Normal Human Breast Research Articles

FT-IR Transflection Micro-Spectroscopy Study on Normal Human Breast Cells after Exposure to a Proton Beam

Fourier transform infrared micro-spectroscopy (μ-FT-IR) is nowadays considered a valuable tool for investigating the changes occurring in human cells after exposure to ionizing radiation. Recently, considerable attention has been devoted to the use of this optical technique in the study of cells exposed to proton beams, that are being increasingly adopted in cancer therapy. Different experimental configurations are used for proton irradiation and subsequent spectra acquisition. To facilitate the use of μ-FT-IR, it may be useful to investigate new experimental approaches capable of speeding up and simplifying the irradiation and measurements phases. Here, we propose the use of low-e-substrates slides for cell culture, allowing the irradiation and spectra acquisition in transflection mode in a fast and direct way. In recent years, there has been a wide debate about the validity of these supports, but many researchers agree that the artifacts due to the presence of the electromagnetic standing wave effects are negligible in many practical cases. We investigated human normal breast cells (MCF-10 cell line) fixed immediately after the irradiation with graded proton radiation doses (0, 0.5, 2, and 4 Gy). The spectra obtained in transflection geometry showed characteristics very similar to those present in the spectra acquired in transmission geometry and confirm the validity of the chosen approach. The analysis of spectra indicates the occurrence of significant changes in DNA and lipids components of cells. Modifications in protein secondary structure are also evidenced.

Knockdown of TRIM66 in MDA-MB-468 triple negative breast cancer cell line suppresses proliferation and promotes apoptosis through EGFR signaling.

Triple negative breast cancer (TNBC) is one ofthemost common malignant tumors in women and thetripartite motif 66 (TRIM66) is closely associated with thebehaviors ofwide variety ofcancer. Thus, in this study, we aimed to explore theeffect ofTRIM66 in MDA-MB-468 TNBC cell line. Western blot and RT-PCR assays were used to detect theexpression level ofTRIM66 in human normal mammary cells and human breast cancer cell lines. We silenced its expression in MDA-MB-468 cells by transient siRNA transfection to ascertain thefunction ofTRIM66 in MDA-MB-468 cells. CCK8 and clonogenicity assays were used to evaluate theability ofcell proliferation. Wound healing and Transwell were used to detect cell invasion and migration. TUNEL was applied to detect theapoptosis level. Western blotting was used to detect theexpression ofrelated proteins. TRIM66 was observed markedly elevated in breast cancer cell line. Knockdown ofTRIM66 inhibited theexpression ofEGFR, P-Jak2, P-STAT3, JAK2, and STAT3 in MDA-MB-468 cells through regulating theepidermal growth factor receptor (EGFR) signaling. Knockdown ofTRIM66 suppresses proliferation, invasion and migration ofMDA-MB-468 cell line and promotes apoptosis in these cells through EGFR signaling.

SnO2-Doped ZnO/Reduced Graphene Oxide Nanocomposites: Synthesis, Characterization, and Improved Anticancer Activity via Oxidative Stress Pathway.

BackgroundTherapeutic selectivity and drug resistance are critical issues in cancer therapy. Currently, zinc oxide nanoparticles (ZnO NPs) hold considerable promise to tackle this problem due to their tunable physicochemical properties. This work was designed to prepare SnO2-doped ZnO NPs/reduced graphene oxide nanocomposites (SnO2-ZnO/rGO NCs) with enhanced anticancer activity and better biocompatibility than those of pure ZnO NPs.Materials and MethodsPure ZnO NPs, SnO2-doped ZnO (SnO2-ZnO) NPs, and SnO2-ZnO/rGO NCs were prepared via a facile hydrothermal method. Prepared samples were characterized by field emission transmission electron microscopy (FETEM), energy dispersive spectroscopy (EDS), field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), ultraviolet-visible (UV-VIS) spectrometer, and dynamic light scattering (DLS) techniques. Selectivity and anticancer activity of prepared samples were assessed in human breast cancer (MCF-7) and human normal breast epithelial (MCF10A) cells. Possible mechanisms of anticancer activity of prepared samples were explored through oxidative stress pathway.ResultsXRD spectra of SnO2-ZnO/rGO NCs confirmed the formation of single-phase of hexagonal wurtzite ZnO. High resolution TEM and SEM mapping showed homogenous distribution of SnO2 and rGO in ZnO NPs with high quality lattice fringes without any distortion. Band gap energy of SnO2-ZnO/rGO NCs was lower compared to SnO2-ZnO NPs and pure ZnO NPs. The SnO2-ZnO/rGO NCs exhibited significantly higher anticancer activity against MCF-7 cancer cells than those of SnO2-ZnO NPs and ZnO NPs. The SnO2-ZnO/rGO NCs induced apoptotic response through the upregulation of caspase-3 gene and depletion of mitochondrial membrane potential. Mechanistic study indicated that SnO2-ZnO/rGO NCs kill cancer cells through oxidative stress pathway. Moreover, biocompatibility of SnO2-ZnO/rGO NCs was also higher against normal breast epithelial (MCF10A cells) in comparison to SnO2-ZnO NPs and ZnO NPs.ConclusionSnO2-ZnO/rGO NCs showed enhanced anticancer activity and better biocompatibility than SnO2-ZnO NPs and pure ZnO NPs. This work suggested a new approach to improve the selectivity and anticancer activity of ZnO NPs. Studies on antitumor activity of SnO2-ZnO/rGO NCs in animal models are further warranted.

Normal breast epithelial MCF-10A cells to evaluate the safety of carbon dots

The human normal breast cell line MCF-10A is being widely used as a model in toxicity studies due to its structural similarity to the normal human mammary epithelium. Over the years, application of carbon dots (C-dots) in biomedicine has been increasing due to their photoluminescence properties, biocompatibility, biosafety and possible applications in bioimaging and as drug carriers. In this work we prepared three different C-dots from the same set of carbon and nitrogen precursors (citric acid and urea, respectively) via three distinct bottom-up synthetic routes and their safety was tested against the normal breast cell line MCF-10A. The characterization results demonstrated a similar size range and composition for all the C-dots. The MCF-10A cells were treated with different concentrations of C-dots for 24, 48 and 72 h to evaluate the cell viability over time. For the 24 h incubation, there were no significant decreases in the viability of the MCF-10A cells. For the 48 h treatment, there was a significant decrease in the viability of the cells treated with calcination-based C-dots, but without significant cellular viability changes for microwave and hydrothermal-based C-dots. For 72 h, cells treated with hydrothermal-based C-dots have the most promising viability profile. Also, compared with paclitaxel, these C-dots have a safety profile very close to that of an antineoplastic in non-tumor cells. Our results suggest that these new C-dots have potential as imaging candidates or biosensing tools as well as drug carriers, and further investigation in animal models is needed for future application in medicine.

Female reproductive tract has low concentration of SARS-CoV2 receptors.

There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2, in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2, Cathepsin B (CTSB), and Cathepsin L (CTSL) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2.

Organotin(IV) complexes derived from 1,4-naphthalenedicarboxylic acid: synthesis, structure, in vitro cytostatic activity

Seven organotin(IV) complexes, [(R3Sn)2L]n (R = Me 1, R = n-Bu 2), [(Ph3Sn)2L] (3), [(R2Sn)2L(μ3-O)]n (R = Me 4, R = n-Bu 5), [R2SnL(1,10-phen)]n (R = Me 6), [(R2SnCl)2L(1,10-phen)2] (R = n-Bu 7) derived from 1,4-naphthalenedicarboxylic acid (H2L) have been synthesized and characterized by elemental analysis, FT-IR, PXRD, NMR and X-ray crystallography. The single crystal diffraction reveals that complexes 1 and 2 represent 2D network structures, which both contain tetranuclear 26-membered macrocycles. Complexes 3 and 7 display dinuclear tin monomers, which can form 1D infinite chain by C-H···O and C-H···π interactions. Meanwhile, complexes 4 and 5 display 2D network containing tetraorganodistannoxane unit, while complex 6 adopts a 1D infinite chain structure, which further constructs 2D supramolecular architecture through C-H···O intermolecular interactions. What's more, in vitro cytostatic activity of the complexes 1-3 against cervical carcinoma cell lines (HeLa), hepatocellular carcinoma cell lines (HepG-2) and human normal breast cell lines (HBL-100) have been investigated, and the results show that organotin derivatives with n-butyl and phenyl group (2 and 3) exhibit significantly higher anticancer activity than complex 1 with methyl group. Meanwhile, organotin(IV) complexes (1-3) display lower cytotoxicity than corresponding organotin(IV) precursors [trimethyltin chloride, bis(tri-n-butyltin) oxide and triphenyltin chloride] in HBL-100 cells. Furthermore, cytostatic assessments of complex 3 against HepG-2 cells reveal that the effect of cytostatic and apoptotic may through ROS-mediated pathway.

Preparation, characterization and in vitro anticancer performance of nanoconjugate based on carbon quantum dots and 5-Fluorouracil

This study is focused on the development of a nanodevice for loading and release of 5-Fluorouracil (5-FU) with a view to improving its therapeutic efficiency, using as strategy the fabrication of a nanoconjugate through drug anchorage on the surface of carbon quantum dots (CQD). Several physicochemical and analytical techniques were employed to obtain information about materials morphology, structure, and optical properties. The results indicated that the interactions between both entities resulted in good physicochemical properties and photostability. Acid pH favored drug release, indicating a tendency to release 5-FU from 5-FU-CQD into the tumor microenvironment. The cytotoxicity of CQD and 5-FU-CQD nanoconjugate was evaluated against normal human lung fibroblast (GM07492A) and human breast cancer (MCF-7) cell lines. The CQD was non-toxic, indicating that these materials are biocompatible and can be used as a nanocarrier for 5-FU in biological systems. For the 5-FU-CQD nanoconjugate, it was observed a reduction in toxicity for normal cells compared to free 5-FU, suggesting that drug anchoring in CQD reduced drug-associated toxicity, while for cancer cells exhibited an antitumor effect equivalent to that of the free drug, opening perspectives for the application of this material in anticancer therapy.

Binding Properties and Biological Activity of a Novel Therapeutic Monoclonal Antibody Directed toward HER2 Antigen

Several biosimilars for Herceptin® (trastuzumab) and other innovator monoclonal antibodies (mAbs) against HER2 molecule have been developed and approved in the last years. Here, it is reported the binding properties of a novel anti-HER2 mAb (called 5G4) as well as the capacity of this mAb to inhibit cell proliferation and to induce Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC). mAb 5G4 was compared with Herceptin® in a panel of human normal tissues, breast tumors and cell lines using immunohistochemistry, immunocytochemistry, western blot, flow cytometry and colorimetric methods. mAb 5G4 showed weak to moderate staining in breast ductal cells (1/2), gastric glandular cells (1/3) and renal tubes (2/3). Additionally, an intense reactivity was evident with mAb 5G4 in HER2-positive breast adenocarcinomas. The tissue staining was slightly more intense with Herceptin® (trastuzumab) that was used as control. Flow cytometry analysis revealed that mAb 5G4 is able to react with HER2 overexpressing cells (SK-BR-3 and SKOV3) comparable to Herceptin®. The relative binding and the anti-proliferative activity ranges of mAb 5G4 using Herceptin® as reference were between 91-115% and between 91-106%, respectively, which are considered comparable. Moreover, preliminary results suggest that the capacity of mAb 5G4 to induce ADCC in SK-BR-3 cells is non-inferior to Herceptin®. Our data permit to consider that mAb 5G4 has similar binding properties to HER2 antigen and biological activity compared with Herceptin®. However, further determinations such as affinity and FcgRIIIa binding activity of mAb 5G4 could be useful to confirm these results.

Identification of tumor-associated macrophage subsets that are associated with breast cancer prognosis.

BackgroundBreast cancer is the leading cause of cancer‐related deaths in women, demanding new treatment options. With the advent of immune checkpoint blockade, immunotherapy emerged as a treatment option. In addition to lymphocytes, tumor‐associated macrophages exert a significant, albeit controversial, impact on tumor development. Pro‐inflammatory macrophages are thought to hinder, whereas anti‐inflammatory macrophages promote tumor growth. However, molecular markers to identify prognostic macrophage populations remain elusive.MethodsWe isolated two macrophage subsets, from 48 primary human breast tumors, distinguished by the expression of CD206. Their transcriptomes were analyzed via RNA‐Seq, and potential prognostic macrophage markers were validated by PhenOptics in tissue microarrays of patients with invasive breast cancer.ResultsNormal human breast tissue contained mainly CD206+ macrophages, while increased relative amounts of CD206− macrophages were observed in tumors. The presence of CD206+ macrophages correlated with a pronounced lymphocyte infiltrate and subsets of CD206+ macrophages, expressing SERPINH1 and collagen 1, or MORC4, were unexpectedly associated with improved survival of breast cancer patients. In contrast, MHCIIhi CD206− macrophages were linked with a poor survival prognosis.ConclusionOur data highlight the heterogeneity of tumor‐infiltrating macrophages and suggest the use of multiple phenotypic markers to predict the impact of macrophage subpopulations on cancer prognosis. We identified novel macrophage markers that correlate with the survival of patients with invasive mammary carcinoma.

Proteomic Exploration of Membrane Curvature Sensors Using a Series of Spherical Supported Lipid Bilayers.

Membrane curvature-sensing (MCS) proteins recognize and regulate the morphologies of biological membranes. As these proteins lack characteristic sequence motifs in their primary structure, they are not instantly recognizable by genomic databases. Overcoming this technological challenge toward the agile identification of new proteins can promote the elucidation of membrane morphological regulation. Here, for the selective identification of MCS proteins, comparative proteomic analysis was performed using different sizes of the spherical supported lipid bilayer (SSLB), which consists of spherical SiO2 particles covered with a lipid bilayer. Because of the presence of SiO2 core, the curvature of the surrounding membrane is well-controlled and stable even on a micron scale. To prove this concept, known membrane curvature-sensing protein domains, Bin/Amphiphysin/Rvs (BAR) and Epsin N-terminal homology (ENTH), were evaluated by performing a binding assay using SSLBs, and the preferential binding to the highly curved membrane was confirmed. Peripheral membrane proteins obtained from normal human dermal fibroblast (NHDF) and human breast cancer (MDA-MB-231) cells were used in shotgun proteomic analysis, and 786 and 949 proteins were identified from SSLBs as lipid membrane binders, respectively. Statistical quantitative analyses of proteins detected from each SSLB with a different size revealed 118 candidate proteins, including 23 proteins unique to MDA-MB-231 cells, as membrane curvature sensors, including some previously reported curvature sensors. Functional clustering analysis based on the KEGG orthology database revealed that the protein-binding property to specific high or low membrane curvature correlated with their functions. Further investigation of candidate proteins will lead to the identification of new MCS proteins as well as cancer biomarkers.

Enhanced cellular uptake and anti-cancer potentials of gold nanoparticles conjugated with cell penetration peptide against lung cancer cells

Gold nanoparticles (GNPs) are often conjugated in the biomedical field with biocompatible peptides, although the effect of biocompatible GNP peptides on cellular responses is still not clearly understood. In the current study, GNPs with / without peptide were used as model probes to investigate the cytotoxicity to the human lung cancer cell line (A457) and human normal breast epithelial cell line (HBL-100). GNPs and GNPs-RGD preparation was confirmed and characterized using UV – VIS spectrophotometer, FE-SEM, FTIR, and TEM. The anticancer effect to A457 cell line was estimated using MTT assay. Our results show that the GNP-RGD had found significant tumor targeted efficacy and decrease in proliferation of A457 cell line compared with HBL-100 which appeared normal growth. Overall, our finding suggests a potential therapeutic effect of GNPs-RGD as a novel anti-cancer drug to be further developed and offer a beneficial targeting therapy.

Boron-Containing Lipids and Liposomes: New Conjugates of Cholesterol with Polyhedral Boron Hydrides.

A series of boron-containing lipids were prepared by reactions of cyclic oxonium derivatives of polyhedron boranes and metallacarboranes (closo-dodecaborate anion, cobalt and iron bis(dicarbollides)) with amine and carboxylic acids which are derived from cholesterol. Stable liposomal formulations, on the basis of synthesized boron-containing lipids, hydrogenated soybean l-α-phosphatidylcholine and (HSPC) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) as excipients, were prepared and then characterized by dynamic light scattering (DLS) that revealed the formation of particles to be smaller than 200 nm in diameter. The resulting liposomal formulations showed moderate to excellent loading and entrapment efficiency, thus justifying the design of the compounds to fit in the lipid bilayer and ensuring ease of in vivo use for future application. The liposomal formulations based on cobalt and iron bis(dicarbollide)-based lipids were found to be nontoxic against both human breast normal epithelial cells MCF-10A and human breast cancer cells MCF-7.

High-resolution label-free imaging of tissue morphology with confocal phase microscopy

Label-free imaging approaches seek to simplify and augment histopathologic assessment by replacing the current practice of staining by dyes to visualize tissue morphology with quantitative optical measurements. Quantitative phase imaging (QPI) operates with visible/UV light and thus provides a resolution matched to current practice. Here we introduce and demonstrate confocal QPI for label-free imaging of tissue sections and assess its utility for manual histopathologic inspection. Imaging cancerous and normal adjacent human breast and prostate, we show that tissue structural organization can be resolved with high spatial detail comparable to conventional hematoxylin and eosin (H&E) stains. Our confocal QPI images are found to be free of halo, solving this common problem in QPI. We further describe a virtual imaging system based on finite-difference time-domain (FDTD) calculations and combine it with numerical tissue phantoms to quantitatively show the absence of halo and the improved clarity in resolving subcellular features with confocal QPI compared to wide-field QPI. Confocal QPI bears the potential to become a common tool for label-free disease diagnosis, while the presented FDTD method provides a flexible platform to evaluate the diagnostic potential of QPI methods.

Breast-Specific Epigenetic Regulation of DeltaNp73 and Its Role in DNA-Damage-Response of BRCA1-Mutated Human Mammary Epithelial Cells.

The function of BRCA1/2 proteins is essential for maintaining genomic integrity in all cell types. However, why women who carry deleterious germline mutations in BRCA face an extremely high risk of developing breast and ovarian cancers specifically has remained an enigma. We propose that breast-specific epigenetic modifications, which regulate tissue differentiation, could team up with BRCA deficiency and affect tissue susceptibility to cancer. In earlier work, we compared genome-wide methylation profiles of various normal epithelial tissues and identified breast-specific methylated gene promoter regions. Here, we focused on deltaNp73, the truncated isoform of p73, which possesses antiapoptotic and pro-oncogenic functions. We showed that the promoter of deltaNp73 is unmethylated in normal human breast epithelium and methylated in various other normal epithelial tissues and cell types. Accordingly, deltaNp73 was markedly induced by DNA damage in human mammary epithelial cells (HMECs) but not in other epithelial cell types. Moreover, the induction of deltaNp73 protected HMECs from DNA damage-induced cell death, and this effect was more substantial in HMECs from BRCA1 mutation carriers. Notably, when BRCA1 was knocked down in MCF10A, a non-malignant breast epithelial cell line, both deltaNp73 induction and its protective effect from cell death were augmented upon DNA damage. Interestingly, deltaNp73 induction also resulted in inhibition of BRCA1 and BRCA2 expression following DNA damage. In conclusion, breast-specific induction of deltaNp73 promotes survival of BRCA1-deficient mammary epithelial cells upon DNA damage. This might result in the accumulation of genomic alterations and allow the outgrowth of breast cancers. These findings indicate deltaNp73 as a potential modifier of breast cancer susceptibility in BRCA1 mutation carriers and may stimulate novel strategies of prevention and treatment for these high-risk women.

Sphingosine Kinase 1 is Associated With Immune Cell–Related Gene Expressions in Human Breast Cancer

BackgroundAlthough previous experiments have implicated sphingosine-1-phosphate (S1P) as a links between immune reactions and cancer progression, the exact mechanism of this interaction has not comprehensively studied in clinical human samples. This study sought to evaluate the S1P regulation by sphingosine kinase 1 (SPHK1), an S1P-producing enzyme, in the immunity/immuno-reactivity of clinical human breast cancer surgical specimens. MethodsS1P levels were examined in tumor, peritumoral, and normal human breast samples using mass spectrometry. Genomics Data Commons data portal of The Cancer Genome Atlas cohort was used to assess the expression of S1P-related and immune-related genes. ResultsS1P levels were significantly higher in tumor samples compared to peritumoral (P < 0.05) or normal human breast samples (P < 0.001). SPHK1 gene expression was elevated in tumoral samples compared to normal breast samples (P < 0.01). Furthermore, the elevated expression of SPHK1 in breast cancer tissue was associated with an increased expression of the different kinds of immune-related genes, such as CD68, CD163, CD4, and FOXP3 (forkhead box P3), in HER2-negative breast cancer. Network analysis showed the central role of SPHK1 in the interaction of S1P signaling and expression of immune cell–related proteins. ConclusionsWe demonstrated that S1P is mainly produced by tumor tissue, rather than peritumoral tissue, in breast cancer patients. Our data revealed the involvement of S1P signaling in the regulation of immune-related genes, suggesting the links between S1P and complicated immune–cancer interactions in breast cancer patients.

Mechanical Responses of Breast Cancer Cells to Substrates of Varying Stiffness Revealed by Single-Cell Measurements.

How cancer cells respond to different mechanical environments remains elusive. Here, we investigated the tension in single focal adhesions of MDA-MB-231 (metastatic breast cancer cells) and MCF-10A (normal human breast cells) cells on substrates of varying stiffness using single-cell measurements. Tension measurements in single focal adhesions using an improved FRET-based tension sensor showed that the tension in focal adhesions of MDA-MB-231 cells increased on stiffer substrates while the tension in MCF-10A cells exhibited no apparent change against the substrate stiffness. Viscoelasticity measurements using magnetic tweezers showed that the power-law exponent of MDA-MB-231 cells decreased on stiffer substrates whereas MCF-10A cells had similar exponents throughout the whole stiffness, indicating that MDA-MB-231 cells change their viscoelasticity on stiffer substrates. Such changes in tension in focal adhesions and viscoelasticity against the substrate stiffness represent an adaptability of cancer cells in mechanical environments, which can facilitate the metastasis of cancer cells to different tissues.

Abstract 1654: Highly multiplexed analysis of FFPE breast tissues using the codex technology

Abstract Highly multiplexed imaging has emerged as a critical tool in understanding the complexities of the tumor microenvironment. This technology enables detection of tens of markers within the same tissue specimen, thereby allowing for key cell identification, quantification and spatial localization. These measurements can in turn provide insights into disease mechanisms and effective treatment modalities. In particular, with the high incidence of breast cancer and the need for early diagnosis and precise treatment regimens, highly multiplexed imaging could improve our understanding of this disease and provide the necessary data for improvements to patient health. Here, we demonstrate the development of a breast cancer specific CODEX panel for the measurement of more than 30 specific markers. The CODEX (Co-Detection by indEXing) platform enables detection of tens of markers within the same tissue specimen through a DNA-based tagging approach, whereby antibodies are labeled with specific oligonucleotide tags (barcodes) and dye-oligonucleotides (reporters) are iteratively hybridized and dehybridized across multiple cycles. This process is completely automated through the CODEX instrument in combination with existing fluorescent microscopes. Using FFPE Human breast cancer tissues at different stages as well as normal FFPE Human breast tissue utilizing the CODEX platform, data was generated for 30+ different biomarkers. This antibody panel was designed to detect cancer cells as well as non-malignant cells as part of tumor microenvironment to enable a better understanding of communication between the cells. The CODEX generated data was analyzed using the CODEX software suite to identify key cell types and analyze the spatial association between them. Upon analysis of data obtained, it was found that the biomarkers expression between the three samples were distinctly different. Using the CODEX technology, we were able to identify and classify key cell types and map the spatial localization of more than 20 key cell types. Citation Format: Nadezhda Nikulina, Oliver Braubach, Sayantani Basak, Maria Elena Gallina, Won-Mean Lee, Joseph Kim, Cassandra Hempel, Edek Williams, Olive Shang, Ben Cheung, Julia Kennedy-Darling. Highly multiplexed analysis of FFPE breast tissues using the codex technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1654.

Abstract 5702: A spatially resolved single cell atlas of the human breast

Abstract The human breast consists of lobules connected to an intricate network of ducts that are surrounded by fatty tissues, designed to produce and transport milk to nourish offspring. Histopathology has identified 10 major cell types based on morphological features but have provided limited information on cell states - the transcriptional programs of cell types that reflect different biological functions. In this study we have generated an unbiased ‘cell atlas' of the normal human breast to define the cell types and cell states using single cell RNA sequencing and spatial transcriptomics methods. We performed 3' microdroplet based single cell and nuclei RNA sequencing of 248,687 stromal cells and 89,301 nuclei from 24 women with pathologically normal breast tissues that were collected from mastectomies and reduction mammoplasties. Unbiased expression analysis identified four major cell types: adipocytes, epithelial cells (luminal and basal), fibroblasts and endothelial cells and defined their transcriptional programs. Additionally, 8 minor cell types were identified in the breast, including macrophages, T-cells, natural killer cells, mast cells, pericytes, apocrine cells, neurons and smooth muscle cells. Our data revealed hundreds of novel markers of these cell types and defined their transcriptional programs and cell states. Most cell types had multiple transcriptional programs including luminal epithelial cells (hormone receptor positive and secretory), basal epithelial cells (myoepithelial or basal), endothelial cells (E1, E2, E3), myeloid cells, T-cells and fibroblasts (F1-F4) and provided insight into developmental lineages. We further delineated the spatial organization of these cell types and cell states within the tissue architecture using spatial transcriptomics assay. The breast cell atlas data provides an invaluable normal reference for the research community to understand how normal cell types are reprogrammed in diseases such as breast cancer. Citation Format: Tapsi Kumar, Quy Nguyen, Kevin Nee, Shanshan Bai, Min Hu, Emi Sei, Anita Wood, Jie Wiley, Hui Chen, Alejandro Contreras, Mediget Teshome, Ken Chen, Bora Lim, Devon Lawson, Nicholas Navin, Kai Kessenbrock. A spatially resolved single cell atlas of the human breast [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5702.

Abstract 5780: Developing a screen for O6-alkylguanines in human DNA using the damage-specific binding protein, Atl1

Abstract Humans are widely exposed to a wide variety of DNA alkylating agents (AAs) either directly, for example via cigarette smoke or indirectly via their formation in the gastrointestinal tract. Certain AAs, such as the tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) are known human carcinogens but the role that other AAs may play in the aetiology of human cancers is still uncertain due to difficulties in assessing human exposure to these agents. The aim of this work was therefore to develop a screening method to assess exposure to AAs by measuring the formation of O6-alkylguanines (O6-AlkGs) in DNA and applying it to the analysis of human DNA samples. To do this we took advantage of the S. pombe alkyltransferase-like protein (Atl1), which binds to all O6-AlkGs so far studied, to develop a slot blot assay. For this, sonicated heat denatured DNA was bound to Hybond-N+ membranes and probed with horseradish peroxidase-conjugated maltose-binding protein-Atl1 fusion protein. The binding was quantitated via peroxidase-mediated enhanced chemiluminescence using TMZ-methylated calf thymus (CT) DNA standards previously calibrated using a tritium-based competition assay. Using 1 μg DNA, the limit of quantitation and detectability were approximately 1.0 and 0.5 fmoles O6-MeG/μg DNA, respectively. Prior treatment of the TMZ-CT DNA standards with the DNA repair protein, O6-methylguanine DNA methyltransferase, reduced the detectable signal to background levels, confirming that the assay detected O6-methylguanine (O6-MeG) in TMZ-CT DNA. Importantly, CT DNA reacted in vitro with the alkylating agents N-ethyl-N-nitrosourea, N-propyl-N-nitrosourea, N-butyl-N-nitrosourea, N-benzyl-N-nitrosourea, N-allyl-N-nitrosourea and potassium diazoacetate all produced strong signals in the assay demonstrating its ability to detect a range of different O6-AlkGs. In addition, a variety of human DNA samples have been analysed in pilot studies: of 14 human placental DNA samples, 8 had O6-AlkG levels in the range of 1.05-1.71 fmoles/μg DNA, 3 had trace levels and in 3 samples there was no detectable signal. Of 5 human breast tumour DNA samples, 4 had O6-AlkGs levels in the range of 1.07-1.30 fmoles/µg DNA and 1 had trace levels. Two DNA samples from normal human breast tissue had no detectable O6-AlkGs but DNA from the tumours of the same patients had levels &amp;gt;1 fmoles/μg DNA. Of 6 DNA samples from whole blood 2 had detectable levels of O6-AlkGs (1.23 &amp; 1.56 fmoles/μg DNA), 3 had trace amounts and 1 had no detectable level. These data thus show the potential usefulness of this slot blot assay as a screening tool to detect and quantify total O6-alkGs adducts in human DNA samples. Citation Format: Hanum Yaakub, Anthony Howell, Geoffrey Margison, Andrew Povey. Developing a screen for O6-alkylguanines in human DNA using the damage-specific binding protein, Atl1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5780.

Abstract 1782: N-myristoyltransferase proteins in breast cancer: Prognostic relevance and validation as a new drug target

Abstract N-myristoyltransferases (NMTs) catalyze the addition of 14-carbon fatty acids to the N-terminus of proteins. This activity regulates numerous membrane-bound signal transduction pathways important in cancer biology, and the pan-NMT inhibitor PCLX-001 is in clinical development as a cancer therapy. The physiologic distribution and relative contributions of the two human NMTs, NMT1 and NMT2, remain poorly understood as previous studies used polyclonal antibodies with potential cross-reactivity. We generated and validated mutually exclusive monoclonal antibodies (mAbs) specific to the human isotypes of NMT1 and NMT2. These mAbs were used to perform an immunohistochemical (IHC) analysis of the abundance and distribution of NMT1 and NMT2 in normal human breast epithelial samples and a large (n=703) cohort of primary breast adenocarcinomas from the BCIRG001 study. While NMT1 protein was readily identifiable in most normal and transformed breast epithelial tissue, NMT1 abundance was associated with higher overall histologic grade, higher Ki67, and lower hormone expression. While NMT2 protein was readily detected in normal breast epithelial tissue, NMT2 protein was undetectable in the majority of malignant breast cancers, but detectable NMT2 protein correlated with significantly poorer overall survival outcomes (hazard ratio for death 1.36; p &amp;lt; 0.029) and significantly worse biological features including younger age, higher histologic grade, lower hormone receptor expression, higher Ki67, and p53 positivity. NMT status was unrelated to HER2 status. NMT1 and NMT2 protein abundances were positively correlated with each other. Treatment of cultured breast cancer cells with the pan-NMT inhibitor PCLX-001 reduced cell viability in vitro. Daily oral administration of PCLX-001 to immunodeficient mice bearing human MDA-MB-231 breast cancer xenografts induced significant dose-dependent tumor growth inhibition in vivo. These results support the further evaluation of NMT immunohistochemistry for selection of patients for NMT inhibitor therapy, and clinical trials of NMT inhibition in breast cancer patients. Citation Format: John R. Mackey, Justine Lai, Utkarsh Chauhan, Wei-Feng Dong, Darryl Glubrect, Sunita Ghosh, Gilbert Bigras, Raymond Lai, Luc G. Berthiaume. N-myristoyltransferase proteins in breast cancer: Prognostic relevance and validation as a new drug target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1782.