Relation of Serum Estrogen Metabolites with Terminal Duct Lobular Unit Involution Among Women Undergoing Diagnostic Image-Guided Breast Biopsy

Higher levels of circulating estrogens and estrogen metabolites (EMs) have been associated with higher breast cancer risk. In breast tissues, reduced levels of terminal duct lobular unit (TDLU) involution, as reflected by higher numbers of TDLUs and acini per TDLU, have also been linked to elevated breast cancer risk. However, it is unknown whether reduced TDLU involution mediates the risk associated with circulating EMs. In a cross-sectional analysis of 94 premenopausal and 92 postmenopausal women referred for clinical breast biopsy at an academic facility in Vermont, we examined the associations of 15 EMs, quantified using liquid chromatography-tandem mass spectrometry, with the number of TDLUs and acini count/TDLU using zero-inflated Poisson regression with a robust variance estimator and ordinal logistic regression models, respectively. All analyses were stratified by menopausal status and adjusted for potential confounders. Among premenopausal women, comparing the highest vs. the lowest tertiles, levels of unconjugated estradiol (risk ratio (RR) = 1.74, 95 % confidence interval (CI) = 1.06-2.87, p trend = 0.03), 2-hydroxyestrone (RR = 1.74, 95 % CI = 1.01-3.01, p trend = 0.04), and 4-hydroxyestrone (RR = 1.74, 95 % CI = 0.99-3.06, p trend = 0.04) were associated with significantly higher TDLU count. Among postmenopausal women, higher levels of estradiol (RR = 2.09, 95 % CI = 1.01-4.30, p trend = 0.04) and 16α-hydroxyestrone (RR = 2.27, 95 % CI = 1.29-3.99, p trend = 0.02) were significantly associated with higher TDLU count. Among postmenopausal women, higher levels of EMs, specifically conjugated estrone and 2- and 4-pathway catechols, were also associated with higher acini count/TDLU. Our data suggest that higher levels of serum EMs are generally associated with lower levels of TDLU involution

An organotypic assay for the quantification and characterization of regenerative primary human mammary epithelial cells

Breast cancer is a heterogeneous disease with a high degree of intra- and intertumoral diversity, which impedes accurate patient stratification, prognosis and optimal treatment. The mammary gland consists of a complex network of epithelial ducts which end in clusters of alveoli, called terminal ductal lobular units (TDLUs) which are the functional units of the mammary gland. Postnatal mammary gland development and homeostasis require an enormous regenerative output, suggesting the existence of tissue stem/progenitor cells and a high degree of cellular plasticity to ensure functional robustness, i.e. the production and secretion of milk during lactation. Therefore, the observed heterogeneity in breast cancer is likely the result of normal mammary gland architecture and functionality. Unfortunately, the identification and characterization of human stem/progenitor cells and the analysis of cellular plasticity are hampered by the limited applicability of currently used murine in vivo assays and the failure of 2D and 3D in vitro assays to recapitulate the histological architecture and functionality of the human mammary gland. Organoid assays span the bridge between standard in vitro culture and in vivo studies by mimicking the stem cell niche during self-renewal or repair in 3D culture. Thereby, organoid assays support the formation of organoids which recapitulate the architecture and functionality of the original tissue. In this study, a novel organotypic assay was developed for the human mammary gland, in which single, freshly isolated human mammary epithelial cells from healthy donors cultured in floating collagen gels generate spheres and branched ductal structures that resemble TDLUs. The TDLU-like organoids were shown to form alveolar buds, express lineage markers at correct positions, and display functionality by contraction. In line with recent literature from transplantation studies, it was found that TDLU-like structure formation is enriched in a CD49fhi/EpCAM− population, commonly referred to as basal, while CD49f+/EpCAM+ LP cells predominantly formed spheres. In addition, by performing limiting dilution analysis, the metalloendopeptidase CD10 was revealed as marker to enrich for TDLU-like structure forming cells within the CD49fhi/EpCAM− population. The use of CD10 further led to identification of stromal cells present within the CD49fhi/EpCAM− population. As expression of CD10 was not restricted to the CD49fhi/EpCAM− cell population, it was shown that a combination of all three markers is required to optimally enrich for TDLU-like structure forming cells. Importantly, the formation of TDLU-like structures was observed only in floating/compliant, but not in attached/rigid collagen gels and required actin-myosin-based cellular contraction. Structures generated in attached collagen gels exhibited aberrant marker expression and invasive phenotype, emphasizing the importance of physical parameters in directing differentiation of the mammary gland and suggesting a role in tumorigenesis. In summary this study describes a defined in vitro assay system to quantify mammary epithelial cells with regenerative potential by limiting dilution, to analyze cellular plasticity, differentiation potential and functionality and to systematically investigate interactions with the physical environment at distinct steps of morphogenesis and the effect on cell fate decisions.Brustkrebs ist durch eine hohe intra- und intertumorale Heterogenität gekennzeichnet, wodurch Stratifizierung sowie optimale Prognose und Behandlung von Patienten erschwert werden. Die adulte humane Brustdrüse besteht aus komplex verzweigten Milchgängen, welche in traubenartigen Strukturen, den so genannten Drüsenlappen (Terminal ductal lobular units, TDLUs), den funktionellen Einheiten der normalen Brustdrüse, enden. Die Entwicklung und Homöostase der Brustdrüse finden hauptsächlich postnatal statt und erfordern eine enorme regenerative Leistung, welche die Existenz von Stamm- oder Progenitorzellen sowie zelluläre Plastizität suggerieren, wodurch auch die Funktion der Brustdrüse, insbesondere die Produktion und Sekretion von Milch während der Laktation, sichergestellt werden kann. Die komplexe Architektur sowie der hohe regenerative Druck, dem die normale Brustdrüse unterliegt, spielen eine wichtige Rolle in der Heterogenität von Brustkrebs. Die heutzutage genutzten in vivo sowie 2D- und 3D-in vitro Modelle scheitern jedoch darin, die Architektur und Funktion der normalen humanen Brustdrüse korrekt nachzubilden, wodurch die Identifizierung und Charakterisierung von humanen Brust Stamm-/Progenitorzellen und die Analyse zellulärer Plastizität erschwert werden. Organoid-Ansätze bilden eine Brücke zwischen standard in vitro und in vivo Modellen, indem sie die Stammzellnische während der Gewebserneuerung/-reparatur nachbilden. Hierdurch fördern Organoid-Modelle die Bildung von Organoiden, die die normale Gewebearchitektur und -funktion widerspiegeln. In dieser Studie wurde ein neuer, organotypischer Assay für die humane Brustdrüse entwickelt, in dem einzelne, frisch isolierte humane Brustepithelzellen verzweigte Strukturen bilden, die den TDLUs ähneln. Die TDLU-ähnlichen Strukturen bilden Alveolen, exprimieren Zelltypmarker an den korrekten Positionen und zeigen Funktionalität, indem sie kontrahieren. In Übereinstimmung mit der aktuellen Literatur, basierend auf murinen Transplantationsstudien, konnten die TDLU-ähnliche Struktur bildenden Zellen einer CD49fhi/EpCAM− Population zugeordnet werden, welche als basal gilt, wohingegen gezeigt wurde, dass CD49f+/EpCAM+ luminale Vorläuferzellen vornehmlich Sphären bilden. Durch die Verwendung von „Limiting Dilution“ konnte zudem gezeigt werden, dass die Expression der Metalloendopeptidase CD10 die TDLU-ähnliche Struktur bildenden Zellen innerhalb der CD49fhi/EpCAM− Population weiter anreichert. Die Verwendung von CD10 führte außerdem zu der Entdeckung, dass die CD49fhi/EpCAM− Population Stromazellen enthält. Da die Expression von CD10 sich nicht auf die CD49fhi/EpCAM− Population beschränkt, wurde bestimmt, dass die Kombination der drei Marker CD49f, EpCAM und CD10 nötig ist, um TDLU-ähnliche Struktur bildende Zellen sauber anzureichern. Es ist beachtenswert, dass die Bildung von TDLU-ähnlichen Strukturen nur in schwimmenden/weichen Collagen-Gelen, jedoch nicht in festen/rigiden Collagen-Gelen beobachtet wurde, sowie Aktin-Myosin-basierte zelluläre Kontraktilität benötigte. So zeigten Strukturen in festen Collagen Gelen abnormale Zellmarker Expression und invasives Wachstum. Die physikalischen Matrixeigenschaften spielen somit eine wichtige Rolle in der Differenzierung der Brustdrüse und der Krebsentwicklung und können in dem hier präsentierten Assay nachgebildet werden. Zusammenfassend beschreibt diese Arbeit einen neuen organotypischen in vitro Assay, der die Quantifizierung von humanen Brustepithelzellen mit regenerativem Potential, die Erforschung von zellulärer Plastizität, Differenzierung und Funktionalität ermöglicht, sowie die Erforschung des Einflusses physikalischer Parameter während verschiedener Schritte der Morphogenese und der Differenzierung von Zellen erlaubt

Association between breast cancer genetic susceptibility variants and terminal duct lobular unit involution of the breast

Terminal duct lobular units (TDLUs) are the predominant source of future breast cancers, and lack of TDLU involution (higher TDLU counts, higher acini count per TDLU and the product of the two) is a breast cancer risk factor. Numerous breast cancer susceptibility single nucleotide polymorphisms (SNPs) have been identified, but whether they are associated with TDLU involution is unknown. In a pooled analysis of 872 women from two studies, we investigated 62 established breast cancer SNPs and relationships with TDLU involution. Poisson regression models with robust variance were used to calculate adjusted per-allele relative risks (with the non-breast cancer risk allele as the referent) and 95% confidence intervals between TDLU measures and each SNP. All statistical tests were two-sided; P<0.05 was considered statistically significant. Overall, 36 SNPs (58.1%) were related to higher TDLU counts although this was not statistically significant (P=0.25). Six of the 62 SNPs (9.7%) were nominally associated with at least one TDLU measure: rs616488 (PEX14), rs11242675 (FOXQ1) and rs6001930 (MKL1) were associated with higher TDLU count (P=0.047, 0.045 and 0.031, respectively); rs1353747 (PDE4D) and rs6472903 (8q21.11) were associated with higher acini count per TDLU (P=0.007 and 0.027, respectively); and rs1353747 (PDE4D) and rs204247 (RANBP9) were associated with the product of TDLU and acini counts (P=0.024 and 0.017, respectively). Our findings suggest breast cancer SNPs may not strongly influence TDLU involution. Agnostic genome-wide association studies of TDLU involution may provide new insights on its biologic underpinnings and breast cancer susceptibility

Novel 3D Ultrasound Elastography Techniques for In Vivo Breast Tumor Imaging and Nonlinear Characterization

Breast cancer comprises about 29% of all types of cancer in women worldwide. This type of cancer caused what is equivalent to 14% of all female deaths due to cancer. Nowadays, tissue biopsy is routinely performed, although about 80% of the performed biopsies yield a benign result. Biopsy is considered the most costly part of breast cancer examination and invasive in nature. To reduce unnecessary biopsy procedures and achieve early diagnosis, ultrasound elastography was proposed.;In this research, tissue displacement fields were estimated using ultrasound waves, and used to infer the elastic properties of tissues. Ultrasound radiofrequency data acquired at consecutive increments of tissue compression were used to compute local tissue strains using a cross correlation method. In vitro and in vivo experiments were conducted on different tissue types to demonstrate the ability to construct 2D and 3D elastography that helps distinguish stiff from soft tissues. Based on the constructed strain volumes, a novel nonlinear classification method for human breast tumors is introduced. Multi-compression elastography imaging is elucidated in this study to differentiate malignant from benign tumors, based on their nonlinear mechanical behavior under compression. A pilot study on ten patients was performed in vivo, and classification results were compared with biopsy diagnosis - the gold standard. Various nonlinear parameters based on different models, were evaluated and compared with two commonly used parameters; relative stiffness and relative tumor size. Moreover, different types of strain components were constructed in 3D for strain imaging, including normal axial, first principal, maximum shear and Von Mises strains. Interactive segmentation algorithms were also evaluated and applied on the constructed volumes, to delineate the stiff tissue by showing its isolated 3D shape.;Elastography 3D imaging results were in good agreement with the biopsy outcomes, where the new classification method showed a degree of discrepancy between benign and malignant tumors better than the commonly used parameters. The results show that the nonlinear parameters were found to be statistically significant with p-value \u3c0.05. Moreover, one parameter; power-law exponent, was highly statistically significant having p-value \u3c 0.001. Additionally, volumetric strain images reconstructed using the maximum shear strains provided an enhanced tumor\u27s boundary from the surrounding soft tissues. This edge enhancement improved the overall segmentation performance, and diminished the boundary leakage effect. 3D segmentation provided an additional reliable means to determine the tumor\u27s size by estimating its volume.;In summary, the proposed elastographic techniques can help predetermine the tumor\u27s type, shape and size that are considered key features helping the physician to decide the sort and extent of the treatment. The methods can also be extended to diagnose other types of tumors, such as prostate and cervical tumors. This research is aimed toward the development of a novel \u27virtual biopsy\u27 method that may reduce the number of unnecessary painful biopsies, and diminish the increasingly risk of cancer

Quantitative Proteomic and Mutational Landscape of Metaplastic Breast Carcinoma and Generation of a 3D Organoid Model of Neoplastic Progression

Triple-negative breast cancer (TNBC) is considered to be the most aggressive and has worse prognosis compared to other breast cancers and accounts for roughly 18% of all epithelial cancers of the breast, or carcinomas. TNBC exhibits complex molecular heterogeneity both inter- and intratumorally and likely consists of several distinct molecular subgroups that are currently unknown. Metaplastic breast carcinoma (MBC) is even more aggressive than triple-negative breast cancer (TNBC) but also typically presents as triple-negative histologically, and is defined by the admixture of both invasive glandular and non-glandular “metaplastic” heterologous elements of spindle, squamous or sarcomatoid subtypes. The protein profiles underpinning the phenotypic diversity and metastatic behavior of MBC are unknown. We present a quantitative multi-subtype proteomic landscape of MBC, non-metaplastic TNBC, and normal breast from small yet well-annotated cohort of 27 patients, and also present the somatic mutational landscape on the same cohort. We used multiplex isobaric tandem mass tag (TMT) labeling for proteomics and quantified 5,798 proteins, and from whole-exome sequencing for genomics analysis we found 980 total somatic mutational variants. MBCs displayed increased epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) signaling, and reduced metabolic pathways compared to TNBC. We discovered subtype-specific profiles among MBCs including distinct upregulated profiles; translation and ribosomal events in spindle, inflammation and apical junctions in squamous, and extracellular matrix in sarcomatoid. Comparison of the proteomes of spindle MBC with MMTV-cre;Ccn6fl/fl spindle MBC mouse tumors revealed a shared spindle-specific signature of 17 upregulated proteins involved in translation (e.g. RPL4,6,18, P3H1, PYCR1). The somatic mutational landscape also revealed MBCs share common TP53 mutations, and in PLEC, MUC17, CRYBG2, and ZNF681. We identified that spindle and squamous MBC exhibit overlapping mutational profiles of genes involved in transcription, RNA metabolic processes and actin filament binding, while sarcomatoid tumors harbor distinct mutations in MAPK, WNT, protocadherin cluster genes, calcium binding and ECM organization. These data identify subtype-specific MBC protein profiles and mutational signatures that identified novel biomarkers for therapy. Three-dimensional (3D) cell culture has been widely used in recent decades, compared with monolayer (2D) culture, because they better mimic the in vivo state. 3D systems utilize different types of gels critical for their success, such as collagen or the reconstituted basement membrane, Matrigel, which has enabled recapitulation of tissue architecture and function that is more physiologic compared to 2D. However, conventional 3D models using gel-embedded platforms have large variability and slow transport of biomolecules to the matrix-encapsulated cells. Here, we developed a highly reproducible, 3D scaffold-free hanging drop method amenable for primary tissues including mouse and human tumors, and our analyses describe a one drop-one organoid format using MCF10A cells, a non-tumorigenic breast cell line. We attained high-yield production of uniform organoids that resemble normal human breast acini, express both mammary gland-specific and progenitor markers, and we developed treatment assays for EMT induction and neoplastic progression delivering rapid quantification of phenotypic and morphological changes. Integration of 3D methods with omics analyses is envisioned to enhance the study of neoplastic progression and generate novel targets of both MBC and TNBC tumors.PHDMolecular & Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155234/1/djomehri_1.pd

Mechanostimulation of integrin αvβ6 and fibronectin in DCISmyoepithelial cells

PhDAlterations to the tumour microenvironment is a common feature of many cancers, including breast cancer, and there is increasing evidence that alterations to the microenvironment, including; increased integrin expression, ECM deposition and protease activity, promote cancer progression. Most invasive breast cancers arise from a preinvasive stage, ductal carcinoma in situ (DCIS). Previous work in our laboratory has shown the microenvironment of DCIS is altered, such that myoepithelial cells (MECs) switch to a tumour-promoting phenotype, associated with upregulation of integrin αvβ6 and fibronectin (FN) expression. Mechanisms by which integrin αvβ6 and FN expression are regulated is unclear. We show DCIS progression into invasion is accompanied by an increase in MEC expression of integrin αvβ6 and periductal FN deposition, and their expression were associated in DCIS. These findings were modelled in isolated primary DCIS-MECs, primary normal MECs and MEC lines, with and without integrin αvβ6 expression, where integrin αvβ6-positive MECs upregulating FN expression. We identified integrin αvβ6-positive DCIS ducts were larger than integrin αvβ6-negative DCIS ducts, and mechanical stretching of primary normal MECs and a normal MEC line led to upregulation of integrin αvβ6 expression and FN deposition in a TGFβ-dependent manner. We further show upregulation of integrin αvβ6 and FN by MECs mediate TGFβ-dependent upregulation of MMP13 which promotes breast cancer cell invasion in vitro. These data show altered tissue mechanics in DCIS and MEC expression of integrin αvβ6 and FN deposition are linked, and implicate TGFβ in their activation. These findings suggest integrin αvβ6 and FN may be used as markers to stratify DCIS patients