHE1 in cell motility. The aim of this study was to determine the roles of NHE1 and of the Na(+), HCO(3)(-) cotransporter NBCn1 in motility of serum-starved MCF-7 breast cancer cells expressing constitutively active ErbB2 (ΔNErbB2). ΔNErbB2 expression elicited NBCn1 upregulation, Ser(703)-phosphorylation of NHE1, and NHE1-inhibitor (EIPA)-sensitive pericellular acidification, in conjunction with increased expression of ß1 integrin and ERM proteins. Active ERM proteins and NHE1 colocalized strongly to invadopodial rosettes, the diameter of which was increased by ΔNErbB2. Adhesion and migration on collagen-I were augmented by ΔNErbB2, unaffected by the NBC inhibitor S0859, and further stimulated by EIPA in a manner potentiated by PI3K-Akt-inhibition. These findings demonstrate that NHE1 inhibition can enhance cancer cell motility, adding an important facet to the understanding of NHE1 in cancer.

Mammary gland stem cells: current status and future challenges.

Distinct subsets of cells, including cells with stem cell-like properties, have been proposed to exist in normal human breast epithelium and breast carcinomas. The cellular origins of epithelial cells contributing to gland development, tissue homeostasis and cancer are, however, still poorly understood. The mouse is a widely used model of mammary gland development, both directly by studying the mouse mammary epithelial cells themselves and indirectly, by studying development, morphogenesis, differentiation and carcinogenesis of xenotransplanted human breast epithelium in vivo. While in early studies, human or mouse epithelium was implanted as fragments into the mouse gland, more recent technical progress has allowed the self-renewal capacity and differentiation potential of distinct cell populations or even individual cells to be interrogated. Here, we review and discuss similarities and differences between mouse and human gland development with particular emphasis on the identity and localization of stem cells, and the influence of the surrounding microenvironment. It is concluded that while recent advances in the field have contributed immense insight into how the normal mammary gland develops and is maintained, significant discrepancies exist between the mouse and human gland which should be taken into consideration in current and future models of mammary stem cell biology.

Epithelial-stromal interaction 1 (EPSTI1) substitutes for peritumoral fibroblasts in the tumor microenvironment.

Tumor cells can activate stroma, yet the implication of this activation in terms of reciprocal induction of gene expression in tumor cells is poorly understood. Epithelial Stromal Interaction 1 (EPSTI1) is an interferon response gene originally isolated from heterotypic recombinant cultures of human breast cancer cells and activated breast myofibroblasts. Here we describe the first immunolocalization of EPSTI1 in normal and cancerous breast tissue, and we provide evidence for a role of this molecule in the regulation of tumor cell properties and epithelial-mesenchymal transition. In general, no EPSTI1 staining was observed in normal breast epithelial cells from reduction mammoplasties (n=25). However, in carcinomas, staining was positive in 22 of 40 biopsies and inversely correlated with the level of differentiation. To address the function of EPSTI1, we expressed EPSTI1 ectopically in one cell line and silenced endogenous EPSTI1 by RNA interference in another. Irrespective of the experimental approach, EPSTI1 expression led to an increase in tumorsphere formation-a property associated with breast stem/progenitor cells. Most remarkably, we show that EPSTI1, by conveying spread of tumor cells, can replace peritumoral activated fibroblasts in a tumor environment assay. These observations implicate EPSTI1 as a hitherto unappreciated regulator of tumor cell properties.

Breast cancer by proxy: can the microenvironment be both the cause and consequence?

Breast cancer is one of the most clear-cut examples of a solid tumor in which systemic cues play a decisive part in its development. The breast tissue is constantly subjected to changes in hormone levels and modifications in the microenvironment. This scenario is even more striking during tumor development because of the dramatic loss or aberration of basement membrane (BM) and myoepithelial cells and the gain of peritumoral myofibroblasts. We suggest that the microenvironment, defined here as all components of the mammary gland other than luminal and/or tumor epithelial cells, might be instrumental in maintaining organ integrity and in promoting, and at times even initiating, breast cancer development. As such, the tumor microenvironment and its constituents, alone or in combination, might serve as promising targets for therapy.

Evidence for a stem cell hierarchy in the adult human breast.

Cellular pathways that contribute to adult human mammary gland architecture and lineages have not been previously described. In this study, we identify a candidate stem cell niche in ducts and zones containing progenitor cells in lobules. Putative stem cells residing in ducts were essentially quiescent, whereas the progenitor cells in the lobules were more likely to be actively dividing. Cells from ducts and lobules collected under the microscope were functionally characterized by colony formation on tissue culture plastic, mammosphere formation in suspension culture, and morphogenesis in laminin-rich extracellular matrix gels. Staining for the lineage markers keratins K14 and K19 further revealed multipotent cells in the stem cell zone and three lineage-restricted cell types outside this zone. Multiparameter cell sorting and functional characterization with reference to anatomical sites in situ confirmed this pattern. The proposal that the four cell types are indeed constituents of an as of yet undescribed stem cell hierarchy was assessed in long-term cultures in which senescence was bypassed. These findings identify an adult human breast ductal stem cell activity and its earliest descendants.

Epithelial progenitor cell lines as models of normal breast morphogenesis and neoplasia.

The majority of human breast carcinomas exhibit luminal characteristics and as such, are most probably derived from progenitor cells within the luminal epithelial compartment. This has been subdivided recently into at least three luminal subtypes based on gene expression patterns. The value of knowing the cellular origin of individual tumours is clear and should aid in designing effective therapies. To do this, however, we need strategies aimed at defining the nature of stem and progenitor cell populations in the normal breast. In this review, we will discuss our technical approach for delineating the origin of the epithelial cell types. A major step forward was the purification of each cell type by the application of immunomagnetic cell sorting based on expression of lineage-specific surface antigens. We then developed chemically defined media that could support either the luminal epithelial or the myoepithelial cell phenotype in primary cultures. Having succeeded in continuous propagation presumably without loss of markers, we could show that a subset of the luminal epithelial cells could convert to myoepithelial cells, signifying the possible existence of a progenitor cell population. By combining the information on marker expression and in situ localization with immunomagnetic sorting and subsequent immortalization, we have identified and isolated a cytokeratin 19-positive suprabasal putative precursor cell in the luminal epithelial compartment and established representative cell lines. This suprabasal-derived epithelial cell line is able to generate both itself and differentiated luminal epithelial and myoepithelial cells, and in addition, is able to form elaborate terminal duct lobular unit (TDLU)-like structures within a reconstituted basement membrane. As more than 90% of breast cancers arise in TDLUs and more than 90% are also cytokeratin 19-positive, we suggest that this cell population contains a breast-cancer progenitor.

Collagen gel contraction serves to rapidly distinguish epithelial- and mesenchymal-derived cells irrespective of alpha-smooth muscle actin expression.

Mesenchymal-like cells in the stroma of breast cancer may arise as a consequence of plasticity within the epithelial compartment, also referred to as epithelial-mesenchymal transition, or by recruitment of genuine mesenchymal cells from the peritumoral stroma. Cells of both the epithelial compartment and the stromal compartment express alpha smooth muscle actin (alpha-sm actin) as part of a myoepithelial or a myofibroblastic differentiation program, respectively. Moreover, because both epithelial- and mesenchymal-derived cells are nontumorigenic, other means of discrimination are warranted. Here, we describe the contraction of hydrated collagen gels as a rapid functional assay for the distinction between epithelial- and mesenchymal-derived stromal-like cells irrespective of the status of alpha-sm actin expression. Three epithelial-derived cell lines and three genuine mesenchymal-derived breast cell lines were plated on top of hydrated collagen lattices. Reduction in gel height was measured every hour for 6 h and after 22 h using an x-y-z automated position table. Significantly, the epithelial-derived cells, irrespective of a high alpha-sm actin expression, had a fivefold lower contractility (10.0% reduction in gel height) than their true mesenchymal counterparts (53.1% reduction in gel height). To test whether at all force generation could be induced in the nonmesenchymal cells by alpha-sm actin, transductions were performed to obtain a tetracycline-dependent expression. Expression under these conditions did not augment contractility. It is concluded that epithelial-derived mesenchymal-like cells are functionally defective within a connective tissue environment irrespective of an apparent contractile phenotype.