A single-cell atlas enables mapping of homeostatic cellular shifts in the adult human breast.

Here we use single-cell RNA sequencing to compile a human breast cell atlas assembled from 55 donors that had undergone reduction mammoplasties or risk reduction mastectomies. From more than 800,000 cells we identified 41 cell subclusters across the epithelial, immune and stromal compartments. The contribution of these different clusters varied according to the natural history of the tissue.

TGFβ-mediated MMP13 secretion drives myoepithelial cell dependent breast cancer progression.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer. Virtually all women with DCIS are treated, despite evidence suggesting up to half would remain with stable, non-threatening, disease. Overtreatment thus presents a pressing issue in DCIS management.

Mechanostimulation of breast myoepithelial cells induces functional changes associated with DCIS progression to invasion.

Women with ductal carcinoma in situ (DCIS) have an increased risk of progression to invasive breast cancer. Although not all women with DCIS will progress to invasion, all are treated as such, emphasising the need to identify prognostic biomarkers. We have previously shown that altered myoepithelial cells in DCIS predict disease progression and recurrence.

An Engineered Human Adipose/Collagen Model for In Vitro Breast Cancer Cell Migration Studies.

Adipocytes are one of the major stromal cell components of the human breast. These cells play a key role in the development of the gland and are implicated in breast tumorigenesis. Frequently, directional stromal collagen I fibers are found surrounding aggressive breast tumors.

The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53.

p53 is an important tumor-suppressor protein that is mutated in more than 50% of cancers. Strategies for restoring normal p53 function are complicated by the oncogenic properties of mutant p53 and have not met with clinical success. To counteract mutant p53 activity, a variety of drugs with the potential to reconvert mutant p53 to an active wildtype form have been developed.

Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors.

Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast tissue.

A 3D in vitro model of the human breast duct: a method to unravel myoepithelial-luminal interactions in the progression of breast cancer.

Background: 3D modelling fulfils a critical role in research, allowing for complex cell behaviour and interactions to be studied in physiomimetic conditions. With tissue banks becoming established for a number of cancers, researchers now have access to primary patient cells, providing the perfect building blocks to recreate and interrogate intricate cellular systems in the laboratory. The ducts of the human breast are composed of an inner layer of luminal cells supported by an outer layer of myoepithelial cells.

Loss of MMP-8 in ductal carcinoma in situ (DCIS)-associated myoepithelial cells contributes to tumour promotion through altered adhesive and proteolytic function.

Background: Normal myoepithelial cells (MECs) play an important tumour-suppressor role in the breast but display an altered phenotype in ductal carcinoma in situ (DCIS), gaining tumour-promoter functions. Matrix metalloproteinase-8 (MMP-8) is expressed by normal MECs but is lost in DCIS. This study investigated the function of MMP-8 in MECs and the impact of its loss in DCIS.

Expression of CDK7, Cyclin H, and MAT1 Is Elevated in Breast Cancer and Is Prognostic in Estrogen Receptor-Positive Breast Cancer.

Purpose: CDK-activating kinase (CAK) is required for the regulation of the cell cycle and is a trimeric complex consisting of cyclin-dependent kinase 7 (CDK7), Cyclin H, and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics.

Rigidity sensing and adaptation through regulation of integrin types.

Tissue rigidity regulates processes in development, cancer and wound healing. However, how cells detect rigidity, and thereby modulate their behaviour, remains unknown. Here, we show that sensing and adaptation to matrix rigidity in breast myoepithelial cells is determined by the bond dynamics of different integrin types.

Adding value to rare tissue samples donated to biobanks: characterisation of breast tissue and primary cell cultures obtained from a female-to-male transgender patient.

Biobanks provide a window of opportunity to store and add value to material from rare cases allowing their future use in biomedical research. One such example is the opportunityto obtain good quality tissue from patients undergoing gender re-assignment. Following patient agreement to donate tissue samples to our biobank we catalogued the histological appearance, defined the expression of the hormone receptors ERα, PR, AR and the proliferation marker Ki67, and generated and characterised primary cell cultures in a female to male (FTM) transgender patient referred to our unit for surgery.

Altered microenvironment promotes progression of preinvasive breast cancer: myoepithelial expression of αvβ6 integrin in DCIS identifies high-risk patients and predicts recurrence.

Purpose: This study investigated the functional and clinical significance of integrin αvβ6 upregulation in myoepithelial cells of ductal carcinoma in situ (DCIS).

Experimental Design: Archival samples of DCIS and DCIS with associated invasion (n = 532) were analyzed for expression of αvβ6 by immunohistochemistry and ability to predict recurrence and progression assessed in an independent, unique cohort of DCIS cases with long-term follow-up. Primary myoepithelial cells and myoepithelial cell lines, with and without αvβ6 expression, were used to measure the effect of αvβ6 on growth and invasion of tumor cell lines in vitro and in a xenograft mouse model.

Selecting radial basis function network centers with recursive orthogonal least squares training.

Recursive orthogonal least squares (ROLS) is a numerically robust method for solving for the output layer weights of a radial basis function (RBF) network, and requires less computer memory than the batch alternative. In this paper, the use of ROLS is extended to selecting the centers of an RBF network. It is shown that the information available in an ROLS algorithm after network training can be used to sequentially select centers to minimize the network output error.

Substrate-induced growth of nanostructured zinc oxide films at room temperature using concepts of biomimetic catalysis.

By kinetically controlled vapor-diffusion catalysis, nanostructured ZnO and Zn5(OH)8(NO3)3*2H2O thin films have been grown on substrates with different chemical compositions and varying degrees of crystallinity. The materials resulting from heterogeneous nucleation under mild conditions (starting from aqueous metal salt precursor solutions at room temperature) were characterized by X-ray diffraction and scanning electron microscopy to determine the influence of the substrates on the overall chemical composition, crystallinity, and morphology of the films.

Kinetically controlled catalytic formation of zinc oxide thin films at low temperature.

We developed a unique method to produce ZnO thin films by kinetically controlled catalytic hydrolysis of a molecular precursor at low temperature, operating in conjunction with the vectorial control of crystal growth. Using a system in which the diffusion of a volatile catalyst into a solution of molecular precursor of the metal oxide limits the rate of hydrolysis and establishes a gradient of catalyst concentration, we investigated the nucleation of textured nanoparticles at the gas-liquid interface and characterized their subsequent growth. Use of this slow diffusion method combined with prediction of molecular species using a partial charge model enables a higher level of organizational control than obtained in other low-temperature synthesis methods, without the use of organic molecules.

Fibroblast growth factor 7, secreted by breast fibroblasts, is an interleukin-1beta-induced paracrine growth factor for human breast cells.

Keratinocyte growth factor/fibroblast growth factor 7 (KGF/FGF7) is known to be a potent growth factor for mammary cells but its origin, cellular targets and mode of action in the breast are unclear. In this study, we carried out studies to determine the localisation of FGF7 and its receptor, and the related growth factor FGF10. We also determined the factors that regulate FGF7 release from stromal cells and the effects of FGF7 on normal and neoplastic breast cells.

Fibroblast growth factor 8 is expressed at higher levels in lactating human breast and in breast cancer.

Fibroblast growth factor 8 can transform NIH3T3 cells and its expression has been found to be associated with breast and prostate cancer. Following our finding that fibroblast growth factor 8 mRNA expression is increased in breast cancer, we have undertaken an immunohistochemistry study of fibroblast growth factor 8 expression in a series of human breast tissues and other normal tissues. Our findings confirm increased expression of fibroblast growth factor 8 in malignant breast tissue but also show significant fibroblast growth factor 8 expression in non-malignant breast epithelial cells.

Altered intracellular localization of fibroblast growth factor receptor 3 in human breast cancer.

Immunohistochemical staining of human breast tissues, using an antibody against fibroblast growth factor receptor 3 [FGFR-3], showed differences in cellular distribution. Both malignant and non-malignant epithelial cells contained FGFR-3 immunoreactivity, but myoepithelial cells and stroma were negative. The staining pattern in malignant epithelial cells was predominantly nuclear, whereas epithelial cells in normal breast tissue showed both cytoplasmic and nuclear elements.

Retroviral infection of the FGF2 gene into MCF-7 cells induces branching morphogenesis, retards cell growth and suppresses tumorigenicity in nude mice.

FGF2 (basic fibroblast growth factor) is a multifunctional growth factor and exhibits diverse function in different cell types. In breast, loss of FGF2 expression is associated with malignant progression. In order to understand the role of FGF2 in maintenance of normal breast structures and control of cell growth, we restored FGF2 expression in the breast cancer cell line MCF-7.

The human mammary gland basement membrane is integral to the polarity of luminal epithelial cells.

We show that myoepithelial cell basement membrane derived E3 and E8 domains of laminin-1 are capable of polarizing luminal epithelial cells with regard to epithelial membrane antigen localization. This event is dependent on the alpha6 integrin and results in aggregation and phosphorylation of the tyrosine residues of the focal adhesion kinase complex. We also demonstrate that uncultured normal luminal epithelial cells synthesize normal levels of beta and gamma laminin chains and reduced levels of alpha chains mRNA in common with malignant epithelial cells.

Increased expression of fibroblast growth factor 8 in human breast cancer.

Fibroblast growth factor 8 (FGF8) is an important developmental protein which is oncogenic and able to cooperate with wnt-1 to produce mouse mammary carcinoma. The level of expression of FGF8 mRNA was measured in 68 breast cancers and 24 non-malignant breast tissues. Elevated levels of FGF8 mRNA were found in malignant compared to non-malignant breast tissues with significantly more malignant tissues expressing FGF8 (P=0.

A paracrine role for myoepithelial cell-derived FGF2 in the normal human breast.

We have studied separated normal human breast epithelial and myoepithelial cells for the presence of basic fibroblast growth factor (FGF2) and its receptors, both low (heparan sulfate proteoglycans) and high affinity (FGFR1), and for the effects of FGF2 on the proliferation of both cell types. Our results indicate that these cells differ markedly in their synthesis and response to FGF2. We found, using PCR of purified cell populations, mRNA for FGF2 only in the myoepithelial cells, whereas immunostaining and Western blotting results demonstrated the presence of FGF2 protein in both epithelial and myoepithelial cells.

The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19.

The aim of this study was to determine whether reverse transcriptase polymerase chain reaction (RT-PCR) for keratin 19 (K19) provides additional information when combined with immunohistochemistry when used to detect micrometastases in blood and bone marrow in patients with primary breast cancer. We studied 78 patients with breast cancer who had no evidence of distant metastases. We collected blood and bone marrow, separated the mononuclear fraction and carried out RT-PCR and immunohistochemistry for K19.

Separated human breast epithelial and myoepithelial cells have different growth factor requirements in vitro but can reconstitute normal breast lobuloalveolar structure.

In order to investigate the specific factors controlling the growth of normal breast cell types, purified populations of human breast epithelial and myoepithelial cells from reduction mammoplasties were grown in primary culture in three defined media and their response to foetal calf serum (FCS), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) measured using MTT growth assays. Epithelial and myoepithelial cells differed markedly in their growth requirements. Whereas epithelial cell survival was dependent on the presence of FCS, myoepithelial cell growth was dramatically inhibited by serum.

Effect of basic fibroblast growth factor-saporin mitotoxin on breast cancer cell lines in vitro.

Fibroblast growth factor receptors are widely expressed on breast cancer cells and we report a preliminary study to determine whether these could be useful as potential targets for delivery of a cytotoxic fibroblast growth factor 2-saporin conjugate. We show that this mitotoxin conjugate can displace I-125-fibroblast growth factor 2 binding though with reduced affinity compared to unlabeled fibroblast growth factor 2. For 4 out of 5 cell lines it is an effective inhibitor of cell growth, and cytotoxic for at least 2 of the lines.