7 research outputs found
Characterization of bone marrow isolated MSCs and fibroblasts.
<p>Phenotypic analysis was performed by flow cytometry was performed on adherent bone marrow cells and Swiss 3T3 fibroblasts with positive populations in red given with S.E.M.(A). Purified MSCs differentiated into adipocytes (B) and osteoblasts (C) within 3 weeks in lineage-specific differentiation media as shown both my staining and RT-PCR (scale bar = 100 µm).</p
Multiple particle tracking microrheology.
<p>The ensemble averaged mean squared displacements (<2(Δt)>>) of 100 nm particles embedded in the cytoplasm of TCM-treated MSCs (A) and 3T3 fibroblasts (B) were evaluated from 0–3 hours. For both cell lines, treatment with TCM reduced the rate of cytoplasmic particle transport in a time-dependent manner. Fluorescent image of 100 nm particles (green) in the cytoplasm of a MSC, which was fixed and stained with phalloidin (red) and DAPI (blue) (C). The phase angle, δ = arctan (G″(ω))/G′(ω)), was used to characterize the viscoelastic nature of the cytoplasm over the course of the experiment (D). The viscoelastic nature of MSCs and 3T3 fibroblasts were similar initially and 3 hours after TCM-treatment; however, MSCs responded much more rapidly to TCM with a 4-fold reduction in δ within 60 minutes. (scale bar = 10 µm)</p
MSCs reorganize their cytoskeleton in response to tumor-secreted soluble factors.
<p>(A) Confocal micrographs of CM and TCM-treated MSCs (a–c) and 3T3 fibroblasts (d–e) stained with Phalloidin (F-actin, red), anti-α-tubulin (microtubules, green), and DAPI (nucleus, blue). The shape and cytoskeletal organization of CM-treated MSCs (a) and CM- (d) and TCM- (e) treated Swiss 3T3 fibroblasts were similar (24 hours after CM or TCM addition); whereas, TCM-treated MSCs were elongated with extended cytoskeletal filaments (b–c). MSC elongation increased between 12- (b) and 24- (c) hours, indicating that cytoskeletal changes may be progressive. Cytoskeletal parameters (B–D) were determined by analysis of confocal images with a custom MATLAB routine. The cell (B) and nuclear (D) shape factors were used to characterize the circularity of an elliptical outline of the cell or nucleus, respectively, with a shape factor of 1 indicating a perfect circle. The stress fiber factor (C) was used to characterize the density of actin stress fibers per cell area. Cytoskeletal changes observed in TCM-treated MSCs (b–c) were confirmed using the cytoskeletal parameters (B–D), which indicated dramatic reductions in cell and nuclear shape factors and stress fiber densities. (scale bars = 10 µm)</p
TCM alters cytoplasmic rheology.
<p>The time-dependent ensemble averaged MSDs of 100 nm particles embedded in the cytoplasm of MSCs and 3T3 fibroblasts were converted to frequency-dependent elastic (G′, solid lines) and visous (G″, dashed lines) moduli using a custom algorithm written for Matlab software. The ensemble-averaged frequency-dependent viscoelasticities of MSCs (A–E, left) and 3T3 fibroblasts (F–J, right) prior to (A,F) and 30 minutes (B,G), 1 hour (C,H), 2 hours (D,I), or 3 hours (E,J) after treatment with TCM. The cytoplasm of MSCs became predominantly elastic within 60 minutes; whereas, 3T3 fibroblasts required 3 hours to undergo a similar change.</p
Effect of TCM on Cell Migration.
<p>Tranwell assays were used to measure the migration of MSCs and 3T3 fibroblasts through 3 µm- (A) and 8 µm- (B) pore transwell inserts toward CM or TCM. The average number of cells per image (n = 9), collected with a 10×-objective, was reported. TCM significantly increased MSC migration, compared to CM, through 3 µm pores within 3 hours and 8 µm pores within 2 hours; however, fibroblast migration was only increased through 8 µm pores within 3 hours. MSCs and 3T3 fibroblasts were then treated with CM or TCM for 1 hour and allowed to migrate through 8 µm-pore transwell inserts toward CM or TCM for 3 hours (C). Pre-treatment with TCM resulted in synergistic effects on chemotactic migration for both cell types.</p
Nuclear Membrane-Targeted Gold Nanoparticles Inhibit Cancer Cell Migration and Invasion
Most cancer patients die from metastasis.
Recent studies have shown that gold nanoparticles (AuNPs) can slow
down the migration/invasion speed of cancer cells and suppress metastasis.
Since nuclear stiffness of the cell largely decreases cell migration,
our hypothesis is that targeting AuNPs to the cell nucleus region
could enhance nuclear stiffness, and therefore inhibit cell migration
and invasion. Our results showed that upon nuclear targeting of AuNPs,
the ovarian cancer cell motilities decrease significantly, compared
with nontargeted AuNPs. Furthermore, using atomic force microscopy,
we observed an enhanced cell nuclear stiffness. In order to understand
the mechanism of cancer cell migration/invasion inhibition, the exact
locations of the targeted AuNPs were clearly imaged using a high-resolution
three-dimensional imaging microscope, which showed that the AuNPs
were trapped at the nuclear membrane. In addition, we observed a greatly
increased expression level of lamin A/C protein, which is located
in the inner nuclear membrane and functions as a structural component
of the nuclear lamina to enhance nuclear stiffness. We propose that
the AuNPs that are trapped at the nuclear membrane both (1) add to
the mechanical stiffness of the nucleus and (2) stimulate the overexpression
of lamin A/C located around the nuclear membrane, thus increasing
nuclear stiffness and slowing cancer cell migration and invasion
Nuclear Membrane-Targeted Gold Nanoparticles Inhibit Cancer Cell Migration and Invasion
Most cancer patients die from metastasis.
Recent studies have shown that gold nanoparticles (AuNPs) can slow
down the migration/invasion speed of cancer cells and suppress metastasis.
Since nuclear stiffness of the cell largely decreases cell migration,
our hypothesis is that targeting AuNPs to the cell nucleus region
could enhance nuclear stiffness, and therefore inhibit cell migration
and invasion. Our results showed that upon nuclear targeting of AuNPs,
the ovarian cancer cell motilities decrease significantly, compared
with nontargeted AuNPs. Furthermore, using atomic force microscopy,
we observed an enhanced cell nuclear stiffness. In order to understand
the mechanism of cancer cell migration/invasion inhibition, the exact
locations of the targeted AuNPs were clearly imaged using a high-resolution
three-dimensional imaging microscope, which showed that the AuNPs
were trapped at the nuclear membrane. In addition, we observed a greatly
increased expression level of lamin A/C protein, which is located
in the inner nuclear membrane and functions as a structural component
of the nuclear lamina to enhance nuclear stiffness. We propose that
the AuNPs that are trapped at the nuclear membrane both (1) add to
the mechanical stiffness of the nucleus and (2) stimulate the overexpression
of lamin A/C located around the nuclear membrane, thus increasing
nuclear stiffness and slowing cancer cell migration and invasion