Healthy Breast Research Articles

Ultra‐wideband microwave imaging for breast tumor screening using step recovery diode‐based pulse generator

SummaryThe feasibility of a multistatic imaging system built on step recovery diode (SRD)‐based pulse generator for microwave breast cancer detection is investigated to develop a low‐cost and portable non‐invasive screening device in the future. The system consists of a pulse generator, an antenna array, and a sampling oscilloscope, in which the pulse generator generates monocycle pulse with a frequency bandwidth from 510 MHz to 6.1 GHz. Experiments are performed on a breast phantom made up of a breast model using 3D printing technology and soybean oil as internal filler, without the usage of any coupling medium. Four identical omnidirectional antennas sequentially transmit ultra‐wideband (UWB) signal and collect signals scattered inside the breast phantom, and then, the interior image of the phantom is reconstructed by background subtraction method and delay and sum (DAS) imaging algorithm. The system can successfully detect and distinguish a variety of test scenarios including cancerous breast phantom containing target with different size and different depth made by iron ball and bacon and healthy breast phantom. The smallest detectable target is 3 mm bacon, with signal to clutter ratio (SCR) and signal to mean ratio (SMR) metrics of 1.63 and 7.79 dB, respectively, and positioning error and relative error of 2.2 mm and 1.7%, respectively. It proves that the proposed time‐domain imaging system is promising for scaling up the detection range of early breast cancer.

Emerging evidence on the role of breast microbiota on the development of breast cancer in high-risk patients.

Cancer is a multi-factorial disease, and the etiology of breast cancer (BC) is due to a combination of both genetic and environmental factors. Breast tissue shows a unique microbiota, Proteobacteria and Firmicutes are the most abundant bacteria in breast tissue, and several studies have shown that the microbiota of healthy breast differs from that of BC. Breast microbiota appears to be correlated with different characteristics of the tumor, and prognostic clinicopathologic features. It also appears that there are subtle differences between the microbial profiles of the healthy control and high-risk patients. Genetic predisposition is an extremely important risk factor for BC. BRCA1/2 germline mutations and Li-Fraumeni syndrome are DNA repair deficiency syndromes inherited as autosomal dominant characters that substantially increase the risk of BC. These syndromes exhibit incomplete penetrance of BC expression in carrier subjects. The action of breast microbiota on carcinogenesis might explain why women with a mutation develop cancer and others do not. Among the potential biological pathways through which the breast microbiota may affect tumorigenesis, the most relevant appear to be DNA damage caused by colibactin and other bacterial-derived genotoxins, β-glucuronidase-mediated estrogen deconjugation and reactivation, and HPV-mediated cancer susceptibility. In conclusion, in patients with a genetic predisposition, an unfavorable breast microbiota may be co-responsible for the onset of BC. Prospectively, the ability to modulate the microbiota may have an impact on disease onset and progression in patients at high risk for BC.

Demethylation-Driven ligase chain reaction for simultaneously sensitive detection of m6A demethylase FTO and m1A demethylase ALKBH3 in breast cancer at Single-Molecule level

RNA demethylases play a crucial role in gene expression regulation by controlling RNA methylation levels, and their abnormal expressions are associated with many human diseases. Herein, we develop a single-molecule detection method for simultaneous quantification of N6-methyladenosine (m6A) demethylase FTO and N1-methyladenosine (m1A) demethylase ALKBH3 based on RNA demethylation-directed multi-cyclic ligase chain reaction (LCR) cascades. We design a ssDNA containing m6A modification as the substrate of FTO and a ssDNA containing m1A modification as the substrate of ALKBH3. FTO and ALKBH3 catalyze the demethylation of m6A and m1A to generate normal adenine (A), respectively. Afterwards, Taq ligase initiates the LCR reaction with the demethylated ssDNAs as the templates to produce abundant biotinylated Cy3/Cy5-labeled signal probes. Subsequently, the resultant biotinylated probes are captured by streptavidin-coated magnetic beads (MBs) to form the MB-ssDNA-Cy3/Cy5 complexes. After magnetic separation, the ssDNAs in the MB-ssDNA-Cy3/Cy5 complexes are digested by DNase I, liberating abundant Cy3 and Cy5 fluorophores that can be counted by single-molecule detection. Remarkably, only a single DNA ligase is employed to achieve target signal conversion and multi-cyclic cascade amplification. This method displays extremely high sensitivity with a limit of detection of 6.33 × 10−18 mol/L for FTO and 1.44 × 10−17 mol/L for ALKBH3. Moreover, it can measure the enzyme kinetic parameters, screen inhibitors, and quantify cellular FTO and ALKBH3 activities with single-cell sensitivity. Furthermore, it can differentiate the FTO and ALKBH3 levels in the tissues of healthy human and breast cancer patients, offering a robust platform for biomedical research and clinical diagnosis.

Immunohistochemical study of the expressed cluster differentiation markers proteins type 20 and 56 in breast tissues from a group of Iraqi patients with breast cancers.

Tumor-infiltrating lymphocytes (TIL) are important immunological components in response to cancers. Patients with higher numbers of TIL in breast cancerous tissues, comprising T- cytotoxic and T - helper cells along with B- and rare natural killer (NK) cells, have more favorable clinical outcomes. To analyze the rate of the expressed surface biomarker proteins of CD20-B cells and CD56- NK cells on the infiltrative lymphocytic subpopulations in a group of breast tumorous tissues (invasive and benign) from female patients in Iraq and explore the relations to the grade of the invasive breast cancerous tissues. One hundred and 75 archived breast tissues were enrolled in this retrospective research: 100 archived breast from female patients with invasive breast cancers (BC) [20 well differentiated BC tissues; 48 moderately differentiated BC and 32 poorly differentiated BC tissues]; 50 tissue biopsies from female patients with benign breast tumors and 25 apparently normal individuals with healthy breast tissues (included as the control group for this study). Immunohistochemistry was achieved for the detection of the expressed surface biomarker proteins related to B cell CD20 and NK cell CD56 present on the infiltrative lymphocytic subpopulations in breast tissues by using specific primary antibodies for these proteins via utilizing an immune-enzymatic antigen detection system. The detection of IHC reactions for the expressed B cell CD20 - cell surface ( CD) biomarker proteins were observed in 53 out of 100 (53.0%) BC tissues, and in 24 out of 50 (48.0%) benign breast tumorous tissues, while CD20- positive cell surface markers was detected in apparently healthy breast tissues of the control group in a percentage of 32.0% (8 out of 25 tissues). Statistical significant differences (P<0.05) between both groups of malignant and benign breast tumors and the control group were found. However, between breast malignant and benign tumor groups, no significant difference was found ( p >0.05). Detection of CD56- IHC reactions revealed in 14% (14 out of 100 BC tissues), in 16% (8 out of 50 benign breast tissues) and none of control breast tissues revealed CD56- IHC reactions. Among all the enrolled groups, no significant differences (P>0.05) were detected. The observed significant rates that showed highly significant differences between both studied groups of breast malignant and benign tumor in comparison to the control group indicate that the CD20- positive infiltrative B cell- lymphocytic subpopulations might contributed in the defense against these subsets of benign and malignant breast tumors. However, the observed rates of NK cell CD56 present on the lymphocytic subpopulations infiltrating the examined malignant and benign breast tumorous tissues seeming to play irrelevant roles in the defense against these studied breast tumor groups.

Ultra Sensitive Breast Cancer Cell Lines Detection Using Dual Nanocavities Engraved Junctionless FET.

This article reports breast cancer cell lines (Hs578T, MDA-MB-231, MCF-7, and T47D) and healthy breast cells (MCF-10A) detection based on the modulation of its electrical properties by deploying dual nanocavity engraved junctionless FET. The device has a dual gate to enhance gate control and has two nanocavities etched under both gates for breast cancer cell lines immobilization. As the cancer cells are immobilized in the engraved nanocavities, which were earlier filled with air, the dielectric constant of the nanocavities changes. This results in the modulation of the device's electrical parameters. This electrical parameters modulation is then calibrated to detect the breast cancer cell lines. The reported device demonstrates a higher sensitivity toward the detection of breast cancer cells. The JLFET device optimization is done for improving the performance by optimizing the nanocavity thickness and the SiO2 oxide length. The variation in the dielectric property of cell lines plays a key role in the detection technique of the reported biosensor. The sensitivity of the JLFET biosensor is analyzed in terms of ∆VTH, ∆ION, ∆gm , and ∆SS . The reported biosensor shows the maximum sensitivity for T47D ( κ = 32 ) breast cancer cell line with ∆VTH = 0.800 V, ∆ION = 0.165 mA/μm, ∆gm = 0.296 mA/V-μm , and ∆SS = 5.41 mV/decade. Moreover, the effect of variation in the occupancy of the cavity by the immobilized cell lines has also been studied and analyzed. With increased cavity occupancy the variation in the device performance parameter enhances Further, the sensitivity of the proposed biosensor is compared with the existing biosensors and it is reported to be highly sensitive as compared to the existing biosensors. Hence, the device can be utilized for array based screening of cell lines of breast cancer and diagnosis with the benefit of easier fabrication and cost effectiveness of the device.

Engineering E2 Bionanoparticles for Targeted Delivery of Chemotherapeutics to Breast Cancer Cells.

Naturally occurring protein nanocages are promising drug carriers as both the interior and exterior can be decorated for drug encapsulation and cell targeting. To provide surface functionalization, we added a SpyTag to E2 nanocages (ST-E2) to enable tunable decoration using the robust SpyCatcher bioconjugation strategy. Additionally, the E2 core was mutated with four phenylalanine substitutions for doxorubicin loading and pH-responsive release. By decorating the exterior with a highly cell-specific epidermal growth factor receptor (EGFR)-targeting protein conjugate, 4GE11-mCherry-SpyCatcher, we demonstrated targeted cell death in inflammatory breast cancer cells compared to healthy breast epithelial cells at concentrations below the IC50 of free doxorubicin.

Early Clinical Experience of Utilizing Online Adaptive Radiotherapy for Accelerated Partial Breast Irradiation with Stereotactic Body Radiation Therapy for Early-Stage Breast Cancer Patients

Accelerated partial breast irradiation (APBI) with stereotactic body radiation therapy (SBRT) is becoming popular for early-stage breast cancer (ESBC) due to decreased treatment time and dose to healthy breast tissue. With variability in lumpectomy cavity size post-surgery, CBCT-guided online adaptive RT (oART) may improve target coverage and reduce normal tissue exposure by accounting for inter- and intrafractional cavity variations. In this study, we report our initial clinical experience utilizing APBI oART for patients with ESBC. A total of 15 patients with 17 ESBC cavities (2 with bilateral disease) were treated with SBRT using oART over one year. Patients were immobilized using standard breast simulation setup with free breathing and breath hold (if left sided). The clinical target volume consisted of an isometric 1 cm expansion off the lumpectomy cavity, which was expanded another 3 mm (cropped 3mm from surface) to create the planning target volume (PTV). 95% of the PTV received prescription dose (30 Gy in 5 fractions). The oART delivery process (supervised by physician and physicist) initially coregisters the daily CBCT to the treatment planning CT with alignment to lumpectomy cavity. Target volumes and organs-at-risk were modified at each treatment and the plan re-optimized. Either the re-optimized oART plan or scheduled plan was chosen for each fraction pending which plan best met prespecified clinical goals. Acute toxicity was assessed at one-month follow-up using CTCAE. Patients were age 56 or older, ECOG 0-1, with no known genetic abnormality, and with anatomic stage 0-1A ESBC. Most tumors were located in the upper outer quadrant (n = 12, 70.6%) at middle or posterior depth (n = 15, 88.2%). Final margins were negative. 88% had tantalum clips placed. One patient had an Oncotype DS score of 34 and received systemic therapy. 82.3% met suitable APBI criteria and 3 met cautionary criteria due to high grade, of which 2 had ipsilateral trimodality treatment 10+ years prior and denied mastectomy at recurrence. Mean time to starting RT after lumpectomy was 2.5 months. Mean PTV to breast ratio was 0.12. From simulation to end of treatment, lumpectomy cavity size decreased a median of 27%. oART plans were chosen 89% of the time. VMAT was utilized 52.9% of the time compared to static-field IMRT (47.1%). Three patients were treated with breath hold. 93% of patients were treated on non-consecutive days with average treatment lasting 9.1 ± 2.2 days. CTCAE grade 1 toxicities were hyperpigmentation (n = 6, 40%), breast pain (n = 2, 13.3%), persistent seroma (n = 1, 6.7%), and fatigue (n = 7, 46.7%). No patients experienced fat necrosis, telangiectasias, breast shrinkage, lymphedema, or CTCAE grade 2+ toxicities. oART for APBI using SBRT for patients with ESBC is clinically feasible and allows for variations in lumpectomy cavity size. Nearly half of patients had no complaints or breast changes at one-month follow-up and the remaining had CTCAE grade 1 toxicities alone.

Breast Cancer Diagnosis Using Extended-Wavelength-Diffuse Reflectance Spectroscopy (EW-DRS)-Proof of Concept in Ex Vivo Breast Specimens Using Machine Learning.

This study aims to investigate the feasibility of using diffuse reflectance spectroscopy (DRS) to distinguish malignant breast tissue from adjacent healthy tissue, and to evaluate if an extended-wavelength range (450-1550 nm) has an advantage over the standard wavelength range (450-900 nm). Multivariate statistics and machine learning algorithms, either linear discriminant analysis (LDA) or support vector machine (SVM) are used to distinguish the two tissue types in breast specimens (total or partial mastectomy) from 23 female patients with primary breast cancer. EW-DRS has a sensitivity of 94% and specificity of 91% as compared to a sensitivity of 40% and specificity of 71% using the standard wavelength range. The results suggest that DRS can discriminate between malignant and healthy breast tissue, with improved outcomes using an extended wavelength. It is also possible to construct a simple analytical model to improve the diagnostic performance of the DRS technique.

B-199 Performance of the MasSpec Pen for Intraoperative Margin Evaluation During Breast Conserving Surgeries

Abstract Background Breast cancer is the second leading cause of cancer deaths among females worldwide. Surgical resection is the most common treatment option for women diagnosed with breast cancers. Many women with in situ disease or early invasive breast cancer are candidates for breast conserving surgery (BCS), which involves removing the lesion of interest and preserving the rest of the breast. One of the greatest challenges a breast cancer surgeon faces is to achieve negative margins while optimizing aesthetic outcomes. Negative margins are highly desirable as they offer the greatest potential for prolonged disease-free survival. Accurate intraoperative surgical margin evaluation is therefore critical during BSC. Molecular technologies offer the opportunity to incorporate cancer-specific biomarkers into clinical decision-making for improved cancer detection. Our team has reported the development of an innovative handheld mass spectrometry (MS) probe, the MasSpec Pen (MSPen), for rapid and gentle molecular analysis of human tissues, including breast cancer tissues. The MSPen uses water to gently extract molecules from tissues upon contact, which are then analyzed by MS and statistical classifiers to provide a predictive classification within seconds. Here, we evaluate the performance of the MSPen for intraoperative detection of breast cancer. Methods The MSPen coupled to an Orbitrap Exploris 120 (Thermo Scientific) is being used to analyze both normal breast and invasive lobular carcinoma (ILC) banked tissues. Tissue samples are first thawed prior to analysis. After analysis, the region analyzed is demarcated, and the samples snap-frozen, sectioned, H&amp;E stained, following evaluation by a pathologist. We will then apply the least absolute shrinkage and selector operator (lasso) method to build classification models for distinguishing normal breast and ILC using the histologically validated mass spectra. The MSPen system is also implemented in the operating room for testing within surgical workflow. Analyses will be carried out during BCS surgeries for patients with ILC. The ILC classifier generated from banked tissues will be used to predict on data obtained from the intraoperative analyses and validated against final histopathological reports. Results To date, we have analyzed 213 banked human breast tissues using the MSPen, including healthy breast (n = 79), invasive ductal carcinoma (IDC) (n = 64), healthy lymph nodes (n = 26), and lymph nodes with metastatic IDC (n = 44). The rich mass spectral data obtained from breast tissues showed high relative abundance of various metabolites and lipid species that have been previously described as potentially diagnostic of breast cancer. Using this data, we built classifiers that discriminate cancer and normal breast tissues with high accuracy (overall 94%) for primary breast IDC. In a pilot clinical study, we demonstrated feasibility for ex vivo and in vivo tissue analysis using the MSPen platform during 29 breast surgeries. We are currently expanding our study to detect ILC using banked tissues and in patient tissues intraoperatively. Conclusion Our study in breast cancer provides evidence that the MSPen enables molecular-based diagnosis of IDC tissues using excised specimens and in in vivo and ex vivo analysis performed by surgeons intraoperatively. Current effort including ILC is expected to show similar capabilities for patients with ILC.

Rapid, Sensitive, and Label-Free Detection of Long Noncoding RNAs in Breast Cancer Tissues by RecJf Exonuclease-Assisted Recombinase Polymerase Amplification.

An abnormal expression level of long noncoding RNAs (lncRNAs) is implicated in multiple cancers, and their sensitive and rapid measurement is pivotal for early cancer diagnosis and cancer treatment. The conventional lncRNA assays often suffer from labor-intensive/time-consuming procedures and limited sensitivity. Herein, we report a simple and sensitive fluorescent biosensor for rapid and label-free measurement of lncRNAs based on recombinase polymerase amplification (RPA) without the involvement of thermal cycling and reverse transcription. Target lncRNAs can bind with the 5'-end of the DNA template to create a DNA-lncRNA hybrid, protecting the DNA template from RecJf exonuclease-mediated degradation. Subsequently, the primers hybridize with the intact DNA templates and are extended to generate the dsDNA products with the assistance of polymerase. The resultant dsDNA products may be amplified by exponential recombinase polymerase amplification to produce abundant dsDNAs, generating a distinct fluorescence signal within 10 min. This biosensor achieves a wide dynamic range from 10-17 to 10-9 M and high sensitivity with a detection limit of 1.23 aM. Moreover, it can distinguish the expressions of lncRNA HOTAIR in the tissues of healthy individuals and breast cancer patients, with broad application prospects in lncRNA-related research and early diagnosis of cancers.

Expression pattern, prognostic value and potential microRNA silencing of FZD8 in breast cancer.

Breast cancer (BC) is one of the most widespread types of cancer affecting females, and therefore, early diagnosis is critical. BC is a complex heterogeneous disease affected by several key pathways. Among these, WNT proteins and their frizzled receptors (FZD) have been demonstrated to be crucial in regulating a number of cellular and molecular events in BC tumorigenesis. The role of the WNT receptor, FZD8, in BC has received minimal attention; for that reason, the present study examined the prognostic value of its protein expression pattern in a BC cohort. FZD8 cytoplasmic expression pattern analysis revealed that ~38% of the primary samples presented with a high expression profile, whereas ~63% of the samples had a low expression profile. Overall, ~46% of the malignant tissues in the lymph node-positive samples exhibited an increased FZD8 cytoplasmic expression, whereas 54% exhibited low expression levels. An increased expression of FZD8 was associated with several clinicopathological characteristics of the patients, including a low survival rate, tumor vascular invasion, tumor size and grade, and molecular subtypes. Affymetrix microarray triple-negative BC datasets were analyzed and compared with healthy breast tissues in order to predict the potential interfering microRNAs (miRNAs) in the WNT/FZD8 signaling pathway. A total of 29 miRNAs with the potential to interact with the WNT/FZD8 signaling pathway were identified, eight of which exhibited a significant prediction score. The target genes for each predicted miRNA were identified. On the whole, the findings of the present study suggest that FZD8 is a potential prognostic marker for BC, shedding some light onto the silencing mechanisms involved in the complex BC signaling.

Fin field-effect-transistor engineered sensor for detection of MDA-MB-231 breast cancer cells: A switching-ratio-based sensitivity analysis.

The present study describes the utilization of a gallium-arsenide gate-stack gate-all-around (GaAs-GS-GAA) fin field-effect transistor (FinFET) to accomplish the electrical identification of the breast cancer cell MDA-MB-231 by monitoring the device switching ratio. The proposed sensor uses four nanocavities carved beneath the gate electrodes for enhanced detection sensitivity. MDA-MB-231 (cancerous) and MCF-10A (healthy) breast cells have a distinct dielectric constant, and it changes when exposed to microwave frequencies spanning across 200 MHz and 13.6 GHz, which modifies the electrical characteristics, allowing for early diagnosis. First, a percentage shift in the primary DC characteristics is presented to demonstrate the advantage of GS-GAA FinFET over conventional FinFET. The sensor measures the switching-ratio-based sensitivity, which comes out to be 99.72% for MDA-MB-231 and 47.78% for MCF-10A. The sensor was tested for stability and reproducibility and found to be repeatable and sufficiently stable with settling times of 55.51, 60.80, and 71.58 ps for MDA-MB-231 cells, MCF-10A cells, and air, respectively. It can distinguish between viable and nonviable cells based on electrical response alterations. The possibility of early detection of cancerous breast cells using Bruggeman's model is also discussed. Further, the impact of biomolecule occupancy and frequency variations on the device sensitivity is carried out. This study also explains how to maximize the sensing performance by adjusting the fin height, fin width, work function, channel doping, temperature, and drain voltage. Lastly, this article compared the proposed breast cancer cell detectors to existing literature to evaluate their performance and found considerable improvement. The findings of this research have the potential to establish GaAs-GS-GAA FinFET as a promising contender for MDA-MB-231 breast cancer cell detection.

Buffalo yogurt fermented with commercial starter and Lactobacillus plantarum originating from breast milk lowered blood pressure in pregnant hypertensive rats

Nutritional therapy, which may have advantages over medication, is being investigated as a novel treatment for pregnancy-induced hypertension. Several studies have shown that probiotic yogurt supplementation during pregnancy has beneficial effects on maternal and fetal health. In this study, fermented buffalo milk was produced with yogurt culture and Lactobacillus plantarum B, a probiotic isolated from healthy breast milk with high angiotensin-converting enzyme inhibitory activity. The fermentation conditions under which the angiotensin-converting enzyme (ACE) inhibitory activity reached 84.51% were optimized by the response surface method as follows: 2 × 106 cfu/mL of L. plantarum B, yogurt culture 2.5 × 105 cfu/mL, and 8 h at 37°C. The distribution of ACE inhibitory peptides from fermented buffalo milk and fermented cow milk were further analyzed by liquid chromatography-mass spectrometry. By searching according to the structural features of ACE inhibitory peptides, 29 and 11 peptides containing ACE inhibitory peptide features were found in fermented buffalo milk and fermented cow milk, respectively. To investigate the in vivo antihypertensive activity of fermented buffalo milk, 18 pregnant rats were divided into 3 groups (n = 6 in each group) and administered 10 mL of normal saline, yogurt (20 mg/kg), or labetalol hydrochloride (4 mg/kg) daily from the beginning of pregnancy to parturition. To induce hypertension, methyl nitrosoarginine (125 mg/kg) was injected subcutaneously every day from d 15 of pregnancy to the day of delivery. Blood pressure was not significantly changed in the yogurt and labetalol groups after induction of hypertension and was lower compared with the normal saline group, but there was no difference between the yogurt and labetalol groups. This implied that the buffalo yogurt had a preventive and antihypertensive effect in the pregnancy-induced hypertensive rat model. Further studies to determine the mechanism of action, as well as a randomized control trial, are warranted.

Modeling of time-dependent elliptic contact parameters for biological nanoparticles with common geometries: Human red blood cell

Extracting mechanical properties of living cells by Atomic Force Microscope (AFM) can cause cell destruction due to uncontrolled forces. In this regard, the reaction of healthy (MCF-7) and cancerous (MCF-10A) breast cells and human Red Blood Cells (RBCs) to contact force was simulated by using different contact theories and applying time-dependent mechanical models. To validate the simulation results, the topography test and nanomanipulation of MCF-7 and MCF-10A using AFM was implemented. Experimental data showed that the average maximum diameter of MCF-7 and MCF-10A is 25.24 and 12.06 μm, respectively. Furthermore, the elastic modulus of the MCF-7 and MCF-10A cells was calculated in the range of 1.23 ± 0.02 kPa, and 1.17 ± 0.02 kPa, respectively. In addition, the contact theories were developed for application in the case of different geometries, and utilized for modeling the contact mechanics of human RBCs. The results showed that Hertz and DMT theories accurately calculate the contact behavior of living cells in small deformations; However, in case of large contact deformations, Tatara and Chang's theories provide more accurate results. Furthermore, the effect of RBC geometry on the contact parameters is negligible due to the significant difference in the dimensions of RBCs in comparison with AFM-tip.

Development of a N6-methyladenosine-directed single quantum dot-based biosensor for sensitive detection of METTL3/14 complex activity in breast cancer tissues

The METTL3/14 complex is an important RNA N6-Methyladenosine (m6A) methyltransferase in organisms, and the abnormal METTL3/14 complex activity is associated with the pathogenesis and various cancers. Sensitive detection of METTL3/14 complex is essential to tumor pathogenesis study, cancer diagnosis, and anti-cancer drug discovery. However, traditional methods for METTL3/14 complex assay suffer from poor specificity, costly antibodies, unstable RNA substrates, and low sensitivity. Herein, we construct a single quantum dot (QD)-based förster resonance energy transfer (FRET) biosensor for sensitive detection of METTL3/14 complex activity. In the presence of METTL3/14 complex, it catalyzes the methylation of adenine in the substrate probe, leading to the formation of m6A that protects the substrate probes from MazF-mediated cleavage. The hybridization of methylated DNA substrate with biotinylated capture probe initiates polymerization reaction to obtain a biotinylated double-stranded DNA (dsDNA) with the incorporation of numerous Cy5 fluorophores. Subsequently, the Cy5-incorporated dsDNA can self-assembly onto the 605QD surface to form the 605QD-dsDNA-Cy5 nanostructure, causing FRET between 605QD donor and Cy5 acceptor. This biosensor has excellent sensitivity with a limit of detection (LOD) of 3.11 × 10−17 M, and it can measure the METTL3/14 complex activity in a single cell. Moreover, this biosensor can be used to evaluate the METTL3/14 complex kinetic parameters and screen potential inhibitors. Furthermore, it can differentiate the METTL3/14 complex expression in healthy human tissues and breast cancer patient tissues, providing a powerful tool for cancer pathogenesis study, clinical diagnosis, prognosis monitoring, and drug discovery.

The mutational landscape of the adult healthy parous and nulliparous human breast

The accumulation of somatic mutations in healthy human tissues has been extensively characterized, but the mutational landscape of the healthy breast is still poorly understood. Our analysis of whole-genome sequencing shows that in line with other healthy organs, the healthy breast during the reproduction years accumulates mutations with age, with the rate of accumulation in the epithelium of 15.24 ± 5 mutations/year. Both epithelial and stromal compartments contain mutations in breast-specific driver genes, indicative of subsequent positive selection. Parity- and age-associated differences are evident in the mammary epithelium, partly explaining the observed difference in breast cancer risk amongst women of different childbearing age. Parity is associated with an age-dependent increase in the clone size of mutated epithelial cells, suggesting that older first-time mothers have a higher probability of accumulating oncogenic events in the epithelium compared to younger mothers or nulliparous women. In conclusion, we describe the reference genome of the healthy female human breast during reproductive years and provide evidence of how parity affects the genomic landscape of the mammary gland.

ECL resonance energy transfer-regulated “off-on” mode biosensor for the detection of miRNA-150-5p in triple negative breast cancer

MiRNAs played critical roles in triple negative breast cancer (TNBC) as potential biomarkers. Herein, an efficient signal “off-on” mode-biosensor based on electrochemiluminescence resonance energy transfer (ECL-RET) was successfully constructed for the miRNA-150-5p determination in TNBC. The ECL-RET regulated-sensing platform consisted of NiMn-LDHs nanoflowers, the artificially assembled phospholipid bilayers and hairpin DNA-labeled Eu-doped MoS2 QDs. Firstly, Eu-doped MoS2 QDs with high quantum efficiency were prepared as the ECL-RET donors. And NiMn-layer double hydroxides (LDHs) nanoflowers with wide UV–vis absorption spectra as the ECL-RET acceptors. Secondly, due to the hairpin DNA structure, the closed distance between ECL-RET donor-acceptor pair can quench the luminescence signal of Eu-doped MoS2 QDs. When miRNA-150-5p was captured, the hairpin DNA structure changed to a rodlike configuration and enlarged the distance between Eu-doped MoS2 QDs and NiMn-LDHs. As a result, the recovery of ECL signal can be observed as a signal “turn off-on” mode. Furthermore, the hydrophilicity of the lipid bilayer can reduce the nonspecific adsorption and improve the flexibility of the hairpin DNA efficiently. Therefore, based on the ECL-RET regulation strategy, the biosensor was employed to detect miRNA-150-5p from 10 fM to 1 nM with a detection limit of 1.5 fM. The constructed biosensor can effectively differentiate TNBC patient tumor and healthy breast fibroadenoma. The ECL-RET regulation strategy provided a new biosensing pathway for ultrasensitive detection of biomolecules and promoted the development of diagnosis and treatment of TNBC.

Immunohistochemical Detection of the Expressed BRCA1 and BRCA2 Proteins in Microenvironment of Malignant Breast Cancerous Tissues Infected with Human Mammary Tumor Virus.

The objective of the study was to examine the prevalence of HMTV infection, its associations with breast malignant tissues, and the expression of BRCA1 and BRCA2 proteins. One hundred archival breast tissues, 40 biopsies from female patients with breast cancer (BC), and 20 healthy breast tissues from the control group were used in the study. Immunohistochemical analysis was conducted to detect the expressed BRCA1 and BRCA2 proteins. Digoxigenin-labeled HMTV probes were used in chromogenic in situ hybridization for the identification of HMTV in breast tumor tissues. The complementary sequence sites of the HMTV probe sequences were stained by NBT/BCIP as blue signals. There were 12 out of 40 (30%) benign breast tumorous tissues and 14 out of 40 (35%) BC tissues, while healthy control breast tissues were 10% (2 out of 20 tissues). Positive immunohistochemical (IHC) reactions for BRCA2 protein were observed in 12 out of 40 BC tissues (30.0%), 25% of benign breast tumorous tissues, and 5% of the control group. A significant (p < 0.05) statistical difference in the percentages of HMTV in the studied groups was found. HMTV might contribute to the development of subsets of benign and malignant breast tumors. The observed rates of defective or mutated BRCA1 and BRCA2 genes in healthy tissues indicate a role in the development of breast tumors.

44425 Characterizing the nipple and areola complex of the healthy and diseased breast using dermoscopy

44425 Characterizing the nipple and areola complex of the healthy and diseased breast using dermoscopy

Tumor-derived exosomes RNA expression profiling identifies the prognosis, immune characteristics, and treatment in HR+/HER2-breast cancer.

Exosomes play crucial roles in intercellular communication and are involved in the onset and progression of various types of cancers, including breast cancer. However, the RNA composition of breast cancer-derived exosomes has not been comprehensively explored. We conducted microarray assays on exosomes isolated from breast cancer and healthy breast epithelial cells from three patients with hormone receptor (HR) +/ human epidermal growth factor receptor (HER2) - breast cancer and identified 817 differentially expressed genes (DEGs). Among these, 315 upregulated tumor-derived exosome genes (UTEGs) were used to classify HR+/HER2- breast cancers into two categories, revealing a difference in survival rates between the groups. We developed and validated a novel prognostic exosome score (ES) model consisting of four UTEGs that provides a refined prognosis prediction in HR+/HER2-breast cancer. ES reflects various immune-related features, including somatic variation, immunogenicity, and tumor immune infiltrate composition. Our findings indicate a considerable positive correlation between the ES and drug sensitivity values for vincristine, paclitaxel, and docetaxel. However, ES was remarkably higher in the endocrine therapy non-responder group than in the responder group. Immunohistochemistry confirmed the remarkable expression of the four model genes in tumor tissues, and their expression in MCF-7 cell exosomes was higher than that in MCF10A cells, as verified via qPCR. In summary, tumor-derived exosome genes provide novel insights into the subtyping, prognosis, and treatment of HR+/HER2-breast cancer.