Luminal Cell Layer Research Articles

Papillomatous breast lesions with atypical columnar cell features

AimsColumnar cell lesions (CCLs) are recognised breast cancer precursor lesions. Intraductal papillomas are usually lined by benign (polyclonal) cells. Although papillomas with monoclonal lesions (atypical ductal hyperplasia (ADH)/ductal carcinoma in...

A Guide Toward Multi-scale and Quantitative Branching Analysis in the Mammary Gland.

The mammary gland consists of a bilayered epithelial structure with an extensively branched morphology. The majority of this epithelial tree is laid down during puberty, during which actively proliferating terminal end buds repeatedly elongate and bifurcate to form the basic structure of the ductal tree. Mammary ducts consist of a basal and luminal cell layer with a multitude of identified sub-lineages within both layers. The understanding of how these different cell lineages are cooperatively driving branching morphogenesis is a problem of crossing multiple scales, as this requires information on the macroscopic branched structure of the gland, as well as data on single-cell dynamics driving the morphogenic program. Here we describe a method to combine genetic lineage tracing with whole-gland branching analysis. Quantitative data on the global organ structure can be used to derive a model for mammary gland branching morphogenesis and provide a backbone on which the dynamics of individual cell lineages can be simulated and compared to lineage-tracing approaches. Eventually, these quantitative models and experiments allow to understand the couplings between the macroscopic shape of the mammary gland and the underlying single-cell dynamics driving branching morphogenesis.

Abstract 3772: Increased mitochondrial gene expression in prostate cancer

Abstract Warburg postulated that aerobic glycolysis, which is commonly observed in cancer, results from crippled mitochondrial function. Yet, most cancers and other rapidly proliferating cells rely on a functioning mitochondrial TCA cycle to generate important biosynthetic intermediates required for cell growth, and on the electron transport chain for pyrimidine synthesis and to recycle electron acceptors. Thus, increases in a number of mitochondrial activities have been proposed to be required for robust proliferation in most cancers. Mitochondrial function is regulated in part through mitochondrial biogenesis, which is influenced by mitochondrial DNA copy number (mtDNAcn). We previously developed an in situ hybridization method to quantify mtDNAcn in specific cell compartments in relation to normal physiological functioning and disease processes. We reported marked cell type heterogeneity across normal tissues including relatively higher mtDNAcn in a number of stem/proliferative compartments in humans and mice (PMID: 32304697). More recently, we combined this in situ approach with immunohistochemistry and reported increased mtDNAcn along with widespread heterogeneity in prostate cancer (DOI: 10.1158/1538-7445.AM2021-2404). We also found increased mtDNAcn in high grade prostatic intraepithelial neoplasia, the main precursor to prostate cancer, as well as precursor lesions in the large intestine and pancreas. To interrogate the functional significance of this mtDNAcn alteration, we sought to determine whether increased and heterogeneous mtDNAcn levels are accompanied by corresponding changes in its gene expression (mtRNA). To establish an initial survey of the landscape of steady state mtRNA levels, we used the RNAscope HiPlex system for multiplex in situ hybridization for 4 different mtRNAs along with in situ hybridization for mtDNA and combined this with immunofluorescence. In normal prostatic epithelium, we observed that the overall mtRNA expression pattern correlated with mtDNAcn, with higher levels in the basal cell layer compared with the luminal cell layer. We also found that, similar to changes in mtDNAcn in invasive prostate cancer, the 4 mtRNAs were upregulated yet heterogeneous in cancer lesions. Among the invasive tumors, cribriform prostate cancer showed distinct mtDNA and mtRNAs expression patterns, with higher signals in the peripheral cells than central cells, suggesting underlying biological differences in this unique histologic pattern. This spatial pattern in cribriform lesions correlated with the pattern of MYC protein expression, which is known to regulate mitochondrial biogenesis. Overall, we present a novel approach that advances the ability to quantify mtDNAcn and mtRNAs simultaneously in specific cell types while preserving the tissue spatial context. These findings suggest that increased mitochondrial function accompanies prostate cancer development and progression. Citation Format: Jiayu Chen, Qizhi Zheng, Jessica L. Hicks, Levent Trabzonlu, Ibrahim Kulac, Alan K. Meeker, Srinivasan Yegnasubramanian, Angelo M. De Marzo. Increased mitochondrial gene expression in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3772.

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.

Peroxidasin Enhances Basal Phenotype and Inhibits Branching Morphogenesis in Breast Epithelial Progenitor Cell Line D492

The human breast is composed of terminal duct lobular units (TDLUs) that are surrounded by stroma. In the TDLUs, basement membrane separates the stroma from the epithelial compartment, which is divided into an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells. Stem cells and progenitor cells also reside within the epithelium and drive a continuous cycle of gland remodelling that occurs throughout the reproductive period. D492 is an epithelial cell line originally isolated from the stem cell population of the breast and generates both luminal and myoepithelial cells in culture. When D492 cells are embedded into 3D reconstituted basement membrane matrix (3D-rBM) they form branching colonies mimicking the TDLUs of the breast, thereby providing a well-suited in vitro model for studies on branching morphogenesis and breast development. Peroxidasin (PXDN) is a heme-containing peroxidase that crosslinks collagen IV with the formation of sulfilimine bonds. Previous studies indicate that PXDN plays an integral role in basement membrane stabilisation by crosslinking collagen IV and as such contributes to epithelial integrity. Although PXDN has been linked to fibrosis and cancer in some organs there is limited information on its role in development, including in the breast. In this study, we demonstrate expression of PXDN in breast epithelium and stroma and apply the D492 cell line to investigate the role of PXDN in cell differentiation and branching morphogenesis in the human breast. Overexpression of PXDN induced basal phenotype in D492 cells, loss of plasticity and inhibition of epithelial-to-mesenchymal transition as is displayed by complete inhibition of branching morphogenesis in 3D culture. This is supported by results from RNA-sequencing which show significant enrichment in genes involved in epithelial differentiation along with significant negative enrichment of EMT factors. Taken together, we provide evidence for a novel role of PXDN in breast epithelial differentiation and mammary gland development.

Reconstruction of the Swine Pulmonary Artery Using a Graft Engineered With Syngeneic Cardiac Pericytes.

The neonatal heart represents an attractive source of regenerative cells. Here, we report the results of a randomized, controlled, investigator-blinded preclinical study, which assessed the safety and effectiveness of a matrix graft cellularized with cardiac pericytes (CPs) in a piglet model of pulmonary artery (PA) reconstruction. Within each of five trios formed by 4-week-old female littermate piglets, one element (the donor) was sacrificed to provide a source of CPs, while the other two elements (the graft recipients) were allowed to reach the age of 10 weeks. During this time interval, culture-expanded donor CPs were seeded onto swine small intestinal submucosa (SIS) grafts, which were then shaped into conduits and conditioned in a flow bioreactor. Control unseeded SIS conduits were subjected to the same procedure. Then, recipient piglets were randomized to surgical reconstruction of the left PA (LPA) with unseeded or CP-seeded SIS conduits. Doppler echocardiography and cardiac magnetic resonance imaging (CMRI) were performed at baseline and 4-months post-implantation. Vascular explants were examined using histology and immunohistochemistry. All animals completed the scheduled follow-up. No group difference was observed in baseline imaging data. The final Doppler assessment showed that the LPA’s blood flow velocity was similar in the treatment groups. CMRI revealed a mismatch in the average growth of the grafted LPA and contralateral branch in both treatment groups. Histology of explanted arteries demonstrated that the CP-seeded grafts had a thicker luminal cell layer, more intraparietal arterioles, and a higher expression of endothelial nitric oxide synthase (eNOS) compared with unseeded grafts. Moreover, the LPA stump adjacent to the seeded graft contained more elastin and less collagen than the unseeded control. Syngeneic CP engineering did not accomplish the primary goal of supporting the graft’s growth but was able to improve secondary outcomes, such as the luminal cellularization and intraparietal vascularization of the graft, and elastic remodeling of the recipient artery. The beneficial properties of neonatal CPs may be considered in future bioengineering applications aiming to reproduce the cellular composition of native arteries.

Multiscale imaging of basal cell dynamics in the functionally mature mammary gland

The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca2+ oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.

Abstract P4-04-04: Loss of epigenetic lineage fidelity with age primes breast epithelia for malignant transformation

Abstract Background: Aging is the largest risk factor for breast cancer, with 80% of new cases diagnosed in women over 50y. However, the molecular mechanism underlying age-associated cancer susceptibility is still not well understood. While aging-associated phenotypes in a number of tissues have been correlated with transcriptional and epigenetic changes, few studies have looked at epigenetic regulation at the resolution of individual cell populations. The bilayered epithelia of the mammary gland is composed of an apical layer of secretory luminal epithelial cells (LEP) surrounded by a basal layer of contractile and tumor suppressive myoepithelial cells (MEP). Breast cancer cells of origin are thought to reside mainly in the luminal or supra-basal regions directly adjacent to the apical surfaces of the MEP. Thus, an understanding of how LEP and MEP interact and maintain lineage-specific expression, and how this is dysregulated with age may elucidate a potential mechanism regulating increased breast cancer susceptibility with age. Previously, we showed that loss of lineage fidelity of LEP and MEP is a key feature of aging. Moreover, this loss of lineage fidelity can be imposed by old MEP onto young LEP in a cell non-autonomous manner (Miyano & Sayaman, et al, Aging 2017). Methods: We present a detailed analysis of genome-wide DNA methylation (Infinium Human Methylation 450K) changes at lineage-specific resolution from FACS-sorted MEP and LEP populations isolated from finite lifespan human mammary epithelial cells (HMEC) derived from reduction mammoplasties of younger (<30y) and older women (>55y). Results: In younger epithelia, maintenance of lineage fidelity is associated with lineage-specific differential methylation (DM) of CpG sites at key regulatory regions. We find that loss of lineage fidelity is recapitulated in genome-wide DNA methylation, with loss of DM at canonical proximal regions associated with transcriptional regulation, specifically annotated CpG island groups, enhancer regions, and CTCF binding sites thought to be involved in chromatin remodeling. Strikingly, DM changes with age occur almost exclusively in LEP population, with older LEP acquiring more MEP-like DNA methylation patterns at the dysregulated sites. Aged LEP and MEP also exhibit significant variability in DNA methylation at both hypo- and hypermethylated regions that show little variance in young samples. Furthermore, predictions of physiologic age using Horvath’s clock show MEP to have older physiologic ages than their isogenic LEP counterparts. Titus et al, found that DM regions (DMRs) between early stage breast tumors and normal-adjacent breast samples in The Cancer Genome Atlas (TCGA) are significantly more enriched in the LUMA, LUMB and HER2 than the Basal molecular subtypes (Sci Reports, 2017). We show that CpG sites associated with the luminal-subtype DMRs are lineage specific and that a significant fraction of the epigenetic changes that occur in these early stage breast cancers are already present during aging. Conclusions: The lineage bias of age-specific of DNA methylation changes suggests that epithelial lineages age via different mechanisms. That LEP are significantly more differentially regulated via DNA methylation with age than MEP raises possibilities that DNA methylation affects aging-associated breast cancer risk in a lineage-specific manner. This lineage bias, along with age-specific changes in breast composition, may underlie the differences in incidence rates of breast cancer subtypes with age and the prevalence of ER+ luminal-subtype breast cancers in women post-menopause. We suggest that these epigenetic changes in the luminal lineage may be priming events that make breast cells more vulnerable to further dysregulation that leads to cancer. Citation Format: Rosalyn W. Sayaman, Masaru Miyano, Mark A. LaBarge. Loss of epigenetic lineage fidelity with age primes breast epithelia for malignant transformation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-04-04.

A microfluidic mammary gland coculture model using parallel 3D lumens for studying epithelial-endothelial migration in breast cancer.

In breast cancer development, crosstalk between mammary epithelial cells and neighboring vascular endothelial cells is critical to understanding tumor progression and metastasis, but the mechanisms of this dynamic interplay are not fully understood. Current cell culture platforms do not accurately recapitulate the 3D luminal architecture of mammary gland elements. Here, we present the development of an accessible and scalable microfluidic coculture system that incorporates two parallel 3D luminal structures that mimic vascular endothelial and mammary epithelial cell layers, respectively. This parallel 3D lumen configuration allows investigation of endothelial-epithelial crosstalk and its effects of the comigration of endothelial and epithelial cells into microscale migration ports located between the parallel lumens. We describe the development and application of our platform, demonstrate generation of 3D luminal cell layers for endothelial cells and three different breast cancer cell lines, and quantify their migration profiles based on number of migrated cells, area coverage by migrated cells, and distance traveled by individual migrating cells into the migration ports. Our system enables analysis at the single-cell level, allows simultaneous monitoring of endothelial and epithelial cell migration within a 3D extracellular matrix, and has potential for applications in basic research on cellular crosstalk as well as drug development.

Case Report: Syringocystadenoma Papilliferum in the Middle Ear

AbstractBackground: Syringocystadenoma papilliferum is a rare adnexal tumor of controversial origin that often occurs as a solitary tumor derived from apocrine and eccrine glands. It is more common in children and adolescents and affects the head and neck regions,although occurrences on other anatom-ical locations have been described. Routine H & E-stained sections revealed cystic epidermal invaginations with papillary projections. The superficial portions of the cyst were lined by stratified keratinizing epithelium, whereas the deeper papillated portion exhibited a double layer of basal-like cells and luminal eosinophilic columnar cells with focal decapitation secretions.Patient and Method: A female patient, aged 29 years, presented to us in the outpatient clinic suffering from left earache and hearing loss since the past 3 months, with gradual onset and a slowly progressive course. On examination, we found a left aural polyp, which was firm and grayish in color, but extremely tender on touch. Biopsy for histopathological examination was consistent with a diagnosis of syringocysta-denoma papilliferum. The patient received regular follow-up and is doing well with regard to her earache with marked hearing improvement. To conclude, solitary lesions seen in unusual locations generate multiple differential diagnoses and should be confirmed by histopathology in order to exclude other pathologies and define the appropriate therapeutic approach.Conclusion: Solitary lesions seen in unusual locations generate multiple differential diagnoses and they must be confirmed by histopathology

Luminal MCF-12A & myoepithelial-like Hs 578Bst cells form bilayered acini similar to human breast.

The epithelium's functional unit is the bilayered acinus, made of a layer of luminal cells, surrounded by a layer of basal cells mainly composed of myoepithelial cells.Aim:The aim of this study was to develop a reproducible and manipulable 3D co-culture model of the bilayered acinus in vitro to study the interactions between the two layers.Materials & methods:Two different combinations of cell lines were co-cultured in Matrigel: SCp2 and SCg6 mice cells, or MCF-12A and Hs 578Bst human cell lines.Results:Confocal microscopy analysis showed that only MCF-12A and Hs 578Bst cells could form some bilayered acini. This in vitro bilayered acini model will allow us to understand the role of interactions between luminal and myoepithelial cells in the normal breast development.

MP65-09 NEAR INFRARED PHOTOIMMUNOTHERAPY USING ANTI-CD47 ANTIBODY FOR HUMAN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2018MP65-09 NEAR INFRARED PHOTOIMMUNOTHERAPY USING ANTI-CD47 ANTIBODY FOR HUMAN BLADDER CANCER Bernhard Kiss, Kelly McKenna, Robert Ertsey, Nynke Sjoerdtje Van den Berg, Kathleen Mach, Eben Rosenthal, and Joseph Liao Bernhard KissBernhard Kiss More articles by this author , Kelly McKennaKelly McKenna More articles by this author , Robert ErtseyRobert Ertsey More articles by this author , Nynke Sjoerdtje Van den BergNynke Sjoerdtje Van den Berg More articles by this author , Kathleen MachKathleen Mach More articles by this author , Eben RosenthalEben Rosenthal More articles by this author , and Joseph LiaoJoseph Liao More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2075AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Near infrared photoimmunotherapy (PIT) is a new molecular-targeted cancer therapy that uses a specific photosensitizer based on a near-infrared (NIR) phthalocyanine dye, IRDye700DX, conjugated to a monoclonal antibody. CD47 is a cell surface protein that elicits a ″don′t eat me signal″ to prevent macrophage engulfment and is expressed on over 80% of bladder cancer tumors but absent on the luminal cell layer of normal bladder urothelium. Thus, targeting CD47 for PIT has the potential to selectively induce cell death in CD47 expressing tumor cells as well as increase phagocytosis of tumor cells. METHODS The anti-CD47 antibody (B6H12) was conjugated to IRDye700DX NHS ester and purified using a dialysis spin column. Anti-CD47-IRDye700 concentration was determined with a Coomassie Plus protein assay. The molecular weight of the conjugated antibody was verified by SDS-page and IRDye700 conjugation verified using the 700nm fluorescence channel of a PEARL imager. Binding of anti-CD47-IRDye700DX to CD47 was verified by ELISA. Three different human urothelial cell lines (UMUC3, HT-1376 and 639V) were used for in vitro NIR-PIT experiments. 639V cells were stably transfected with GFP using a lentiviral vector. After plating in a 96well plate and incubation with 10 µg/ml of anti-CD47-IRDye700 or unconjugated anti-CD47 antibody (1h on ice), cells were irradiated with a red light-emitting diode (LED) at 670-710nm wavelength at different energy levels. The cytotoxic effects of NIR-PIT with anti-CD47-IRDye700DX were determined by propidium iodide staining and flow cytometry. To evaluate macrophage activity, a phagocytosis assay using human macrophages and analyzed by flow cytometry was performed. RESULTS Cancer-cell death significantly increased with anti-CD47-IRDye700DX in a light-dose dependent manner in all 3 human bladder cancer cell lines. Significant cancer-cell death was first observed with 2J exposure and when exposed to 40J of NIR light over 90% of 639V and UMUC3 cells and over 50% of HT1376 died. Phagocytosis of cancer-cells incubated with anti-CD47-IRDye700DX and irradiated with 8J was significantly increased compared to anti-CD47-antibody alone (p=0.0002). There was no cytotoxicity associated with anti-CD47 antibody alone with NIR light, with NIR light alone or with anti-CD47-IRDye700DX without NIR light. CONCLUSIONS NIR-PIT with anti-CD47-IRDye700 shows significantly increased cancer-cell cytotoxicity and significantly increased cancer-cell phagocytosis making this combination a highly promising novel approach for molecular targeted bladder cancer therapy. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e862 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Bernhard Kiss More articles by this author Kelly McKenna More articles by this author Robert Ertsey More articles by this author Nynke Sjoerdtje Van den Berg More articles by this author Kathleen Mach More articles by this author Eben Rosenthal More articles by this author Joseph Liao More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Expression profile of polycomb group proteins in odontogenic keratocyst and ameloblastoma

Polycomb group (PcG) proteins are repressive chromatin modifiers required for proliferation and development. PcG proteins form two large repressive complexes, namely, Polycomb Repressive Complex 1 and 2. These proteins have been shown to drive tumorigenesis by repressing cell-type specific sets of target genes. Using immunohistochemistry, we investigated the expression patterns of five human PcG proteins, including Bmi-1, Ring1b, Mel-18, Ezh2, and Suz12, in various cellular components of odontogenic keratocysts (OKCs), ameloblastomas and, pericoronal follicles (PFs). In OKCs, expression of PcG proteins were found in the majority of cases while the expression pattern was relatively different for each PcG proteins. All PcG proteins were strongly expressed in the basal cells while some proteins showed variable expression in the parabasal and luminal cell layer of OKCs. In ameloblastomas, almost all PcG proteins showed a similar expression pattern of moderate to strong staining in the peripheral ameloblast-like cells and metaplastic squamous cells. Some of the central stellate reticulum-like cells also showed positive reaction to most PcG proteins. In PFs, most PcG proteins were intensely expressed in odontogenic epithelium lining the follicles, except Mel-18 and Suz12. The present study provides the initial evidence regarding epigenetic involvement by PcG proteins in these odontogenic lesions. Although these proteins are known to be in the same repressive group proteins, differential expression patterns of these proteins in OKCs and ameloblastomas indicates that these proteins may play different roles in pathogenesis of these odontogenic lesions.

Immunohistochemical expression profiles of MUC1 and MUC2 mucins in urothelial tumors of bladder.

Mucins may show aberrant expression, localization, and/or glycosylation in multiple malignancies. However, information regarding expression of these mucins is mostly unknown in urothelial tumors. This study was conducted for examining the expressions of membrane associated and secreted mucin (MUC1) and a secreted gel-forming mucin (MUC2) in urothelial tumors of the urinary bladder. Archival transurethral resection materials of 97 urothelial carcinoma cases were reexamined light microscopically and graded according to the 2004 WHO Classification. Pathological stage was given as pTa, pT1, and pT2. Demonstrative sections were recut for immunohistochemistry for MUC1 and MUC2. The results were statistically analyzed, and P < 0.05 was considered statistically significant. The positivity for MUC1 and MUC2 was 89.7% and 44.3%, respectively. Independent from pathological stage of the tumor, MUC1 expression showed statistically significant correlation with tumor grade (P < 0.05). We did not find any correlation between pathological stage and MUC1 and MUC2 expression (P > 0.05). MUC1 staining pattern in papillary urothelial neoplasm of low malignant potential cases was more commonly apical and superficial (luminal cell layer only). Intermediate cells ± basal cells or isolated cells or islands of tumor cells with cytoplasmic and/or circumferential membrane positivity for MUC1 and MUC2 were more commonly observed in both low- and high-grade carcinomas. The difference between groups in terms of MUC1 and MUC2 staining was statistically significant (P < 0.05). The staining patterns of both mucins are different between urothelial papillary tumors and may be used to make a differentiation, especially for low-grade papillary urothelial lesions. This difference may also be important in the carcinomatous transformation of urothelial neoplastic and preneoplastic lesions.

Lobular carcinoma in situ and invasive lobular breast cancer are characterized by enhanced expression of transcription factor AP-2β

Transcription factor AP-2β (TFAP2B) regulates embryonic organ development and is overexpressed in alveolar rhabdomyosarcoma, a rare childhood malignancy. Gene expression profiling has implicated AP-2β in breast cancer (BC). This study characterizes AP-2β expression in the mammary gland and in BC. AP-2β protein expression was assessed in the normal mammary gland epithelium, in various reactive, metaplastic and pre-invasive neoplastic lesions and in two clinical BC cohorts comprising >2000 patients. BCs from various genetically engineered mouse (GEM) models were also evaluated. Human BC cell lines served as functional models to study siRNA-mediated inhibition of AP-2β. The normal mammary gland epithelium showed scattered AP-2β-positive cells in the luminal cell layer. Various reactive and pre-invasive neoplastic lesions, including apocrine metaplasia, usual ductal hyperplasia and lobular carcinoma in situ (LCIS) showed enhanced AP-2β expression. Cases of ductal carcinoma in situ (DCIS) were more often AP-2β-negative (P<0.001). In invasive BC cohorts, AP-2β-positivity was associated with the lobular BC subtype (P<0.001), loss of E-cadherin (P<0.001), a positive estrogen receptor (ER) status (P<0.001), low Ki67 (P<0.001), low/intermediate Oncotype DX recurrence scores (P<0.001), and prolonged event-free survival (P=0.003). BCs from GEM models were all AP-2β-negative. In human BC cell lines, AP-2β expression was independent from ER-signaling. SiRNA-mediated inhibition of AP-2β diminished proliferation of lobular BC cell lines in vitro. In summary, AP-2β is a new mammary epithelial differentiation marker. Its expression is preferentially retained and enhanced in LCIS and invasive lobular BC and has prognostic implications. Our findings indicate that AP-2β controls tumor cell proliferation in this slow-growing BC subtype.

Histone 2B-GFP Label-Retaining Prostate Luminal Cells Possess Progenitor Cell Properties and Are Intrinsically Resistant to Castration.

SummaryThe existence of slow-cycling luminal cells in the prostate has been suggested, but their identity and functional properties remain unknown. Using a bigenic mouse model to earmark, isolate, and characterize the quiescent stem-like cells, we identify a label-retaining cell (LRC) population in the luminal cell layer as luminal progenitors. Molecular and biological characterizations show that these luminal LRCs are significantly enriched in the mouse proximal prostate, exhibit relative dormancy, display bipotency in both in vitro and in vivo assays, and express a stem/progenitor gene signature with resemblance to aggressive prostate cancer. Importantly, these LRCs, compared with bulk luminal cells, maintain a lower level of androgen receptor (AR) expression and are less androgen dependent and also castration resistant in vivo. Finally, analysis of phenotypic markers reveals heterogeneity within the luminal progenitor cell pool. Our study establishes luminal LRCs as progenitors that may serve as a cellular origin for castration-resistant prostate cancer.

Vitamin D receptor expression in canine mammary gland and relationship with clinicopathological parameters and progesterone/oestrogen receptors.

The vitamin D receptor (VDR) belongs to the nuclear class II receptor family. VDR is a ligand transcription factor and mediates the actions of calcitriol, the active product of vitamin D synthesis. Nowadays, it is known that the biological actions of calcitriol include the capacity to modulate cancer features, such as proliferation and differentiation, apoptosis, angiogenesis, invasion and metastasis. VDR expression has been demonstrated in human breast cancer and vitamin D has emerged as a promising targeted therapy. We analyse the VDR expression in normal and neoplastic canine mammary tissue samples and its relationship with clinicopathological parameters and progesterone/oestrogens receptors (PR/ER). Expression of VDR, Ki67 (to evaluate the proliferation index, PI), PR and ER was assessed in 50 mammary gland tissue samples from 41 female dogs by immunohistochemistry. VDR-positive staining was found in the nuclei of both myoepithelial and luminal epithelial cell layers. VDR expression was higher in normal mammary tissue (37/37 cases, 100%) then followed by benign tumours (6/15 cases, 40%) and malignant tumours (9/34 cases, 26.5%) (P = .001). Female dogs aged ≥10 years had lower VDR expression compared with dogs younger (P = .017). Relationship between VDR and breed, number of tumours, tumour size, histologic subtype, histologic grade of malignancy, PI and PR and ER expression was not observed. Studies with more samples are necessary to further evaluate the possible role of VDR in the biological behaviour of canine mammary tumours, and to corroborate the possibility to use the dog as model for human breast cancer.

Process of tight junction recovery in the injured vocal fold epithelium: Morphological and paracellular permeability analysis

The vocal fold epithelium that includes tight junction (TJ)-based barrier function protects underlying connective tissues from external insults. TJs play an important role to control paracellular permeability of not only solutes but also ions, and preserve the vocal fold homeostasis. However, the distribution of TJs and paracellular diffusion barrier across the entire vocal fold epithelium are still unknown. The aim of this study was to identify the distribution of TJs in the vocal fold epithelium and to characterize the recovery process of TJ-based paracellular diffusion barrier in a rat model of vocal fold injury. Animal experiments with controls. Normal and vocal fold-injured rats were used. Larynges were harvested for immunohistochemical examination of TJ proteins. For functional analysis, a tracer permeability assay was performed using EZ-Link Sulfo-NHS-LC-Biotin. TJ proteins occludin and zonula occludens 1 signals were localized to the junctional regions of the most luminal cell layers of the vocal fold epithelium. The injured region had been recovered with epithelium at 5 days postinjury, but the paracellular diffusion barrier assays revealed that biotinylation reagents diffused into the lamina propria at 5 days postinjury, and were blocked at the epithelium at 14 and 28 days postinjury. It was strongly suggested that TJs in the vocal fold epithelium exist at the junctional regions of the first layer of stratified squamous epithelium. TJ-based paracellular diffusion barrier following vocal fold injury is recovered by 14 days postinjury, and this period corresponds with the time course of structural changes in the regenerating epithelium layer. NA. Laryngoscope, 128:E150-E156, 2018.

Epithelial Markers aSMA, Krt14, and Krt19 Unveil Elements of Murine Lacrimal Gland Morphogenesis and Maturation.

As an element of the lacrimal apparatus, the lacrimal gland (LG) produces the aqueous part of the tear film, which protects the eye surface. Therefore, a defective LG can lead to serious eyesight impairment. Up to now, little is known about LG morphogenesis and subsequent maturation. In this study, we delineated elements of the cellular and molecular events involved in LG formation by using three epithelial markers, namely aSMA, Krt14, and Krt19. While aSMA marked a restricted epithelial population of the terminal end buds (TEBs) in the forming LG, Krt14 was found in the whole embryonic LG epithelial basal cell layer. Interestingly, Krt19 specifically labeled the presumptive ductal domain and subsequently, the luminal cell layer. By combining these markers, the Fucci reporter mouse strain and genetic fate mapping of the Krt14+ population, we demonstrated that LG epithelium expansion is fuelled by a patterned cell proliferation, and to a lesser extent by epithelial reorganization and possible mesenchymal-to-epithelial transition. We pointed out that this epithelial reorganization, which is associated with apoptosis, regulated the lumen formation. Finally, we showed that the inhibition of Notch signaling prevented the ductal identity from setting, and led to a LG covered by ectopic TEBs. Taken together our results bring a deeper understanding on LG morphogenesis, epithelial domain identity, and organ expansion.

Label retention and stem cell marker expression in the developing and adult prostate identifies basal and luminal epithelial stem cell subpopulations

BackgroundProstate cancer is the second most frequent cancer among males worldwide, and most patients with metastatic disease eventually develop therapy-resistant disease. Recent research has suggested the existence of cancer stem-like cells, and that such cells are behind the therapy resistance and progression.MethodsHere, we have taken advantage of the relatively quiescent nature of stem cells to identify the slow-cycling label-retaining stem cell (LRC) populations of the prostate gland. Mice were pulsed with bromodeoxyuridine (BrdU) during prostate organogenesis, and the LRC populations were then identified and characterized in 5-day-old and in 6-month-old adult animals using immunohistochemistry and immunofluorescence.ResultsQuantification of LRCs in the adult mouse prostate showed that epithelial LRCs were significantly more numerous in prostatic ducts (3.7 ± 0.47% SD) when compared to the proximal (1.4 ± 0.83%) and distal epithelium (0.48 ± 0.08%) of the secretory lobes. LRCs were identified in both the basal and epithelial cell layers of the prostate, and LRCs co-expressed several candidate stem cell markers in a developmental and duct/acini-specific manner, including Sca-1, TROP-2, CD133, CD44, c-kit, and the novel prostate progenitor marker cytokeratin-7. Importantly, a significant proportion of LRCs were localized in the luminal cell layer, the majority in ducts and the proximal prostate, that co-expressed high levels of androgen receptor in the adult prostate.ConclusionsOur results suggest that there are separate basal and luminal stem cell populations in the prostate, and they open up the possibility that androgen receptor-expressing luminal stem-like cells could function as cancer-initiating and relapse-responsible cells in prostate cancer.