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Abstract Background NAD(P)-dependent steroid dehydrogenase-like protein (NSDHL), which is involved in breast tumor growth and metastasis, has been implicated in the maintenance of cancer stem cells. However, its role in regulating breast cancer stem-like cells (BCSCs) remains unclear. We have previously reported the clinical significance of NSDHL in patients with estrogen receptor-positive (ER +) breast cancer. This study aimed to elucidate the molecular mechanisms by which NSDHL regulates the capacity of BCSCs in the ER + human breast cancer cell line, MCF-7. Methods NSDHL knockdown suppressed tumor spheroid formation in MCF-7 human breast cancer cells grown on ultralow-attachment plates. RNA sequencing revealed that NSDHL knockdown induced widespread transcriptional changes in the MCF-7 spheroids. TGF-β signaling pathway was the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (fold change ≥ 2, P ≤ 0.05) identified in NSDHL-knockdown MCF-7 spheroids compared with the control. In orthotopic tumor models injected with NSDHL-knockdown MCF-7 spheroids, tumor initiation and growth were strongly suppressed compared with those in the control. Results BCSC populations with CD44+/CD24- and CD49f+/EpCAM + phenotypes and high ALDH activity were decreased in NSDHL-knockdown MCF-7 spheroids and xenograft tumors relative to controls, along with decreased secretion of TGF-β1 and 3, phosphorylation of Smad2/3, and expression of SOX2. In RNA-sequencing data from The (TCGA) database, a positive correlation between the expression of NSDHL and SOX2 was found in luminal-type breast cancer specimens (n = 998). Our findings revealed that NSDHL plays an important role in maintaining the BCSC population and tumor-initiating capacity of ER-positive MCF-7 spheroids, suggesting that NSDHL is an attractive therapeutic target for eliminating BCSCs, thus preventing breast cancer initiation and progression. Conclusions Our findings suggest that NSDHL regulates the BCSC/tumor-initiating cell population in MCF-7 spheroids and xenograft tumors.

Abstract Background Heterogeneous tumor cells are thought to be a significant factor in the failure of endocrine therapy in estrogen receptor-positive (ER+) cancers. Culturing patient-derived breast cancer cells (PDBCCs) provides an invaluable tool in pre-clinical and translational research for the heterogeneity of cancer cells. This study aimed to investigate the effects of different media components and culture methods on the BCSC-associated immunophenotypes and gene expression in ER + PDBCCs. Methods Ten patients with ER + breast cancer were employed in this study, six of whom had neoadjuvant chemotherapy and four of whom did not. PDBCCs were isolated by enzymatic methods using collagen I and hyaluronidase. PDBCCs were grown as monolayers in mediums with different compositions and as multicellular spheroid in a suspended condition. Collagen I-coated plate and ultralow attachment plate coated with polymer-X were used for monolayer and spheroid culture. Flow cytometry, immunofluorescent staining, RT-PCR, and RNA-sequencing were employed to examine the immunophenotype and genetic profile of PDBCCs. Results More than 95% of PDBCCs sustain EpCAM high/+/fibroblast marker- phenotypes in monolayer conditions by subculturing 3–4 times. A83-01 removal induced senescent cells with high β-galactosidase activity. PDBCCs grown as monolayers were characterized by the majority of cells having an EpCAM+/CD49f + phenotype. Compared to full media in monolayer culture, EGF removal increased EpCAM+/CD49f − phenotype (13.8-fold, p = 0.028), whereas R-spondin removal reduced it (0.8-fold, p = 0.02). A83-01 removal increased EpCAM+/CD24 + phenotype (1.82-fold, p = 0.023) and decreased EpCAM low/-/CD44+/CD24- phenotype (0.45-fold, p = 0.026). Compared to monolayer, spheroid resulted in a significant increase in the population with EpCAM-/CD49+ (14.6-fold, p = 0.006) and EpCAM low/-/CD44+/CD24- phenotypes (4.16-fold, p = 0.022) and ALDH high activity (9.66-fold, p = 0.037). ALDH1A and EMT-related genes were upregulated. In RNA-sequencing analysis between spheroids and monolayers, a total of 561 differentially expressed genes (2-fold change, p

Abstract Background The cholesterol biosynthesis pathway is typically upregulated in breast cancer. The role of NAD(P)-dependent steroid dehydrogenase-like (NSDHL) gene, which is involved in cholesterol biosynthesis, in breast cancer remains unknown. This study aimed to uncover the role of NSDHL in the growth and metastasis of breast cancer. Methods After NSDHL knockdown by transfection of short interfering RNA into human breast cancer cell lines (MCF-7, MDA-MB-231 and BT-20) and human breast epithelial cell line (MCF10A), cell proliferation assay, cell cycle analysis, three-dimensional cell culture, clonogenic assay, transwell migration and invasion assays, and wound healing assay were performed. Erlotinib was used as the target drug for epidermal growth factor receptor. Immunodeficient mice (NOD.Cg-Prkdcscid Il2rgtm1wjl /SzJ) were used as orthotropic breast tumor models by injecting them with NSDHL-knockdown MDA-MB-231 cells using lentivirus-carrying NSDHL short hairpin RNA. Clinical data from 3951 breast cancer patients in Gene Expression Omnibus databases were used to investigate the potential prognostic role of NSDHL by survival analysis. Results NSDHL knockdown in BT-20, and MDA-MB-231 resulted in a significant decrease in their viability, colony formation, migration, and invasion abilities (p

We here evaluated the therapeutic effect of tumor cell-derived exosomes (TEXs)-stimulated dendritic cells (DCs) in a syngeneic orthotopic breast tumor model. The DC line DC2.4 and breast cancer cell line E0771 originally isolated from C57BL/6 mice were used. E0771 cells stably expressing the exosomal CD63-RFP or luciferase (Luc) and DC2.4 cells stably expressing GFP were produced using lentivirus. TEXs were purified from conditioned medium of E0771/CD63-RFP cells. Breast tumor model was established by injecting E0771/Luc cells into mammary gland fat pad of mice. TEXs contained immune modulatory molecules such as HSP70, HSP90, MHC I, MHC II, TGF-β, and PD-L1. TEXs were easily taken by DC2.4 cells, resulting in a significant increase in the in vitro proliferation and migration abilities of DC2.4 cells, accompanied by the upregulation of CD40. TEX-DC-treated group exhibited a decreased tumor growth compared with control group. CD8+ cells were more abundant in the tumors and lymph nodes of TEX-DC-treated group than in those of control group, whereas many CD4+ or FOXP3+ cells were localized in those of control group. Our results suggest a potential application of TEX-DC-based cancer immunotherapy.

A total of 20 Lactobacillus strains isolated from fermented dates were tested for their probiotic potential by comparing their pH stability, resistance to low pH, and ability to tolerate bile salts. Out of 20 strains, 3 strains named as Lactobacillus pentosus KAU001, Lactiplantibacillus pentosus KAU002, and Lactiplantibacillus plantarum KAU003 had a high tolerance of acids and bile salts and the capability to adhere to the intestinal wall. In addition, the three isolates were tested for their anti-oxidation, anti-glucosidase inhibition, cholesterol-lowering, and anti-inflammation properties. Among them, strain KAU001 and KAU002 inhibited α-glucosidase, lowered cholesterol level, inhibited nitric oxide production, and showed a higher anti-oxidative ability that was significantly better than strain KAU003. Both strains also significantly inhibited the release of inflammatory mediators such as TNF-α, IL-6 and IL-10 induced by LPS on RAW 264.7 macrophages (p < 0.001). The results indicated that KAU001 and KAU002 have the highest probiotic potential, potentially modulating metabolic health and reducing pro-inflammatory cytokines in response to allergic reactions.

Abstract The need for basement membrane extract (BME) with undefined constituents, such as Matrigel, for intestinal stem cell (ISC) culture in traditional systems poses a significant barrier that must be overcome for the development of clinical-grade, scalable, ready-to-use ISCs. Here, we propose a functional polymer-based xenogeneic-free dish for the culture of intestinal stem cells (XF-DISC), ensuring substantially prolonged maintenance of ISCs derived from 3-dimensional human intestinal organoids (ISCs3D-hIO). XF-DISC enables remarkable expandability, exhibiting a 24-fold increase in cell numbers within 30 days, with long-term maintenance of ISCs3D-hIO for more than 30 consecutive passages (>210 days). In addition, XF-DISC is fully compatible with a cell banking system. Notably, human pluripotent stem cell-derived ISCs3D-hIO cultured on XF-DISC are successfully transplanted into intestinal injury and inflammation mouse models, leading to engraftment and regeneration of damaged mouse intestinal epithelium. As a reliable and scalable xenogeneic-free ISC3D-hIO culture method, XF-DISC is highly promising for the development of regenerative ISC therapy for human intestinal diseases.

Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.

Active surveillance (AS) has been widely adopted as an alternative to immediate surgery owing to the indolent nature and favorable outcomes of papillary thyroid microcarcinoma (PTMC). AS is generally recommended for tumors measuring ≤1 cm without aggressive cytological subtypes, risk of gross extrathyroidal extension (ETE), lymph node metastasis (LNM), or distant metastasis. AS requires careful patient selection based on various patient and tumor characteristics, and ultrasound (US) findings. Moreover, during AS, regular US is performed to monitor any signs of tumor progression, including tumor growth, new US features of potential gross ETE, and LNM. Therefore, appropriate imaging-based assessment plays a crucial role in determining whether AS or surgery should be pursued. However, detailed recommendations concerning US evaluation are currently insufficient, necessitating the formulation of this guideline. The Korean Society of Thyroid Radiology has developed a consensus statement for low-risk PTMC, covering US assessment methods when considering AS as a management option and conducting follow-up imaging tests during AS. This guideline aims to provide optimal scientific evidence and expert opinion consensus regarding a standardized US-based assessment protocol for low-risk PTMC.

Accurate and reliable bearing-fault diagnosis is important for ensuring the efficiency and safety of industrial machinery. This paper presents a novel method for bearing-fault diagnosis using Mel-transformed scalograms obtained from vibrational signals (VS). The signals are windowed and pass through a Mel filter bank, converting them into a Mel spectrum. These scalograms are subsequently fed into an autoencoder comprising convolutional and pooling layers to extract robust features. The classification is performed using an artificial neural network (ANN) optimized with the FOX optimizer, which replaces traditional backpropagation. The FOX optimizer enhances synaptic weight adjustments, leading to superior classification accuracy, minimal loss, improved generalization, and increased interpretability. The proposed model was validated on a laboratory dataset obtained from a bearing testbed with multiple fault conditions. Experimental results demonstrate that the model achieves perfect precision, recall, F1-scores, and an AUC of 1.00 across all fault categories, significantly outperforming comparison models. The t-SNE plots illustrate clear separability between different fault classes, confirming the model’s robustness and reliability. This approach offers an efficient and highly accurate solution for real-time predictive maintenance in industrial applications.