Betül Çelebi-Saltik

Investigating the interaction of cancer stem cells (CSCs) with the tumor microenvironment provides critical insights into their roles in promoting angiogenesis and metastasis through various cellular and molecular adaptation mechanisms, including survival, maintenance of stemness, dormancy, and treatment response. In the present study, we aimed to evaluate the biological responses of CSCs following targeted treatment with phenolic oleuropein (OLE), a glycosylated seco-iridoid compound, within a three-dimensional (3D) microenvironment. CD44⁺/CD24⁻/low CSCs were isolated from the MCF-7 cell line using magnetic-activated cell sorting (MACS). The expression levels of stemness-related genes (OCT3/4, NANOG, SOX2) and drug resistance markers (ABCG1, ABCG2) were assessed in both MCF-7 cells and CSCs using RT-qPCR and the spheroid structures were further evaluated using the TUNEL assay to assess apoptotic activity. CSCs exhibited significantly elevated expression of ABCG1 and stemness markers (OCT3/4, NANOG, SOX2) compared to parental MCF-7 cells. Following OLE treatment, both static and dynamic cultures demonstrated increased expression of OCT3/4, NANOG, SOX2, SURVIVIN, CYCLIN-D1, and p21. Notably, while untreated cells expressed ABCG1 and ABCG2, these genes were not detectable in OLE-treated cells under either culture condition. Collectively, these findings underscore the anti-proliferative and pro-apoptotic effects of OLE on CSCs and highlight its potential to counteract drug resistance mechanisms within a biomimetic 3D microenvironment.

This study evaluated the efficacy of niclosamide after the invasion of aggressive TNBC breast CSCs into a 3D bone-mimicking model. Initially, the optional dose required for triggering apoptosis was determined for MDA-MB-231 CSCs (CD44+ and CD24−). Our findings revealed that approximately 50% of the cells showed apoptotic properties, as assessed with Annexin V/7AAD assay and WST-1 at IC₅₀ = 100 μM (6 h). Additionally, this treatment suppressed p-STAT3 protein levels and increased Bax levels (p < 0.05), as determined by Western Blot. The expression of genes associated with metastasis and cell migration (CXCR4, MMP2, MMP9), drug resistance (ABCG1, ABCG2), stemness (OCT4, NANOG) and cell cycle and proliferation (CYCLIN D1) was found to be significantly suppressed (p < 0.05). Therefore, after validating the efficacy of the 100 μM dose on CSCs, cell cycle, ELISA, Western Blot, and RT-qPCR analyses were conducted in the 3D model. It was found that the cells were arrested in the G0–G1 phase (p < 0.05). 100 μM Niclosamide suppressed the levels of EMT markers, Vimentin (p > 0.05) and ZEB1 (p < 0.05). Additionally, RT-qPCR results indicated a significant downregulation of CXCR4, ABCG1, ABCG2, MMP2, OCT4, CCND1, AXIN2, and LGR5 gene expressions following niclosamide treatment in both CD133+ and CD133– groups (p < 0.05). The increase in the Bax protein, a key player in apoptosis induction, along with the decrease in the anti-apoptotic protein Bcl-2, suggests the activation of cell death mechanisms. Notably, its targeted impact on the CD44+/CD24– population suggests that niclosamide could enhance the sensitivity of CSCs to treatment, thereby preventing tumor recurrence.

This study evaluated the efficacy of temsirolimus in a 3D model mimicking breast cancer stem cell (CSC) metastasis to bone. A composite material (collagen/PGA/Na₂SiO₃) is used to create a scaffold with bone marrow mesenchymal stem cells (BM‐MSCs) and human umbilical vein endothelial cells (HUVECs). MSCs maintained over 80% viability for 21 days on the scaffold. Calcium analysis shows increased calcium release in both control (3‐fold, 2.7‐fold) and osteogenic (2.3‐fold, 2.5‐fold) mediums on days 14 and 21. Gene expression analysis reveals higher levels of Osteopontin (OPN) (6‐fold), Osteocalcin (OCN) (12‐fold), and RUNX Family transcription factor 2 (RUNX2) (1.8‐fold) in the osteogenic medium (p < 0.05). The impact of 5 µM temsirolimus treatment for 6 hours is assessed under control and dynamic culture conditions, reducing mammalian target of rapamycin (mTOR)‐related protein levels (phospho (p)‐mTOR and p‐AKT) in CSCs. The composite material is characterized through viscosity and compression testing, confirming its suitability for supporting osteogenic differentiation and cell viability (WST‐1, Alizarin Red Staining, Calcium Assay, RT‐qPCR). Gene expression of CSCs, selected based on CD133 expression, shows elevated stemness‐associated genes (OCT4, NANOG), EMT (MMP2, CXCR4, CDH1, CDH2), and drug resistance (ABCG1, ABCG2) in the CD133⁺ group in dynamic condition. Temsirolimus treatment reduces the expression of these genes by 21‐fold to 7.52‐fold (p < 0.05). These findings suggest temsirolimus as a promising therapeutic for CSC metastasis to bone.[Correction added on April 8, 2025, after first online publication: the word CHD is updated to CDH in this version.]

Telocytes have some cytoplasmic extensions called telopodes, which are thought to play a role in mitochondrial transfer in intercellular communication. Besides, it is hypothesized that telocytes establish cell membrane-mediated connections with breast cancer cells in coculture and may contribute to the survival of neoplastic cell clusters together with other stromal cells. The aim of this study is to investigate the contribution of telocytes and telocyte-derived mitochondria, which have also been identified in breast tumors, to the tumor development of breast cancer stem cells (CSCs) via miR-146a-5p. The isolation/characterization of telocytes from bone marrow mononuclear cells and the isolation of mitochondria from these cells were performed, respectively. In the next step, CSCs were isolated from the MDA-MB-231 cell line and were characterized. Then, miR-146a-5p expressions of CSCs were inhibited by anti-miR-146a-5p. The epithelial-mesenchymal transition (EMT) was determined by evaluating changes in vimentin protein levels and was evaluated by analyzing BRCA1, P53, SOX2, E-cadherin, and N-cadherin gene expression changes. Our results showed that miR-146a promoted stemness and oncogenic properties in CSCs. EMT (N-cadherin, vimentin, E-cadherin) and tumorigenic markers (BRCA1, P53, SOX2) of CSCs decreased after miR-146a inhibition. Bone marrow-derived telocytes and mitochondria derived from telocytes favored the reduction of CSC aggressiveness following this inhibition.

Bone tissue is composed of organic minerals and cells. It has the capacity to heal for certain minor damages, but when the bone defects surpass the critical threshold, they need fixing. Bone regeneration through natural and synthetic biodegradable materials requires various steps, such as manufacturing methods and materials selection. A successful biodegradable bone graft should have a high surface area/ volume ratio, strength, and a biocompatible, porous structure capable of promoting cell adhesion, proliferation, and differentiation. Considering these requirements, the electrospinning technique is promising for creating functional nano-sized scaffolds. The multi-axial methods, such as coaxial and triaxial electrospinning, are the most popular techniques to produce double or tri-layered scaffolds, respectively. Recently, stem cell culture on scaffolds and the application of osteogenic differentiation protocols on these scaffolds have opened new possibilities in the field of biomaterials research. This review discusses an overview of the progress in coaxial and triaxial technology through biodegradable composite bone materials. The review also carefully elaborates the osteogenic differentiation using stem cells and their performance with nano-sized scaffolds.

Background: Cancer stem cells (CSCs) are a subpopulation of cancer cells that are believed to be responsible for tumor initiation, progression, metastasis, and resistance to conventional therapies. Oleuropein as a natural compound found in olive leaves and olive oil, has potential therapeutic effects in cancer treatment, particularly in targeting CSCs. It induces apoptosis in CSCs while sparing normal cells, inhibit proliferation, migration, and invasion, and suppress the self-renewal ability of CSCs. Additionally, oleuropein has shown synergistic effects with conventional chemotherapy drugs, enhancing their efficacy against CSCs. Objectives: This study aims to selectively target therapeutically resistant cancer stem cells (CSCs) within a heterogeneous tumor population by utilizing oleuropein (OLE) encapsulated in methacrylated alginate (OLE-mALG) within an in vivo-like microenvironment. Purpose: This study aims to target therapeutically resistant cancer stem cells (CSCs) with oleuropein (OLE) encapsulated in the methacrylated alginate (OLE-mALG) in a heterogeneous tumor population with an in vivo-like microenvironment. Methods: Co-culture of CSCs with non-tumorogenic MCF-12 A cells was performed, the 3D breast cancer model was supported with methocel/matrigel/collagen-I, and vascularization was ensured with human umbilical vein endothelial cells (HUVEC). Then, OLE-loaded methacrylated alginate microparticles (mALG) were formed by dual crosslinking in the presence of both ionic and visible light obtained with a droplet based microfluidic system. The characterization and effectiveness of the produced OLE-mALG were evaluated by the FTIR, swelling/degradation/release analysis. Before producing OLE loaded mALG microparticles, a preliminary study was carried out to determine the effective dose of OLE for cells and the duration of OLE action on MCF-7, CSCs and MCF-12 A. Subsequently, CSC viability (WST-1), apoptosis (Bcl-2, Bax, caspase-3, caspase-9), stemness (OCT3/4, NANOG, SOX2), EMT profile (E-cadherin, Vimentin, Slug) and proliferation (SURVIVIN, p21, CYCLIN D1) after OLE-mALG treatment were all evaluated in the 3D model. Results: OLE was encapsulated in mALG with an efficiency of 90.49% and released 73% within 7 h. OLE-mALG induced apoptosis through the decrease in anti-apoptotic Bcl-2 and an increase in pro-apoptotic Bax, caspase-3, and caspase-9 protein levels. While Vimentin and Slug protein levels decreased after 200 µg/mL OLE-mALG treatment to 3D breast cancer culture, E-cadherin levels increased. OLE-mALG treatment to CSC co-culture led to a decrease in proliferation by triggering p21/SURVIVIN expressions, and also resulted in an increase in stemness genes (OCT3/4/NANOG/SOX2). Conclusion: 200 µg/mL OLE-loaded mALG microparticles suppressed epithelial-to-mesenchymal transition by suppressing Vimentin and Slug protein levels, and increased E-cadherin levels in the 3D breast cancer model we created with CSCs, MCF-12 A and HUVECs. This complex system may allow the use of personalized cells for rapid drug screening in preclinical studies compared to animal experiments. OLE-mALG showed apoptotic and metastasis suppressive properties in cancer cells and it was concluded that it can be used in combination with or alternatively with chemotherapeutic agents to target breast cancer stem cells. Graphical