-
Physical Biology Nov 2022Tumor-associated collagen signature-3 (TACS-3) is a prognostic indicator for breast cancer survival. It is characterized by highly organized, parallel bundles of...
Tumor-associated collagen signature-3 (TACS-3) is a prognostic indicator for breast cancer survival. It is characterized by highly organized, parallel bundles of collagen fibers oriented perpendicular to the tumor boundary, serving as directional, confining channels for cancer cell invasion. Here we design a TACS-3-mimetic anisotropic, confined collagen I matrix and examine the relation between anisotropy of matrix, directed cellular migration, and anisotropy of cell membrane-the first direct contact between TACS-3 and cell-using Michigan Cancer Foundation-7 (MCF-7) cells as cancer-model. Using unidirectional freezing, we generated ∼50m-wide channels filled with collagen I. Optical tweezer (OT) microrheology shows that anisotropic confinement increases collagen viscoelasticity by two orders of magnitude, and the elastic modulus is significantly greater along the direction of anisotropic confinement compared to that along the orthogonal direction, thus establishing matrix anisotropy. Furthermore, MCF-7 cells embedded in anisotropic collagen I, exhibit directionality in cellular morphology and migration. Finally, using customized OT to trap polystyrene probes bound to cell-membrane (and not to ECM) of either free cells or cells under anisotropic confinement, we quantified the effect of matrix anisotropy on membrane viscoelasticity, both in-plane and out-of-plane, vis-à-vis the membrane. Both bulk and viscous modulus of cell-membrane of MCF-7 cells exhibit significant anisotropy under anisotropic confinement. Moreover, the cell membrane of MCF-7 cells under anisotropic confinement is significantly softer (both in-plane and out-of-plane moduli) despite their local environment being five times stiffer than free cells. In order to test if the coupling between anisotropy of extracellular matrix and anisotropy of cell-membrane is regulated by cell-cytoskeleton, actin cytoskeleton was depolymerized for both free and confined cells. Results show that cell membrane viscoelasticity of confined MCF-7 cells is unaffected by actin de-polymerization, in contrast to free cells. Together, these findings suggest that anisotropy of ECM induces directed migration and correlates with anisotropy of cell-membrane viscoelasticity of the MCF-7 cells in an actin-independent manner.
Topics: Humans; Anisotropy; MCF-7 Cells; Actins; Collagen; Cell Membrane
PubMed: 36354019
DOI: 10.1088/1478-3975/ac9bc1 -
Scientific Reports Dec 2023Epithelial cells undergoing EMT experience significant alterations at transcriptional and morphological levels. However, changes in the cytoskeleton, especially...
Epithelial cells undergoing EMT experience significant alterations at transcriptional and morphological levels. However, changes in the cytoskeleton, especially cytoskeleton dynamics are poorly described. Addressing the question we induced EMT in three cell lines (MCF-7, HaCaT and A-549) and analyzed morphological and cytoskeletal changes there using immunostaining and life cell imaging of cells transfected with microtubule and focal adhesion markers. In all studied cell lines, cell area after EMT increased, MCF-7 and A-549 cells became elongated, while HaCaT cells kept the aspect ratio the same. We next analyzed three components of the cytoskeleton: microtubules, stress fibers and focal adhesions. The following changes were observed after EMT in cultured cells: (i) Organization of microtubules becomes more radial; and the growth rate of microtubule plus ends was accelerated; (ii) Actin stress fibers become co-aligned forming the longitudinal cell axis; and (iii) Focal adhesions had decreased area in all cancer cell lines studied and became more numerous in HaCaT cells. We conclude that among dynamic components of the cytoskeleton, the most significant changes during EMT happen in the regulation of microtubules.
Topics: Cell Adhesion; Cytoskeleton; Microtubules; Actins; Focal Adhesions; Actin Cytoskeleton
PubMed: 38092761
DOI: 10.1038/s41598-023-48279-y -
Analytical Chemistry Jan 2020Profiling the kinetics of cell-matrix adhesion is of great importance to understand many physiological and pathological processes such as morphogenesis, tissue...
Profiling the kinetics of cell-matrix adhesion is of great importance to understand many physiological and pathological processes such as morphogenesis, tissue homeostasis, wound healing, and tumorigenesis. Here, we developed a novel digital acoustofluidic device for parallel profiling cell-matrix adhesion at single-cell level. By introduction of localized and uniform acoustic streaming into an open chamber microfluidic device, the adherent cells within the open chamber can be detached by the streaming-induced Stokes drag force. By digital regulation of pulsed acoustic power from a low level to high levels, the hundreds of adherent cells can be ruptured from the fibronectin-coated substrate accordingly, and their adhesive forces (from several pN to several nN) and kinetics can be determined by the applied power and cell incubation time. As a proof-of-concept application for studying cancer metastasis, we applied this technique to measure the adhesion strength and kinetics of human breast cancer cells to extracellular matrix such as fibronectin and compared their metastatic potentials by measuring the rupture force of cancer cells representing malignant (MCF-7 cells and MDA-MB-231 cells) and nonmalignant (MCF-10A cells) states. Our acoustofluidic device is simple, easy to operate, and capable of measuring, in parallel, hundreds of individual cells' adhesion forces with a resolution at the pN level. Thus, we expect this device could be widely used for both fundamental cell biology research as well as development of cancer diagnostics and tissue engineering technologies.
Topics: Acoustics; Cell Adhesion; Cell-Matrix Junctions; Cells, Cultured; Equipment Design; Humans; Kinetics; MCF-7 Cells; Microfluidic Analytical Techniques
PubMed: 31880433
DOI: 10.1021/acs.analchem.9b05065 -
Biochemical and Biophysical Research... Aug 2020Breast cancer is the most frequent female malignancy in the world. In this regard, cancer detection by assessing the biomechanical properties of cells is a promising...
Breast cancer is the most frequent female malignancy in the world. In this regard, cancer detection by assessing the biomechanical properties of cells is a promising method in oncology. Cell state can be identified by studying viscosity behavior; however, a more complex understanding of cells requires a profound insight into the solidity and fluidity of cells via the characterization of cell viscoelasticity. The present study aimed to compare the viscoelasticity of healthy human breast epithelial cells (MCF-10A) with that of cancerous cells (MCF 7). The experiment included the addition of nano magnetic particles (NMP) to the cell culture environment and placement of the Petri Dishes under a microscope after the completion of primary culture stages and, ultimately, adoption of a magnetic tweezer technique to perform a creep test. A viscoelastic model of cells was suggested with discrete differential equations for both groups of healthy and cancerous cells after obtaining information about cell membrane movements and performing image processes on these data. A comparison of cell stiffness was made under two conditions of static and dynamic. According to the findings, cancerous static stiffness was lower than that of healthy cells by a factor of 3.5. The creep test results showed that MCF 7 cells would exhibit solid-like behavior. At a higher gel point frequency, these cells emerged more solidity compared to their corresponding healthy cells. The obtained results revealed the clear changes in cancerous cells' viscoelastic properties and the potential alterations of their cytoskeleton.
Topics: Biomechanical Phenomena; Breast; Breast Neoplasms; Cell Line; Elasticity; Epithelial Cells; Female; Humans; MCF-7 Cells; Viscosity
PubMed: 32703447
DOI: 10.1016/j.bbrc.2020.06.010 -
Archives of Razi Institute 2021Conventional cancer treatments are costly and have different serious side effects for patients. Natural herbal treatments are widely accepted among people because of...
Conventional cancer treatments are costly and have different serious side effects for patients. Natural herbal treatments are widely accepted among people because of their minimal side effects, although there is little scientific knowledge about them. One of these remedies utilizes the root of that has been used for years in Iran to treat different chronic genital diseases. The current study examined the effects of methanolic and ethanolic extracts of (induction of necrosis and apoptosis) on breast cancer (MCF-7), ovarian cancer (A2780), and human cervix cancer (HeLa) cell lines in comparison with normal breast cells. These effects were determined to be morphological alterations in cell light microscopy, by flow cytometry (staining with annexin V and propidium iodide), and by measuring live cells and inhibition concentrations by MTT assay. IC50 of on the MCF-7 cell line (methanolic extract) was 400 µg/ml and for A2780 was 250 µg/ml. The IC50 amount of on the MCF-7 cell line (ethanolic extract) was 750 µg/ml and 1500 for A2780. Results demonstrated that apoptosis and necrosis occurred in MCF-7 and A2780 following the addition of ethanolic and methanolic extracts of to the medium. These findings confirmed the anti-cancer effects of mehthanolic extracts of root and its safety for normal cells; thus, it can be applied in cancer therapy as a novel medication.
Topics: Cell Line, Tumor; Female; HeLa Cells; Humans; MCF-7 Cells; Ovarian Neoplasms; Plant Extracts
PubMed: 34824753
DOI: 10.22092/ari.2020.351952.1545 -
Toxicology in Vitro : An International... Sep 2022The present study investigates the mechanisms underlying the in vitro antitumoral activity of cirsimarin (CIR 10 to 320 μM), a flavone extracted from the aerial parts...
The present study investigates the mechanisms underlying the in vitro antitumoral activity of cirsimarin (CIR 10 to 320 μM), a flavone extracted from the aerial parts of Scoparia dulcis L., on MCF-7 cells cultured in 2D and multicellular tumor spheroids (3D). CIR (from 40 μM) decreased cell viability in the resazurin assay and colony formation in the 2D model. In the same way, in the 3D model, CIR (from 40 μM) induced cell death (triple staining assay) and decreased spheroid integrity after 16 days with no induction of intracellular reactive species (CM-HDCFDA). In 2D, CIR decreased the invasion (transwell) and horizontal migration (wound healing), while in 3D, CIR diminished cell migration (ECM® gel) and induced DNA damage (comet assay) possibly related to cell death. CIR mediated antitumoral effects in 3D spheroids by negative modulation of genes associated with cell proliferation (CCND1, CCNA2, CDK2, CDK4, and TNF) and death (BCL-XL, BAX, CASP9, and BIRC5). BIRC5 and CDKs inhibitors have been proposed as versatile anticancer drugs, which makes our results quite interesting. TNF negative modulation may also be related to the downregulation of MMP9 and MMP11 and anti-migration/invasion of MCF-7 cells cultured in 2D and 3D models. These are relevant properties for long-term strategies to avoid metastasis and improve the prognosis of breast cancer.
Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Flavones; Glycosides; Humans; MCF-7 Cells; Spheroids, Cellular
PubMed: 35710092
DOI: 10.1016/j.tiv.2022.105416 -
PloS One 2021The nucleus-to-cytoplasm ratio (N:C) can be used as one metric in histology for grading certain types of tumor malignancy. Current N:C assessment techniques are...
The nucleus-to-cytoplasm ratio (N:C) can be used as one metric in histology for grading certain types of tumor malignancy. Current N:C assessment techniques are time-consuming and low throughput. Thus, in high-throughput clinical contexts, there is a need for a technique that can assess cell malignancy rapidly. In this study, we assess the N:C ratio of four different malignant cell lines (OCI-AML-5-blood cancer, CAKI-2-kidney cancer, HT-29-colon cancer, SK-BR-3-breast cancer) and a non-malignant cell line (MCF-10A -breast epithelium) using an imaging flow cytometer (IFC). Cells were stained with the DRAQ-5 nuclear dye to stain the cell nucleus. An Amnis ImageStreamX® IFC acquired brightfield/fluorescence images of cells and their nuclei, respectively. Masking and gating techniques were used to obtain the cell and nucleus diameters for 5284 OCI-AML-5 cells, 1096 CAKI-2 cells, 6302 HT-29 cells, 3159 SK-BR-3 cells, and 1109 MCF-10A cells. The N:C ratio was calculated as the ratio of the nucleus diameter to the total cell diameter. The average cell and nucleus diameters from IFC were 12.3 ± 1.2 μm and 9.0 ± 1.1 μm for OCI-AML5 cells, 24.5 ± 2.6 μm and 15.6 ± 2.1 μm for CAKI-2 cells, 16.2 ± 1.8 μm and 11.2 ± 1.3 μm for HT-29 cells, 18.0 ± 3.7 μm and 12.5 ± 2.1 μm for SK-BR-3 cells, and 19.4 ± 2.2 μm and 10.1 ± 1.8 μm for MCF-10A cells. Here we show a general N:C ratio of ~0.6-0.7 across varying malignant cell lines and a N:C ratio of ~0.5 for a non-malignant cell line. This study demonstrates the use of IFC to assess the N:C ratio of cancerous and non-cancerous cells, and the promise of its use in clinically relevant high-throughput detection scenarios to supplement current workflows used for cancer cell grading.
Topics: Cell Nucleus; Cytoplasm; Flow Cytometry; HT29 Cells; Humans; Image Cytometry; Neoplasms
PubMed: 34166419
DOI: 10.1371/journal.pone.0253439 -
International Journal of Molecular... Jul 2023In healthy tissues, cells are in mechanical homeostasis. During cancer progression, this equilibrium is disrupted. Cancer cells alter their mechanical phenotype to a...
In healthy tissues, cells are in mechanical homeostasis. During cancer progression, this equilibrium is disrupted. Cancer cells alter their mechanical phenotype to a softer and more fluid-like one than that of healthy cells. This is connected to cytoskeletal remodeling, changed adhesion properties, faster cell proliferation and increased cell motility. In this work, we investigated the mechanical properties of breast cancer cells representative of different breast cancer subtypes, using MCF-7, tamoxifen-resistant MCF-7, MCF10A and MDA-MB-231 cells. We derived viscoelastic properties from atomic force microscopy force spectroscopy measurements and showed that the mechanical properties of the cells are associated with cancer cell malignancy. MCF10A are the stiffest and least fluid-like cells, while tamoxifen-resistant MCF-7 cells are the softest ones. MCF-7 and MDA-MB-231 show an intermediate mechanical phenotype. Confocal fluorescence microscopy on cytoskeletal elements shows differences in actin network organization, as well as changes in focal adhesion localization. These findings provide further evidence of distinct changes in the mechanical properties of cancer cells compared to healthy cells and add to the present understanding of the complex alterations involved in tumorigenesis.
Topics: Humans; Female; Cell Line, Tumor; Cytoskeleton; MCF-7 Cells; Actins; Tamoxifen; Breast Neoplasms; Microscopy, Atomic Force
PubMed: 37569585
DOI: 10.3390/ijms241512208 -
Journal of the American Chemical Society Jan 2020Effective and cell-type-specific delivery of CRISPR/Cas9 gene editing elements remains a challenging open problem. Here we report the development of biomimetic cancer...
Effective and cell-type-specific delivery of CRISPR/Cas9 gene editing elements remains a challenging open problem. Here we report the development of biomimetic cancer cell coated zeolitic imidazolate frameworks (ZIFs) for targeted and cell-specific delivery of this genome editing machinery. Coating ZIF-8 that is encapsulating CRISPR/Cas9 (CC-ZIF) with a cancer cell membrane resulted in the uniformly covered C-ZIF. Incubation of C-ZIF with MCF-7, HeLa, HDFn, and aTC cell lines showed the highest uptake by MCF-7 cells and negligible uptake by the healthy cells (i.e., HDFn and aTC). As to genome editing, a 3-fold repression in the EGFP expression was observed when MCF-7 were transfected with C-ZIF compared to 1-fold repression in the EGFP expression when MCF-7 were transfected with C-ZIF. In vivo testing confirmed the selectivity of C-ZIF to accumulate in MCF-7 tumor cells. This supports the ability of this biomimetic approach to match the needs of cell-specific targeting, which is unquestionably the most critical step in the future translation of genome editing technologies.
Topics: Animals; Biomimetics; CRISPR-Cas Systems; HeLa Cells; Heterografts; Humans; MCF-7 Cells; Metal-Organic Frameworks; Mice
PubMed: 31931564
DOI: 10.1021/jacs.9b11638 -
Molecular Pharmaceutics Aug 2020The design, synthesis, characterization, and biological activity of a series of platinum(IV) prodrugs containing the axial ligand...
The design, synthesis, characterization, and biological activity of a series of platinum(IV) prodrugs containing the axial ligand 3-(4-phenylquinazoline-2-carboxamido)propanoate () are reported. is a derivative of the quinazolinecarboxamide class of ligands that binds to the translocator protein (TSPO) at the outer mitochondrial membrane. The cytotoxicities of ,,-[Pt(NH)Cl()(OH)] (), ,,-[Pt(NH)Cl()(BZ)] (), -[Pt(DACH)(OX)()(OH)] (), and -[Pt(DACH)(OX)()(BZ)] () (DACH: ,-diaminocyclohexane, BZ: benzoate, OX: oxalate) in MCF-7 breast cancer and noncancerous MCF-10A epithelial cells were assessed and compared with those of cisplatin, oxaliplatin, and the free ligand . Moreover, the cellular uptake, ROS generation, DNA damage, and the effect on the mitochondrial function, mitochondrial membrane potential, and morphology were investigated. Molecular interactions of in the TSPO binding site were studied using molecular docking. The results showed that complex is the most effective Pt(IV) complex and exerts a multimodal mechanism involving DNA damage, potent ROS production, loss of the mitochondrial membrane potential, and mitochondrial damage.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Cisplatin; DNA Damage; Epithelial Cells; Humans; Ligands; MCF-7 Cells; Mitochondria; Mitochondrial Membranes; Organoplatinum Compounds; Oxaliplatin; Prodrugs; Reactive Oxygen Species
PubMed: 32628022
DOI: 10.1021/acs.molpharmaceut.0c00417