Configurational entropy is an intrinsic driver of tissue structural heterogeneity.

Tissues comprise ordered arrangements of cells that can be surprisingly disordered in their details. How the properties of single cells and their microenvironment contribute to the balance between order and disorder at the tissue-scale remains poorly understood. Here, we address this question using the self-organization of human mammary organoids as a model. We find that organoids behave like a dynamic structural ensemble at the steady state. We apply a maximum entropy formalism to derive the ensemble distribution from three measurable parameters - the degeneracy of structural states, interfacial energy, and tissue activity (the energy associated with positional fluctuations). We link these parameters with the molecular and microenvironmental factors that control them to precisely engineer the ensemble across multiple conditions. Our analysis reveals that the entropy associated with structural degeneracy sets a theoretical limit to tissue order and provides new insight for tissue engineering, development, and our understanding of disease progression.

Early growth response 2 (EGR2) is a novel regulator of the senescence programme.

Senescence, a state of stable growth arrest, plays an important role in ageing and age-related diseases in vivo. Although the INK4/ARF locus is known to be essential for senescence programmes, the key regulators driving p16 and ARF transcription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up-regulated during senescence, and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates the ARF and p16 promoters and directly binds to both the ARF and p16 promoters. Loss of EGR2 down-regulates p16 levels and increases the pool of p16- p21- 'reversed' cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a direct transcriptional activator of the p16/pRB and ARF/p53/p21 pathways in senescence and a novel marker of senescence.

Evidence for accelerated aging in mammary epithelia of women carrying germline BRCA1 or BRCA2 mutations.

During aging in the human mammary gland, luminal epithelial cells lose lineage fidelity by expressing markers normally expressed in myoepithelial cells. We hypothesize that loss of lineage fidelity is a general manifestation of epithelia that are susceptible to cancer initiation. In the present study, we show that histologically normal breast tissue from younger women who are susceptible to breast cancer, as a result of harboring a germline mutation in BRCA1, BRCA2 or PALB2 genes, exhibits hallmarks of accelerated aging. These include proportionately increased luminal epithelial cells that acquired myoepithelial markers, decreased proportions of myoepithelial cells and a basal differentiation bias or failure of differentiation of cKit+ progenitors. High-risk luminal and myoepithelial cells are transcriptionally enriched for genes of the opposite lineage, inflammatory- and cancer-related pathways. We have identified breast-aging hallmarks that reflect a convergent biology of cancer susceptibility, regardless of the specific underlying genetic or age-dependent risk or the associated breast cancer subtype.

Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures.

The ability to culture normal human mammary epithelial cells (HMEC) greatly facilitates experiments that seek to understand both normal mammary cell biology and the many differences between normal and abnormal human mammary epithelia. To maximize in vivo relevance, the primary cell culture conditions should maintain cells in states that resemble in vivo as much as possible. Towards this goal, we compared the properties of HMEC strains from two different reduction mammoplasty tissues that were grown in parallel using different media and culture conditions. Epithelial organoids were initiated into three different media: two commonly used serum-free-media, MCDB 170-type (e.g. MEGM) and WIT-P, and a low stress media, M87A. Growth, lineage heterogeneity, p16 protein expression, and population doublings to senescence were measured for each culture condition. MCDB 170 caused rapid senescence and loss of heterogeneity within 2 to 3 passages, but some cultures went through the 1 to 2 month process of selection to generate clonal finite post-selection post-stasis cells. WIT-P caused impressive expansion of luminal cells in 2nd passage followed by their near complete disappearance by passage 4 and senescence shortly thereafter. M87A supported as much as twice the number of population doublings compared to either serum-free medium, and luminal and myoepithelial cells were present for as many as 8 passages. Thus, of the three media compared, WIT-P and MCDB 170 imposed rapid senescence and loss of lineage heterogeneity, phenotypes consistent with cells maintained in high-stress conditions, while M87A supported cultures that maintained multiple lineages and robust growth for up to 60 population doublings. In conjunction with previous studies examining the molecular properties of cultures grown in these media, we conclude that M87A medium is most able to support long-term culture of multiple lineages similar to in vivo conditions, thereby facilitating investigations of normal HMEC biology relevant to the mammary gland in situ.

Microenvironment-Induced Non-sporadic Expression of the AXL and cKIT Receptors Are Related to Epithelial Plasticity and Drug Resistance.

The existence of rare cancer cells that sporadically acquire drug-tolerance through epigenetic mechanisms is proposed as one mechanism that drives cancer therapy failure. Here we provide evidence that specific microenvironments impose non-sporadic expression of proteins related to epithelial plasticity and drug resistance. Microarrays of robotically printed combinatorial microenvironments of known composition were used to make cell-based functional associations between microenvironments, which were design-inspired by normal and tumor-burdened breast tissues, and cell phenotypes. We hypothesized that specific combinations of microenvironment constituents non-sporadically impose the induction of the AXL and cKIT receptor tyrosine kinase proteins, which are known to be involved in epithelial plasticity and drug-tolerance, in an isogenic human mammary epithelial cell (HMEC) malignant progression series. Dimension reduction analysis reveals type I collagen as a dominant feature, inducing expression of both markers in pre-stasis finite lifespan HMECs, and transformed non-malignant and malignant immortal cell lines. Basement membrane-associated matrix proteins, laminin-111 and type IV collagen, suppress AXL and cKIT expression in pre-stasis and non-malignant cells. However, AXL and cKIT are not suppressed by laminin-111 in malignant cells. General linear models identified key factors, osteopontin, IL-8, and type VIα3 collagen, which significantly upregulated AXL and cKIT, as well as a plasticity-related gene expression program that is often observed in stem cells and in epithelial-to-mesenchymal-transition. These factors are co-located with AXL-expressing cells in situ in normal and breast cancer tissues, and associated with resistance to paclitaxel. A greater diversity of microenvironments induced AXL and cKIT expression consistent with plasticity and drug-tolerant phenotypes in tumorigenic cells compared to normal or immortal cells, suggesting a reduced perception of microenvironment specificity in malignant cells. Microenvironment-imposed reprogramming could explain why resistant cells are seemingly persistent and rapidly adaptable to multiple classes of drugs. These results support the notion that specific microenvironments drive drug-tolerant cellular phenotypes and suggest a novel interventional avenue for preventing acquired therapy resistance.

Chromatin remodeling regulates catalase expression during cancer cells adaptation to chronic oxidative stress.

Regulation of ROS metabolism plays a major role in cellular adaptation to oxidative stress in cancer cells, but the molecular mechanism that regulates catalase, a key antioxidant enzyme responsible for conversion of hydrogen peroxide to water and oxygen, remains to be elucidated. Therefore, we investigated the transcriptional regulatory mechanism controlling catalase expression in three human mammary cell lines: the normal mammary epithelial 250MK primary cells, the breast adenocarcinoma MCF-7 cells and an experimental model of MCF-7 cells resistant against oxidative stress resulting from chronic exposure to H2O2 (Resox), in which catalase was overexpressed. Here we identify a novel promoter region responsible for the regulation of catalase expression at -1518/-1226 locus and the key molecules that interact with this promoter and affect catalase transcription. We show that the AP-1 family member JunB and retinoic acid receptor alpha (RARα) mediate catalase transcriptional activation and repression, respectively, by controlling chromatin remodeling through a histone deacetylases-dependent mechanism. This regulatory mechanism plays an important role in redox adaptation to chronic exposure to H2O2 in breast cancer cells. Our study suggests that cancer adaptation to oxidative stress may be regulated by transcriptional factors through chromatin remodeling, and reveals a potential new mechanism to target cancer cells.

Rapid Organoid Reconstitution by Chemical Micromolding.

Purified populations of cells can be reconstituted into organoids that recapitulate aspects of their in vivo structure and function. These organoids are useful as models of healthy and diseased tissue in the basic sciences, in vitro screens, and regenerative medicine. Existing strategies to reconstitute organoids from purified cells face obstacles with respect to cell-viability, multicellular connectivity, scalability, and compatibility with subsequent experimental or analytical techniques. To address these challenges, we developed a strategy for rapidly casting populations of cells into microtissues of prescribed size and shape. This approach begins by chemically remodeling the adhesive properties of living cells with membrane-anchored ssDNA with modest annealing kinetics. Populations of complementary labeled cells are then combined into microwells that rapidly mold the DNA-adhesive cell populations into 3D aggregates of uniform size and shape. Once formed, aggregates are removed from the molds in the presence of "capping" oligonucleotides that block hybridization of residual surface DNA between aggregates in suspension. Finally, transfer of aggregates to biomimetic gels for 3D culture completes the process of reconstitution. This strategy of chemical micromolding allows for control over aggregate internal topology and does not perturb the natural process of self-organization in primary human mammary epithelial cells.

184AA3: a xenograft model of ER+ breast adenocarcinoma.

Despite the prevalence and significant morbidity resulting from estrogen receptor positive (ER(+)) breast adenocarcinomas, there are only a few models of this cancer subtype available for drug development and arguably none for studying etiology. Those models that do exist have questionable clinical relevance. Given our goal of developing luminal models, we focused on six cell lines derived by minimal mutagenesis from normal human breast cells, and asked if any could generate clinically relevant xenografts, which we then extensively characterized. Xenografts of one cell line, 184AA3, consistently formed ER(+) adenocarcinomas that had a high proliferative rate and other features consistent with "luminal B" intrinsic subtype. Squamous and spindle cell/mesenchymal differentiation was absent, in stark contrast to other cell lines that we examined or others have reported. We explored intratumoral heterogeneity produced by 184AA3 by immunophenotyping xenograft tumors and cultured cells, and characterized marker expression by immunofluorescence and flow cytometry. A CD44(High) subpopulation was discovered, yet their tumor forming ability was far less than CD44(Low) cells. Single cell cloning revealed the phenotypic plasticity of 184AA3, consistent with the intratumoral heterogeneity observed in xenografts. Characterization of ER expression in cultures revealed ER protein and signaling is intact, yet when estrogen was depleted in culture, and in vivo, it did not impact cell or tumor growth, analogous to therapeutically resistant ER(+) cancers. This model is appropriate for studies of the etiology of ovarian hormone independent adenocarcinomas, for identification of therapeutic targets, predictive testing, and drug development.

A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers.

Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the gene expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'-deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. The epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis.

The senescent methylome and its relationship with cancer, ageing and germline genetic variation in humans.

BACKGROUND: Cellular senescence is a stable arrest of proliferation and is considered a key component of processes associated with carcinogenesis and other ageing-related phenotypes. Here, we perform methylome analysis of actively dividing and deeply senescent normal human epithelial cells. RESULTS: We identify senescence-associated differentially methylated positions (senDMPs) from multiple experiments using cells from one donor. We find that human senDMP epigenetic signatures are positively and significantly correlated with both cancer and ageing-associated methylation dynamics. We also identify germline genetic variants, including those associated with the p16INK4A locus, which are associated with the presence of in vivo senDMP signatures. Importantly, we also demonstrate that a single senDMP signature can be effectively reversed in a newly-developed protocol of transient senescence reversal. CONCLUSIONS: The senDMP signature has significant potential for understanding some of the key (epi)genetic etiological factors that may lead to cancer and age-related diseases in humans.

Programmed synthesis of three-dimensional tissues.

Reconstituting tissues from their cellular building blocks facilitates the modeling of morphogenesis, homeostasis and disease in vitro. Here we describe DNA-programmed assembly of cells (DPAC), a method to reconstitute the multicellular organization of organoid-like tissues having programmed size, shape, composition and spatial heterogeneity. DPAC uses dissociated cells that are chemically functionalized with degradable oligonucleotide 'Velcro', allowing rapid, specific and reversible cell adhesion to other surfaces coated with complementary DNA sequences. DNA-patterned substrates function as removable and adhesive templates, and layer-by-layer DNA-programmed assembly builds arrays of tissues into the third dimension above the template. DNase releases completed arrays of organoid-like microtissues from the template concomitant with full embedding in a variety of extracellular matrix (ECM) gels. DPAC positions subpopulations of cells with single-cell spatial resolution and generates cultures several centimeters long. We used DPAC to explore the impact of ECM composition, heterotypic cell-cell interactions and patterns of signaling heterogeneity on collective cell behaviors.

Delineating transcriptional networks of prognostic gene signatures refines treatment recommendations for lymph node-negative breast cancer patients.

The majority of women diagnosed with lymph node-negative breast cancer are unnecessarily treated with damaging chemotherapeutics after surgical resection. This highlights the importance of understanding and more accurately predicting patient prognosis. In the present study, we define the transcriptional networks regulating well-established prognostic gene expression signatures. We find that the same set of transcriptional regulators consistently lie upstream of both 'prognosis' and 'proliferation' gene signatures, suggesting that a central transcriptional network underpins a shared phenotype within these signatures. Strikingly, the master transcriptional regulators within this network predict recurrence risk for lymph node-negative breast cancer better than currently used multigene prognostic assays, particularly in estrogen receptor-positive patients. Simultaneous examination of p16(INK4A) expression, which predicts tumours that have bypassed cellular senescence, revealed that intermediate levels of p16(INK4A) correlate with an intact pRB pathway and improved survival. A combination of these master transcriptional regulators and p16(INK4A), termed the OncoMasTR score, stratifies tumours based on their proliferative and senescence capacity, facilitating a clearer delineation of lymph node-negative breast cancer patients at high risk of recurrence, and thus requiring chemotherapy. Furthermore, OncoMasTR accurately classifies over 60% of patients as 'low risk', an improvement on existing prognostic assays, which has the potential to reduce overtreatment in early-stage patients. Taken together, the present study provides new insights into the transcriptional regulation of cellular proliferation in breast cancer and provides an opportunity to enhance and streamline methods of predicting breast cancer prognosis.

Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity.

Developing tissues contain motile populations of cells that can self-organize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its self-organization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue-ECM boundary, rather than by differential homo- and heterotypic energies of cell-cell interaction. Surprisingly, interactions with the tissue-ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell-cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell-cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell-ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer.

Formation of spatially and geometrically controlled three-dimensional tissues in soft gels by sacrificial micromolding.

Patterned three-dimensional (3D) cell culture models aim to more accurately represent the in vivo architecture of a tissue for the purposes of testing drugs, studying multicellular biology, or engineering functional tissues. However, patterning 3D multicellular structures within very soft hydrogels (<500 Pa) that mimic the physicochemical environment of many tissues remains a challenge for existing methods. To overcome this challenge, we use a Sacrificial Micromolding technique to temporarily form spatially and geometrically defined 3D cell aggregates in degradable scaffolds before transferring and culturing them in a reconstituted extracellular matrix. Herein, we demonstrate that Sacrificial Micromolding (1) promotes cyst formation and proper polarization of established epithelial cell lines, (2) allows reconstitution of heterotypic cell-cell interactions in multicomponent epithelia, and (3) can be used to control the lumenization-state of epithelial cysts as a function of tissue size. In addition, we discuss the potential of Sacrificial Micromolding as a cell-patterning tool for future studies.

Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations.

Telomerase reactivation and immortalization are critical for human carcinoma progression. However, little is known about the mechanisms controlling this crucial step, due in part to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. We achieved efficient non-clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the 2 main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier was bypassed using shRNA to p16(INK4); replicative senescence due to critically shortened telomeres was bypassed in post-stasis HMEC by c-MYC transduction. Thus, 2 pathologically relevant oncogenic agents are sufficient to immortally transform normal HMEC. The resultant non-clonal immortalized lines exhibited normal karyotypes. Most human carcinomas contain genomically unstable cells, with widespread instability first observed in vivo in pre-malignant stages; in vitro, instability is seen as finite cells with critically shortened telomeres approach replicative senescence. Our results support our hypotheses that: (1) telomere-dysfunction induced genomic instability in pre-malignant finite cells may generate the errors required for telomerase reactivation and immortalization, as well as many additional "passenger" errors carried forward into resulting carcinomas; (2) genomic instability during cancer progression is needed to generate errors that overcome tumor suppressive barriers, but not required per se; bypassing the senescence barriers by direct targeting eliminated a need for genomic errors to generate immortalization. Achieving efficient HMEC immortalization, in the absence of "passenger" genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression, and exploration of agents that could prevent immortalization.

Age-related dysfunction in mechanotransduction impairs differentiation of human mammary epithelial progenitors.

Dysfunctional progenitor and luminal cells with acquired basal cell properties accumulate during human mammary epithelial aging for reasons not understood. Multipotent progenitors from women aged <30 years were exposed to a physiologically relevant range of matrix elastic modulus (stiffness). Increased stiffness causes a differentiation bias towards myoepithelial cells while reducing production of luminal cells and progenitor maintenance. Lineage representation in progenitors from women >55 years is unaffected by physiological stiffness changes. Efficient activation of Hippo pathway transducers YAP and TAZ is required for the modulus-dependent myoepithelial/basal bias in younger progenitors. In older progenitors, YAP and TAZ are activated only when stressed with extraphysiologically stiff matrices, which bias differentiation towards luminal-like phenotypes. In vivo YAP is primarily active in myoepithelia of younger breasts, but localization and activity increases in luminal cells with age. Thus, aging phenotypes of mammary epithelia may arise partly because alterations in Hippo pathway activation impair microenvironment-directed differentiation and lineage specificity.

Cellular senescence mediated by p16INK4A-coupled miRNA pathways.

p16 is a key regulator of cellular senescence, yet the drivers of this stable state of proliferative arrest are not well understood. Here, we identify 22 senescence-associated microRNAs (SA-miRNAs) in normal human mammary epithelial cells. We show that SA-miRNAs-26b, 181a, 210 and 424 function in concert to directly repress expression of Polycomb group (PcG) proteins CBX7, embryonic ectoderm development (EED), enhancer of zeste homologue 2 (EZH2) and suppressor of zeste 12 homologue (Suz12), thereby activating p16. We demonstrate the existence of a tight positive feedback loop in which SA-miRNAs activate and re-enforce the expression of other SA-miRNA members. In contrast, PcG members restrain senescence by epigenetically repressing the expression of these SA-miRNAs. Importantly, loss of p16 leads to repression of SA-miRNA expression, intimately coupling this effector of senescence to the SA-miRNA/PcG self-regulatory loop. Taken together, our findings illuminate an important regulatory axis that underpins the transition from proliferation to cellular senescence.

Aging phenotypes in cultured normal human mammary epithelial cells are correlated with decreased telomerase activity independent of telomere length.

BACKGROUND: Shortening of telomeres, which are essential for maintenance of genomic integrity, is a mechanism commonly associated with the aging process. Here we ascertained whether changes in telomere lengths or telomerase activity correlated with age in normal human mammary epithelial cells (HMEC), or with phenotypes of aging in breast. Accordingly, flow cytometry fluorescence in situ hybridization (flowFISH) was used to determine relative telomere lengths (RTL), and telomerase activity was measured by the telomeric repeat amplification protocol (TRAP), in a collection of 41 primary HMEC strains established from women aged 16 to 91 years. RESULTS: RTL measurements of HMEC strains that were heterogeneous with respect to lineage composition revealed no significant associations between telomere length with age, maximum observed population doublings, or with lineage composition of the strains. However, within strains, luminal epithelial and cKit-expressing epithelial progenitor cells that were flow cytometry-enriched from individual HMEC strains exhibited significantly shorter telomeres relative to isogenic myoepithelial cells (P < 0.01). In unsorted strains, detectable telomerase activity did not correlate with RTL. Telomerase activity declined with age; the average age of strains that exhibited TRAP activity was 29.7 ± 3.9y, whereas the average age of strains with no detectable TRAP activity was 49.0 ± 4.9y (P < 0.01). Non-detectable TRAP activity also was correlated with phenotypes of aging previously described in HMEC strains; increased proportions of CD227-expressing luminal epithelial cells (P < 0.05) and cKit-expressing progenitor cells (P < 0.05). CONCLUSIONS: Telomere shortening did not correlate with the chronological ages of HMEC strains, whereas decreased telomerase activity correlated with age and with lineage distribution phenotypes characteristic of aging.

Processing of human reduction mammoplasty and mastectomy tissues for cell culture.

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries. The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor). Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities. Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.