Human Mammary Gland Research Articles

Collective cell migration during human mammary gland organoid morphogenesis.

Organ morphogenesis is driven by cellular migration patterns, which become accessible for observation in organoid cultures. We demonstrate here that mammary gland organoids cultured from human primary cells, exhibit oscillatory and collective migration patterns during their development into highly branched structures, as well as persistent rotational motion within the developed alveoli. Using high-resolution live-cell imaging, we observed cellular movement over the course of several days and subsequently characterized the underlying migration pattern by means of optical flow algorithms. Confined by the surrounding collagen matrix, characteristic correlated back-and-forth movements emerge due to a mismatch between branch invasion and cell migration speeds throughout the branch invasion phase. In contrast, alveolar cells exhibit continuous movement in the same direction. By modulating cell-cell adhesions, we identified collective migration as a prerequisite for sustaining these migration patterns both during the branching elongation process and after alveolus maturation.

ODP575 Single-cell analysis reveals heterogeneity of mouse mammary gland fibroblasts with discrete functions, estrogen responses, differentiation, and communications with epithelium.

Abstract Mammary gland is an essential organ for milk production. It continues to develop after birth under the influence of ovarian hormones, especially estrogen. Among its cellular components, fibroblasts are recognized as an important player for mammary gland organization. Although human mammary gland fibroblasts have been classified into two subtypes based on histological localizations and different marker expressions (i. e., interlobular and intralobular fibroblasts), their detailed characteristics and functions are still limited. Moreover, despite the importance of experimental mouse models for studying mammary gland biology, heterogeneity of mouse mammary gland fibroblasts has not been fully defined. Here, we performed single-cell RNA sequencing (scRNA-seq) to determine cellular populations and their potential contributions to mammary gland organization. Based on the comprehensive gene expression profiles at a single cell level, we identified two major clusters of the mouse mammary gland fibroblasts. One of the major clusters showed the highly specific expression of Dpp4 gene, which is a known marker for human interlobular fibroblasts, and the upregulation of the inflammatory response gene signature. The other major cluster was consisted of three subclusters without Dpp4 expression, which suggested the distinct contributions to adipogenesis and/or ECM modeling in mammary gland stroma. Through the histological validation, we defined these two major subsets as "DPP4+" and "DPP4-" fibroblasts. Since our scRNA-seq was performed on two different ovarian hormone-depleted mouse models followed with hormone treatments, we evaluated the effect of the hormones, especially estrogen. The results demonstrated that the estrogen treatment altered the gene expression profiles of the DPP4+ and a subcluster of the DPP4- fibroblasts in a population-specific manner. Estrogen upregulated interferon-regulated genes and ECM genes in these fibroblast populations, respectively, but not typical estrogen-regulated genes, even in the ERα+ DPP4+ fibroblasts. To define these mammary gland fibroblasts better, we integrated our datasets with a recently established "steady-state mouse fibroblasts atlas" (1) as well as four other scRNA-seq datasets containing normal mouse mammary gland fibroblasts. Using this integrated dataset, we revealed the directional differentiation among the mammary gland fibroblasts, going from the fibroblasts and branching toward the DPP4- fibroblasts with more specialized functions. Moreover, the cell-cell interaction analysis combined with a mammary epithelium dataset from our previous publication (2) indicated possible cell-type-specific communications between mammary gland fibroblasts and epithelial cells. In conclusion, this study provided a comprehensive view of mouse mammary gland fibroblast biology at the single-cell level. Our findings would provide fundamental insights for further investigations on the roles of different fibroblast populations in mammary gland organization and pathological conditions, such as breast cancer. Reference: (1) Buechler et al., Nature. 2021 May;593(7860): 575–579. (2) Saeki et al., Commun Biol. 2021 Jun;4(1): 1–16. Presentation: No date and time listed

Human dihydrofolate reductase inhibition effect of 1-Phenylpyrazolo[3,4–d]pyrimidines: Synthesis, antitumor evaluation and molecular modeling study

A new series of pyrazolo[3,4–d]pyrimidine analogues bearing different amino acid conjugates 10a-m were synthesized with the aim to evaluate their antitumor effect through simultaneous inhibition of human dihydrofolate reductase (hDHFR). All novel compounds were tested to screen their enzyme inhibition activity against (hDHFR) beside their in vitro cytotoxicity against six human MTX resistant cancer cell lines namely, human prostate cancer (PC-3), pancreatic human cancer cell lines (BxPC-3), colorectal carcinoma (HCT-116), human hepatocellular carcinoma (HepG-2), cervical carcinoma (HeLa), and mammary gland breast cancer (MCF-7), besides normal immortalized pancreatic cell line (HPDE). Compounds 10e, 10f, 10g inhibited DHFR at considerable low (IC50 < 1 µM) in comparison to MTX (IC50 = 5.61 µM) beside their characteristic cytotoxic effects on different resistant cancer cell lines. Flow cytometry was done for the most active candidate compound 10e against MCF-7 breast cancer cell line. The results illustrated that compound 10e induced apoptosis and arrested MCF-7 cell cycle in the G1/S phase. Western blot for visualization and quantification was used to confirm the capability of compound 10e to induce the expression of proapoptotic caspases and Bax proteins in MCF-7 breast cancer cell line beside its ability to reduce the expression of antiapoptotic Bcl-2 protein. Molecular modeling studies demonstrated that compound 10e elucidated binding energy of (S= − 8.4390 Kcal/mol) that exceed that of the normal ligand MTX (S= − 8.3951Kcal/mol) in addition to several favorable binding interactions with the active site residues.

Andrographolide suppresses breast cancer progression by modulating tumor-associated macrophage polarization through the Wnt/β-catenin pathway.

Andrographolide(ADE) has been demonstrated to inhibit tumor growth through direct cytotoxicity on tumor cells. However, its potential activity on tumor microenvironment (TME) remains unclear. Tumor-associated macrophages (TAMs), composed mainly of M2 macrophages, are the key cells that create an immunosuppressive TME by secretion of cytokines, thus enhancing tumor progression. Re-polarized subpopulations of macrophages may represent vital new therapeutic alternatives. Our previous studies showed that ADE possessed anti-metastasis and anoikis-sensitization effects. Here, we demonstrated that ADE significantly suppressed M2-like polarization and enhanced M1-like polarization of macrophages. Moreover, ADE inhibited the migration of M2 and tube formation in HUVECs under M2 stimulation. In vivo studies showed that ADE restrained the growth of MDA-MB-231 and HCC1806 human breast tumor xenografts and 4T-1 mammary gland tumors through TAMs. Wnt5a/β-catenin pathway and MMPs were particularly associated with ADE's regulatory mechanisms to M2 according to RNA-seq and bioinformatics analysis. Moreover， western blot also verified the expressions of these proteins were declined with ADE exposure. Among the cytokines released by M2, PDGF-AA and CCL2 were reduced. Our current findings for the first time elucidated that ADE could modulate macrophage polarization and function through Wnt5a signaling pathway, thereby playing its role in inhibition of triple-negative breast cancer.

Abstract 5682: Accelerated biological age is a driver of cancer susceptibility in genetic high risk breast tissue

Abstract 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. We show that histologically normal breast tissue from younger women who are susceptible to breast cancer because they harbor a germline mutation in BRCA1, BRCA2, or PALB2 genes, exhibit 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. Genetically high risk luminal epithelial cells also show evidence of accelerated age, by as much as four decades compared to their chronological age, using a breast specific biological clock comprised of measurements of methylation and expression of the luminal-specific ELF5 transcription factor. 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. Citation Format: Masaru Miyano, Sundus Shalabi, Rosalyn W. Sayaman, Martha Stampfer, Victoria E. Seewaldt, Mark A. LaBarge. Accelerated biological age is a driver of cancer susceptibility in genetic high risk breast tissue [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 5682.

Antiproliferative and cytotoxic effects of bioactive compounds isolated from Onosma bourgaei.

Onosma species have been used commonly for traditional medicine for years due to their bioactive compounds content. Onosma bourgaei aerial part was extracted with hexane and methanol successively. The methanol extract was subjected to chromatographic techniques to isolate allantoin (1), 3,4-dihydroxybenzaldehyde (2), luteolin-7-O-glucoside (3), apigenin-7-O-β-glucoside (4), diosmetin-7-O-β-glucoside (5), rosmarinic acid (6), and globoidnan A (7). The structure of isolated compounds were identified by spectroscopic techniques such as 1D-NMR, 2D-NMR, FTIR, and LC-TOF/MS/MS. Antiproliferative activity of extract and natural compounds were carried out using HeLa (human epithelial cervix adenocarcinoma, ATCC® CCL-2™), HT29 (human colorectal adenocarcinoma, ATCC® HTB38™), MCF7 (human mammary gland adenocarcinoma, ATCC® HTB22™), and A549 (human lung carcinoma, ATCC® CCL185™) cancerous cells and normal cells, FL (human epithelial amnion cell, ATCC® CCL62™). Lactate dehydrogenase (LDH) was performed for cytotoxicity. The compounds, 4, 6, and 7 displayed the strong antiproliferative activity against corresponding cell lines. Apigenin-7-O-β-glucoside (4) revealed the excellent activity on HeLa, HT29, A549, and MCF6 cancer cell lines with the values of (IC50, µM) 167.3, 196.8 181.1, and 203.5, respectively, compared standard compound, cisplatin.

Comparative analysis of the molecular subtype landscape in canine and human mammary gland tumors

Breast cancers in humans belong to one of several intrinsic molecular subtypes each with different tumor biology and different clinical impact. Mammary gland tumors in dogs are proposed as a relevant comparative model for human breast cancer; however, it is still unclear whether the intrinsic molecular subtypes have the same significance in dogs and humans. Using publicly available data, we analyzed gene expression and whole-exome sequencing data from 158 canine mammary gland tumors. We performed molecular subtyping using the PAM50 method followed by subtype-specific comparisons of gene expression characteristics, mutation patterns and copy number profiles between canine tumors and human breast tumors from The Cancer Genome Atlas (TCGA) breast cancer cohort (n = 1097). We found that luminal A canine tumors greatly resemble luminal A human tumors both in gene expression characteristics, mutations and copy number profiles. Also, the basal-like canine and human tumors were relatively similar, with low expression of luminal epithelial markers and high expression of genes involved in cell proliferation. There were, however, distinct differences in immune-related gene expression patterns in basal-like tumors between the two species. Characteristic HER2-enriched and luminal B subtypes were not present in the canine cohort, and we found no tumors with high-level ERBB2 amplifications. Benign and malignant canine tumors displayed similar PAM50 subtype characteristics. Our findings indicate that deeper understanding of the different molecular subtypes in canine mammary gland tumors will further improve the value of canines as comparative models for human breast cancer.

Nitroimidazole-sulfonamides as carbonic anhydrase IX and XII inhibitors targeting tumor hypoxia: Design, synthesis, molecular docking and molecular dynamics simulation

In the present investigation, we focused on the development of dual drugs able to inhibit carbonic anhydrase enzyme (CA) by harboring sulfonamide group and to increase the sensitivity of hypoxic tumors to radio and chemotherapies by incorporating 5-nitroimidazole scaffold. The synthesized compounds were evaluated as cytotoxic agents under oxic and hypoxic conditions against three cell lines including human colorectal carcinoma (HCT116), epithelioid carcinoma (Hela), and mammary gland carcinoma (MCF-7) cells. Compounds 13a and 15a,b exhibited the highest cytotoxic activity against the three cell lines under both oxic (IC50 = 6.18 - 23.06 µM) and hypoxic (IC50 = 5.63 - 19.78 µM) conditions. The capability of compounds 13a and 15b to augment the lethal effect of ɤ-irradiation was evaluated. Both compounds increased the sensitivity of cancerous HCT116 cells to the cell-killing effect of ionizing radiation (IC50 = 4.18 and 2.53 µM, respectively). Sulfonamide derivatives 13a,b, 15a,b, and 17a,b were evaluated for their inhibitory effect against the tumor-associated hCA IX and XII isoforms in comparison to acetazolamide. Molecular docking and molecular dynamics simulation were performed for some sulfonamides on isoforms hCA IX and XII to understand the elements governing the inhibitory effect and the stability of interaction under dynamic conditions.

ОПРЕДЕЛЕНИЕ СОДЕРЖАНИЯ ХИМИЧЕСКИХ ЭЛЕМЕНТОВ В ЗДОРОВОЙ ТКАНИ МОЛОЧНОЙ ЖЕЛЕЗЫ ЧЕЛОВЕКА МЕТОДОМ НЕЙТРОННО-АКТИВАЦИОННОГО АНАЛИЗА

Purpose: To develop the method of chemical element (ChE) content measurement in samples of breast tissue (BT) using the activation by neutrons of nuclear reactor combined with the high-resolution spectrometry of gamma-radiation of short-lived radionuclides (INAA-SLR), and to investigate ChE contents in normal human mammary gland. Material and methods: In the samples of BT taken from female with intact mammary glands (mostly died from trauma, n = 38) the contents of calcium (Ca), chlorine (Cl), iodine (I), potassium (K), magnesium (Mg), manganese (Mn), sodium (Na), and strontium (Sr) were measured. To determine these element contents, the method of INAA-SLR was developed. Results: The accuracy of the developed method and the reliability of the results obtained in the study were confirmed by the measurements of international certified reference material IAEA H-4 Animal Muscles and the good agreement with data of its certificate. The main statistic parameters, including, arithmetic mean, standard deviation, standard error of mean, minimum and maximum values, median, percentiles with 0.025 and 0.975 levels was calculated for ChE contents in the normal BT. It was shown, for example, that means of mass fractions ± standard errors of means (M±SEM, mg/kg dry tissue) in normal human mammary gland were: Ca 128±14, Cl 1014±146, I 0,82±0,11, K 196±20, Mg 80,0±9,4, Mn 0,27±0,04, Na 665±71 и Sr 5,20±0,75. It was found that the Ca, Cl, I, K, Mg, Mn, Na и Sr contents in human mammary gland very differ from the normal level of these elements in whole blood, muscle and adipose tissue, particularly in contents of I and Sr. Conclusion: The method of INAA-SLR allow to obtain the representative data on Ca, Cl, I, K, Mg, Mn, Na и Sr contents in human BT specimen. It was shown, that such trace elements as I and Sr may be directly involved in the mammary gland function.

Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Agents.

A series of cyclic peptides, [(DipR)(WR)4], [(DipR)2(WR)3], [(DipR)3(WR)2], [(DipR)4(WR)], and [DipR]5, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as hydrophobic residues, were synthesized and evaluated for their molecular transporter efficiency. The in vitro cytotoxicity of the synthesized peptides was determined in human epithelial ovary adenocarcinoma cells (SK-OV-3), human lymphoblast peripheral blood cells (CCRF-CEM), human embryonic epithelial kidney healthy cells (HEK-293), human epithelial mammary gland adenocarcinoma cells (MDA-MB-468), pig epithelial kidney normal cells (LLC-PK1), and human epithelial fibroblast uterine sarcoma cells (MES-SA). A concentration of 5–10 µM and 3 h incubation were selected in uptake studies. The cellular uptake of a fluorescent-labeled phosphopeptide, stavudine, lamivudine, emtricitabine, and siRNA was determined in the presence of peptides via flow cytometry. Among the peptides, [DipR]5 (10 µM) was found to be the most efficient transporter and significantly improved the uptake of F’-GpYEEI, i.e., by approximately 130-fold after 3 h incubation in CCRF-CEM cells. Confocal microscopy further confirmed the improved delivery of fluorescent-labeled [DipR]5 (F’-[K(DipR)5]) alone and F’-GpYEEI in the presence of [DipR]5 in MDA-MB-231 cells. The uptake of fluorescent-labeled siRNA (F’-siRNA) in the presence of [DipR]5 with N/P ratios of 10 and 20 was found to be 30- and 50-fold higher, respectively, compared with the cells exposed to F’-siRNA alone. The presence of endocytosis inhibitors, i.e., nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin, did not completely inhibit the cellular uptake of F’-[K(DipR)5] alone or F’-GpYEEI in the presence of [DipR]5, suggesting that a combination of mechanisms contributes to uptake. Circular dichroism was utilized to determine the secondary structure, while transmission electron microscopy was used to evaluate the particle sizes and morphology of the peptides. The data suggest the remarkable membrane transporter property of [DipR]5 for improving the delivery of various small molecules and cell-impermeable negatively charged molecules (e.g., siRNA and phosphopeptide).

Genetic and genomic analysis of age at first insemination in Israeli dairy cattle

We performed a genetic analysis of age at first insemination, including estimation of the heritability and genetic correlations with other economic traits, and the consequences of including this trait in the Israeli selection index. The genetic factors affecting age at first insemination were determined via GWAS. Five data sets were analyzed. Data sets 1, 2, and 3 were used to compute variance components among age at first insemination, first calving age, days from first insemination to calving, and the 9 traits included in the Israel breeding index. Heritabilities for age at first insemination, calving age, and days from first insemination to calving in Israeli Holsteins as computed by REML individual animal model analyses of 273,239 Israeli Holstein cows were 0.072, 0.042, and 0.014. The estimated genetic correlation between the first 2 traits was 0.88. In addition to the fact that heritability of age at first insemination is 1.7 times the heritability for calving, the former trait has the advantage that the number of records is greater, and the records are generated earlier. Absolute values of the genetic and residual correlations between age at first insemination and the 9 traits included in the Israeli index were all less than 0.2. Data set 4 included first insemination dates of 1,181,600 calves born from 1985 through 2018. Genetic evaluations were computed by a single trait animal model. Annual phenotypic and genetic trends for age at first calving for calves born since 1985 were "positive," that is, economically negative, at 0.320 ± 0.003 and 0.169 ± 0.005 d, respectively. Applying the GCTA-GREML software, 54% of variance in the transmitting ability of 1,585 sires could be explained by considering all 40,498 markers included in the GWAS analysis. The significant markers were mainly associated with milk production genes. The SNP UA-IFASA-8854 on chromosome 11 had the lowest probability value, 1.2 × 10-24. This marker is located between the genes RETSAT and ELMOD3, both of which are overexpressed in human mammary glands. The gene RETSAT is reported to be essential for lipid accumulation and adipogenesis promotion. Gene enrichment analysis found that genes in the genomic region flanking significant markers are associated with vasopressin receptor activity, which was shown to mediate puberty in humans. If age at first insemination is included in the index with a weighting to account for 9% of the index, reductions of 2.8 and 2.6 d for age at first insemination and first calving age after 10 yr of selection are predicted, as compared with reductions of 1.4 and 1.1 d with the current index. Gains for the other index traits are only marginally affected. We suggest selection on age at first insemination as an alternative to selection for early calving.

Bisphenol A replacement chemicals, BPF and BPS, induce protumorigenic changes in human mammary gland organoid morphology and proteome

SignificanceBisphenol A (BPA), found in many plastic products, has weak estrogenic effects that can be harmful to human health. Thus, structurally related replacements-bisphenol S (BPS) and bisphenol F (BPF)-are coming into wider use with very few data about their biological activities. Here, we compared the effects of BPA, BPS, and BPF on human mammary organoids established from normal breast tissue. BPS disrupted organoid architecture and induced supernumerary branching. At a proteomic level, the bisphenols altered the abundance of common targets and those that were unique to each compound. The latter included proteins linked to tumor-promoting processes. These data highlighted the importance of testing the human health effects of replacements that are structurally related to chemicals of concern.

Modulation of Cell-Cycle Progression by Hydrogen Peroxide-Mediated Cross-Linking and Degradation of Cell-Adhesive Hydrogels.

The cell cycle is known to be regulated by features such as the mechanical properties of the surrounding environment and interaction of cells with the adhering substrates. Here, we investigated the possibility of regulating cell-cycle progression of the cells on gelatin/hyaluronic acid composite hydrogels obtained through hydrogen peroxide (H2O2)-mediated cross-linking and degradation of the polymers by varying the exposure time to H2O2 contained in the air. The stiffness of the hydrogel varied with the exposure time. Human cervical cancer cells (HeLa) and mouse mammary gland epithelial cells (NMuMG) expressing cell-cycle reporter Fucci2 showed the exposure-time-dependent different cell-cycle progressions on the hydrogels. Although HeLa/Fucci2 cells cultured on the soft hydrogel (Young’s modulus: 0.20 and 0.40 kPa) obtained through 15 min and 120 min of the H2O2 exposure showed a G2/M-phase arrest, NMuMG cells showed a G1-phase arrest. Additionally, the cell-cycle progression of NMuMG cells was not only governed by the hydrogel stiffness, but also by the low-molecular-weight HA resulting from H2O2-mediated degradation. These results indicate that H2O2-mediated cross-linking and degradation of gelatin/hyaluronic acid composite hydrogel could be used to control the cell adhesion and cell-cycle progression.

Biomedical applications of L-alanine produced by Pediococcus acidilactici BD16 (alaD+).

L-alanine possesses extensive physiological functionality and tremendous pharmacological significance, therefore could be considered as potential ingredient for food, pharmaceutical, and personal care products. However, therapeutic properties of L-alanine still need to be addressed in detail to further strengthen its utilization as a viable ingredient for developing natural therapeutics with minimum side effects. Thus, the present study was aimed to explore the anticipated therapeutic potential of L-alanine, produced microbially using a lactic acid bacterial strain Pediococcus acidilactici BD16 (alaD+) expressing L-alanine dehydrogenase enzyme. The anticipated therapeutic potential of L-alanine was assessed in terms of anti-proliferative, anti-bacterial, and anti-urolithiatic properties. Anti-bacterial assays revealed that L-alanine successfully inhibited growth and in vitro proliferation of important human pathogens including Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Streptococcus mutans, and Vibrio cholerae in a concentration-dependent manner. Current investigation has also revealed its significant anti-proliferative potential against human lung adenocarcinoma (A549; IC50 7.32μM) and mammary gland adenocarcinoma (MCF-7; IC50 8.81μM) cells. The anti-urolithiatic potential of L-alanine was augmented over three different phases, viz., nucleation inhibition, aggregation inhibition, and oxalate depletion. Further, an in vitro cell culture-based kidney stone dissolution model using HEK293-T cells was also established to further strengthen its anti-urolithiatic potential. This is probably the first in vitro cell culture-based model which experimentally validates the immense therapeutic efficacy of L-alanine in treating urolithiasis disease. KEY POINTS: • Assessment of therapeutic potential of L-alanine produced by LAB. • L-alanine exhibited significant anti-proliferative and anti-bacterial activities. • L-alanine as potential anti-urolithiatic agent.

Role of ADCC, CDC, and CDCC in Vaccine-Mediated Protection against Her2 Mammary Carcinogenesis.

Amplification or mutation of the Her2 oncoantigen in human mammary glands leads to the development of an aggressive breast carcinoma. Several features of this breast carcinoma are reproduced in mammary carcinomas that spontaneously arise in female transgenic mice bearing the activated rat Her2 oncogene under transcriptional control of the mouse mammary tumor virus promoter-BALB-neuT (neuT) mice. We previously demonstrated that carcinoma progression in neuT mice can be prevented by DNA vaccination with RHuT, a plasmid coding for a chimeric rat/human Her2 protein. RHuT vaccination exerts an antitumor effect, mostly mediated by the induction of a strong anti-rat Her2 antibody response. IgG induced by RHuT vaccine mainly acts by blocking Her2 signaling, thus impairing cell cycle progression and inducing apoptosis of cancer cells, but other indirect effector mechanisms could be involved in the antibody-mediated protection. The recruitment of cells with perforin-dependent cytotoxic activity, able to perform antibody-dependent cellular cytotoxicity, has already been investigated. Less is known about the role of the complement system in sustaining antitumor response through complement-dependent cytotoxicity and cellular cytotoxicity in vaccinated mice. This work highlights that the weight of such mechanisms in RHuT-induced cancer protection is different in transplantable versus autochthonous Her2+ tumor models. These results may shed new light on the effector mechanisms involved in antibody-dependent anti-cancer responses, which might be exploited to ameliorate the therapy of Her2+ breast cancer.

Stem-like breast cancer cells in the activated state resist genetic stress via TGFBI-ZEB1

Breast cancer cells with stem-like properties are critical for tumor progression, yet much about these cells remains unknown. Here, we characterize a population of stem-like breast cancer cells expressing the integrin αvβ3 as transcriptionally related to activated stem/basal cells in the normal human mammary gland. An unbiased functional screen of genes unique to these cells identified the matrix protein TGFBI (BIG-H3) and the transcription factor ZEB1 as necessary for tumorsphere formation. Surprisingly, these genes were not required for cell proliferation or survival, but instead maintained chromosomal stability. Consistent with this finding, CRISPR deletion of either gene synergized with PARP inhibition to deplete αvβ3+ stem-like cells, which are normally resistant to this therapy. Our findings highlight a critical role for TGFBI-ZEB1 protection against genetic stress as a key attribute of activated stem-like cells and suggest that disrupting this ability may enhance their “BRCAness” by increasing sensitivity to PARP inhibitors.

Reverse Ordered Sequential Mechanism for Lactoperoxidase with Inhibition by Hydrogen Peroxide.

Lactoperoxidase (LPO, FeIII in its resting state in the absence of substrates)—an enzyme secreted from human mammary, salivary, and other mucosal glands—catalyzes the oxidation of thiocyanate (SCN−) by hydrogen peroxide (H2O2) to produce hypothiocyanite (OSCN−), which functions as an antimicrobial agent. The accepted catalytic mechanism, called the halogen cycle, comprises a two-electron oxidation of LPO by H2O2 to produce oxoiron(IV) radicals, followed by O-atom transfer to SCN−. However, the mechanism does not explain biphasic kinetics and inhibition by H2O2 at low concentration of reducing substrate, conditions that may be biologically relevant. We propose an ordered sequential mechanism in which the order of substrate binding is reversed, first SCN− and then H2O2. The sequence of substrate binding that is described by the halogen cycle mechanism is actually inhibitory.

Surface-tension-induced budding drives alveologenesis in human mammary gland organoids.

Organ development involves complex shape transformations driven by active mechanical stresses that sculpt the growing tissue 1,2. Epithelial gland morphogenesis is a prominent example where cylindrical branches transform into spherical alveoli during growth3-5. Here we show that this shape transformation is induced by a local change from anisotropic to isotropic tension within the epithelial cell layer of developing human mammary gland organoids. By combining laser ablation with optical force inference and theoretical analysis, we demonstrate that circumferential tension increases at the expense of axial tension through a reorientation of cells that correlates with the onset of persistent collective rotation around the branch axis. This enables the tissue to locally control the onset of a generalized Rayleigh-Plateau instability, leading to spherical tissue buds6. The interplay between cell motion, cell orientation and tissue tension is a generic principle that may turn out to drive shape transformations in other cell tissues.

Hybrid Cyclic-Linear Cell-Penetrating Peptides Containing Alternative Positively Charged and Hydrophobic Residues as Molecular Transporters

The cell membrane properties create a significant obstacle in intracellular delivery of cell-impermeable and negatively charged molecules. Herein, we report the synthesis and biological evaluation of a novel series of hybrid cyclic-linear peptides containing alternative positive and hydrophobic amino acids on the ring and side chain [(RW)5]K(RW)X (X = 1-5) to compare their molecular transporter efficiency. The peptides were synthesized through Fmoc solid-phase peptide synthesis. In vitro cytotoxicity of the peptides showed that the peptides did not exhibit any significant cytotoxicity at the concentration of 10 μM in human leukemia carcinoma cell line (CCRF-CEM), human ovarian adenocarcinoma cells (SK-OV-3), human epithelial embryonic kidney healthy (HEK-293), and human epithelial mammary gland adenocarcinoma cells (MDA-MB-231) after 3 h incubation. The cellular uptake of a fluorescence-labeled phosphopeptide (F'-GpYEEI) and anti-human immunodeficiency virus (HIV) drugs (lamivudine (F'-3TC), emtricitabine (F'-FTC), Stavudine (F'-d4T)), where F' is carboxyfluorescein, was measured in the presence of the peptides in CCRF-CEM and SK-OV-3 cells. Among all peptides, [(RW)5K](RW)5 (10 μM) was the most efficient transporter that improved the cellular uptake of F'-GpYEEI (2 μM) by 18- and 11-fold in CCRF-CEM and SK-OV-3, respectively, compared with F'-GpYEEI alone. Fluorescence-activated cell sorting (FACS) analysis results indicated that the cellular uptake of fluorescence-labeled peptide (F'-[(RW)5K](RW)5) was only partially inhibited by chlorpromazine as an endocytosis inhibitor after 3 h incubation in MDA-MB-231 cells. These data suggest the potential of this series of hybrid cyclic-linear peptides as cell-penetrating peptides and molecular transporters.

Label-free isolation and cultivation of patient-matched human mammary epithelial and stromal cells from normal breast tissue.

Breast cancer is primarily derived from mammary epithelial cells, the main cell type in human mammary glands. The majority of knowledge gained thus far around breast cancer has come from research using immortalized epithelial cell lines. The use of primary cells derived from breast tissue can be used in research to provide more biological relevance representative of the heterogeneous nature of breast cancer development and metastasis in its natural microenvironment. However, the successful isolation and propagation of human primary mammary gland cells can be costly and difficult due to their complex in vivo microenvironment and sensitivity when isolated. Here, we present a gentle isolation method for viable human mammary epithelial cells (hMECs) and donor-matched human mammary fibroblasts (hMFbs) from human mammary gland tissue. We isolated, expanded and passaged the hMECs and hMFbs in vitro and characterized cultures using cell-specific markers. A total of four primary cell lines were isolated and established from normal breast tissue and characterized through various markers, including pan cytokeratin (panCK), CK14, CD44, CD31, fibronectin and vimentin by immunofluorescence. To determine functional potential for subsequent studies, epithelial cells were examined via Matrigel® assays to assess spheroid development. Both cell type cultures expressed lineage specific markers with hMECs but not hMFbs forming spheroid structures in 3D Matrigel® assays. Our analyses confirm the successful isolation of two different cell phenotypes from normal breast tissues. This robust technique provides an inexpensive and accessible approach for mammary cell isolation.