21 research outputs found
Mesenchymal stem cell-derived extracellular vesicles may promote breast cancer cell dormancy
Disseminated breast cancer cells have the capacity to metastasise to the bone marrow and
reside in a dormant state within the mesenchymal stem cell (MSC) niche. Research has
focussed on paracrine signalling factors, such as soluble proteins, within the microenvironment.
However, it is now clear extracellular vesicles (EVs) secreted by resident MSCs into this
microenvironment also play a key role in the initiation of dormancy. Dormancy encourages
reduced cell proliferation and migration, whilst upregulating cell adhesion, thus retaining the
cancer cells within the bone marrow microenvironment. Here, MCF7 breast cancer cells were
treated with MSC-derived EVs, resulting in reduced migration in 2D and 3D culture, with
reduced cell proliferation and enhanced adhesion, collectively supporting cancer cell dormancy
Mesenchymal stem cell-derived extracellular vesicles may promote breast cancer cell dormancy
Disseminated breast cancer cells have the capacity to metastasise to the bone marrow and
reside in a dormant state within the mesenchymal stem cell (MSC) niche. Research has
focussed on paracrine signalling factors, such as soluble proteins, within the microenvironment.
However, it is now clear extracellular vesicles (EVs) secreted by resident MSCs into this
microenvironment also play a key role in the initiation of dormancy. Dormancy encourages
reduced cell proliferation and migration, whilst upregulating cell adhesion, thus retaining the
cancer cells within the bone marrow microenvironment. Here, MCF7 breast cancer cells were
treated with MSC-derived EVs, resulting in reduced migration in 2D and 3D culture, with
reduced cell proliferation and enhanced adhesion, collectively supporting cancer cell dormancy
Submerged growth media have different effects on differentiation of ovine tracheal epithelial cells.
<p>Ovine tracheal epithelial cells were cultured to confluency in SGM or AEGM and at ALI for 21 days in ALI medium. (A) Immunofluorescent staining with anti-β-tubulin (green), rhodamine-phalloidin (red) and DAPI (blue). (B) Scanning electron microscopy. (C) Immunofluorescent staining with anti-ZO-1 (green) and DAPI (blue). (D) Haematoxylin and eosin-stained histological sections. (E) Quantitation of ciliation as percentage of total area from β-tubulin staining. (F) Trans-epithelial electrical resistance measurements. Data shown are from a single representative animal with mean +/- standard deviation from three inserts displayed. (G) Quantitation of ciliation by counting ciliated cells in H&E-stained sections. (E, G) Five images from each of three inserts were analysed and data displayed is mean +/- standard deviation from four animals. Statistical significance was assessed by Student’s <i>t</i>-test (E, F and G). Significance values are indicated by one (<i>P</i><0.05), two (<i>P</i><0.01) or three (<i>P</i><0.001) asterisks.</p
Epidermal growth factor plays diverse roles in the differentiation of ovine tracheal epithelial cells.
<p>Ovine tracheal epithelial cells were cultured at ALI for 21 days with the indicated concentrations of EGF. (A) Immunofluorescent staining with anti-β-tubulin (green), rhodamine-phalloidin (red) and DAPI (blue). (B) Scanning electron microscopy. (C) Immunofluorescent staining with anti-ZO-1 (green) and DAPI (blue). (D) Haematoxylin and eosin-stained histological sections. (E) Quantitation of ciliation as percentage of total area from β-tubulin staining. (F) Trans-epithelial electrical resistance measurements. Data shown are from a single representative animal with mean +/- standard deviation from three inserts displayed. (G) Cell layer thickness measured from three points per field in H&E-stained sections. (H) Cell layer thickness as determined by counting nuclei at three points per field in H&E-stained sections. (I) Quantitation of ciliation by counting ciliated cells in H&E-stained sections. (E, G, H, I) Five images from each of three inserts were analysed and data displayed is mean +/- standard deviation from four animals. Statistical significance was assessed by Student’s <i>t</i>-test (E, G, H and I) or one-way ANOVA with Dunnet’s post-test (F). Significance values are indicated by one (<i>P</i><0.05), two (<i>P</i><0.01) or three (<i>P</i><0.001) asterisks. Black asterisks indicate all samples were significantly different to untreated control in panel F.</p
Ovine tracheal epithelial cell cultures display a time-dependent increase in apical surface ciliation.
<p>(A) Ovine tracheal epithelial cell cultures were grown at an ALI for the indicated number of days (relative to establishment of the ALI), fixed and immunostained using an anti-β tubulin antibody to detect cilia and rhodamine-phalloidin to stain the actin cytoskeleton. (B) Z-stack orthogonal representation of 21-day post-ALI tissue layer. (C) 3-dimensional representation of the Z-stack in panel B. (D) Ciliation was quantified by measuring the area above a manual fluorescence intensity threshold in ImageJ. For each time point, five regions on three independent cell cultures were measured. Results displayed are the mean +/- standard deviation from tissue layers derived from three animals.</p
Ovine tracheal epithelial cell cultures display stable barrier function and junctional integrity.
<p>(A) Ovine tracheal epithelial cell cultures were grown at ALI for the indicated number of days (relative to establishment of the ALI) and tissue layers were fixed and immunostained using an anti-ZO1 antibody at the indicated time points (relative to establishment of the ALI). (B) Orthogonal representation of ALI culture at 24 days post-ALI. (C) 3-dimensional model of the Z-stack shown in panel B. (D) TEER measurements from four independent cell culture inserts at each time-point. Results for ALI cultures derived from three independent animals are shown (mean +/- standard deviation).</p
Ultrastructural analysis of the apical surface of ovine tracheal epithelial cell cultures by SEM.
<p>Ovine tracheal epithelial cell cultures were grown at an ALI for the indicated number of days (relative to establishment of the ALI), fixed and processed for SEM. (A) Images were taken at 1500Ă— magnification. (B) Images were taken at 5000Ă— magnification. Ciliated epithelial cells were observed from day 12 onwards.</p
Retinoic acid is required for <i>in vitro</i> differentiation of ovine tracheal epithelial cells.
<p>Ovine tracheal epithelial cells were cultured at ALI for 21 days with the indicated concentrations of retinoic acid. (A) Immunofluorescent staining with anti-β-tubulin (green), rhodamine-phalloidin (red) and DAPI (blue). (B) Scanning electron microscopy. (C) Immunofluorescent staining with anti-ZO-1 (green) and DAPI (blue). (D) Haematoxylin and eosin-stained histological sections. (E) Quantitation of ciliation as percentage of total area from β-tubulin staining. (F) Trans-epithelial electrical resistance measurements. Data shown are from a single representative animal with mean +/- standard deviation from three inserts displayed. (G) Cell layer thickness measured from three points per field in H&E-stained sections. (H) Cell layer thickness as determined by counting nuclei at three points per field in H&E-stained sections. (I) Quantitation of ciliation by counting ciliated cells in H&E-stained sections. (E, G, H, I) Five images from each of three inserts were analysed and data displayed is mean +/- standard deviation from four animals. Statistical significance was assessed by Student’s <i>t</i>-test (E, G, H and I) or one-way ANOVA with Dunnet’s post-test (F). Significance values are indicated by one (<i>P</i><0.05), two (<i>P</i><0.01) or three (<i>P</i><0.001) asterisks. Black asterisks indicate all samples were significantly different to untreated control in panel F.</p
Ovine tracheal epithelial cells differentiate optimally at sub-ambient oxygen tension.
<p>Ovine tracheal epithelial cells were cultured in an atmosphere of 7, 14 or 21% O<sub>2</sub>. (A) Immunofluorescent staining with anti-β-tubulin (green), rhodamine-phalloidin (red) and DAPI (blue). (B) Scanning electron microscopy. (C) Immunofluorescent staining with anti-ZO-1 (green) and DAPI (blue). (D) Haematoxylin and eosin-stained histological sections. (E) Quantitation of ciliation as percentage of total area from β-tubulin staining. (F) Trans-epithelial electrical resistance measurements. Data shown are from a single representative animal with mean +/- standard deviation from three inserts displayed. (G) Quantitation of ciliation by counting ciliated cells in H&E-stained sections. (E, G) Five images from each of three inserts were analysed and data displayed is mean +/- standard deviation from four animals. Statistical significance was assessed by Student’s <i>t</i>-test (E and G) or one-way ANOVA with Dunnet’s post-test (F). Significance values are indicated by one (<i>P</i><0.05), two (<i>P</i><0.01) or three (<i>P</i><0.001) asterisks.</p
Mucus production by differentiated ovine tracheal epithelial cell cultures.
<p>(A) Ovine tracheal epithelial cell cultures were grown at ALI for the indicated number of days (relative to establishment of the ALI), fixed and processed for SEM. (B) Ovine tracheal epithelial cell cultures were grown for the indicated number of days, fixed and stained with jacalin-FITC (green), rhodamine-phalloidin (red) and DAPI (blue). Mucus globules are indicated by white arrows, carpets of amorphous mucus are indicated by white arrowheads and jacalin-labelled mucin-positive cells are indicated by yellow arrows.</p