12 research outputs found
Tissue Factor-Expressing Tumor Cells Can Bind to Immobilized Recombinant Tissue Factor Pathway Inhibitor under Static and Shear Conditions <i>In Vitro</i>
<div><p>Mammary tumors and malignant breast cancer cell lines over-express the coagulation factor, tissue factor (TF). High expression of TF is associated with a poor prognosis in breast cancer. Tissue factor pathway inhibitor (TFPI), the endogenous inhibitor of TF, is constitutively expressed on the endothelium. We hypothesized that TF-expressing tumor cells can bind to immobilized recombinant TFPI, leading to arrest of the tumor cells under shear <i>in vitro</i>. We evaluated the adhesion of breast cancer cells to immobilized TFPI under static and shear conditions (0.35 – 1.3 dyn/cm<sup>2</sup>). We found that high-TF-expressing breast cancer cells, MDA-MB-231 (with a TF density of 460,000/cell), but not low TF-expressing MCF-7 (with a TF density of 1,400/cell), adhered to recombinant TFPI, under static and shear conditions. Adhesion of MDA-MB-231 cells to TFPI required activated factor VII (FVIIa), but not FX, and was inhibited by a factor VIIa-blocking anti-TF antibody. Under shear, adhesion to TFPI was dependent on the TFPI-coating concentration, FVIIa concentration and shear stress, with no observed adhesion at shear stresses greater than 1.0 dyn/cm<sup>2</sup>. This is the first study showing that TF-expressing tumor cells can be captured by immobilized TFPI, a ligand constitutively expressed on the endothelium, under low shear <i>in vitro</i>. Based on our results, we hypothesize that TFPI could be a novel ligand mediating the arrest of TF-expressing tumor cells in high TFPI-expressing vessels under conditions of low shear during metastasis.</p></div
Adhesion of tumor cells to protein-immobilized microfluidic channels under low shear (0.35dyn/cm<sup>2</sup>).
<p>Microfluidic channels were incubated with Protein G (100μg/ml), then anti-TF IgG (100μg/ml), or an anti-His antibody (100μg/ml) followed by TFPI (100μg/ml). Isotype IgG (100μg/ml) and anti-His IgG (100μg/ml) antibodies were used as negative control for anti-TF IgG and TFPI respectively. Tumor cells (1x10<sup>6</sup>cells/mL, pre-treated with 10nM FVIIa and 10nM FX for TFPI-coated channels) were introduced into the channels at 0.35dyn/cm<sup>2</sup> for 30 minutes, and non-specifically adhered cells were removed at 2.0dyn/cm<sup>2</sup>. The entire channel was imaged to quantify the number of adherent cells. <b>A.</b> Representative bright field images of adherent tumor cells on channels immobilized with Protein G (negative control), anti-TF IgG and TFPI showing that more MDA-MB-231 than MCF-7 cells were bound to both anti-TF IgG- and TFPI-coated channels. <b>B.</b> The number of adherent cells was counted and normalized by the channel area. MDA-MB-231 showed significantly higher adhesion to TFPI- and anti-TF IgG-coated channels than MCF-7 (* p < 0.05, n = 4 for anti-TF IgG, n = 3 for TFPI). Significantly more MDA-MB-231 bound to TFPI- and anti-TF IgG-coated channels than negative controls (** p<0.05). <b>C.</b> MDA-MB-231 cells were pretreated with 50μg/ml anti-TF IgG (TF9-5B7 which blocks FVIIa binding to TF, or TF9-10H10 which does not block FVIIa binding to TF). The positive control had no antibody pretreatment, and isotype IgG pretreatment (50μg/ml) was used as a negative control. Blocking FVIIa binding to TF with TF9-5B7 antibody significantly decreased adhesion to TFPI-coated channels (* p < 0.05, n = 4). The observed decrease in MDA-MB-231 adhesion with the TF9-10H10 antibody, albeit not significant with this stringent statistical test, could be due to steric hindrance of TFPI binding to the TF/FVIIa/FXa complex on the tumor cells.</p
Schematic of microfluidic channel.
<p>The microfluidic channel consisted of four branches (120x120μm), which allowed for four simultaneous experiments under different coating conditions or cell treatments. The indicated region of interest (along the length of the 4 branches) is where adherent cells are quantified. Cell suspensions were introduced at the inlet and the outlet was connected to a syringe pump.</p
Effect of shear, TFPI-coating concentration and FVIIa concentration in MDA-MB-231 adhesion to protein-immobilized channels under shear.
<p><b>A.</b> Microfluidic channels were immobilized with different concentrations of anti-TF IgG antibody (20–100μg/mL), and MDA-MB-231 cells were introduced at a shear of 0.35 and 0.60dyn/cm<sup>2</sup>. Adhesion of MDA-MB-231 cells to anti-TF IgG antibody reached a plateau at 50μg/mL at both shear stresses (n = 3). <b>B.</b> Microfluidic channels were immobilized with different concentrations of TFPI (5–100μg/mL), and MDA-MB-231 cells (pretreated with 10nM FVIIa and FX) were introduced at a shear of 0.35 and 0.60dyn/cm<sup>2</sup>. The adhesion of MDA-MB-231 increased with increasing TFPI concentration (n = 3). <b>Inset.</b> When FVIIa concentration was increased from 10nM to 100nM at a shear of 0.60dyn/cm<sup>2</sup>, the adhesion of MDA-MB-231 to TFPI-coated channels increased (n = 3). <b>C.</b> Microfluidic channels were immobilized with 100μg/mL TFPI, and MDA-MB-231 cells (pretreated with 10nM or 100nM FVIIa, and 10nM FX) were introduced at a range of shear stresses (0.35–1.3dyn/cm<sup>2</sup>). The adhesion of MDA-MB-231 decreased with increasing shear. Increasing the concentration of FVIIa from 10nM to 100nM increased adhesion of MDA-MB-231 to immobilized TFPI at 0.35 and 0.60dyn/cm<sup>2</sup>. A few tumor cells bound at 1.3dyn/cm<sup>2</sup> with the higher, but not the lower, FVIIa concentration (n = 3).</p
Effect of FVIIa and FX in adhesion of MDA-MB-231 to TFPI-immobilized channels (0.35dyn/cm<sup>2</sup>).
<p><b>A.</b> MDA-MB-231 cells were treated with different combinations of FVIIa (10nM) and FX (10nM) before introduction into TFPI-immobilized channels. Adhesion of MDA-MB-231 to immobilized TFPI in microfluidic channels was abolished when FVIIa was absent (* p<0.05, n = 6). <b>B.</b> MDA-MB-231 cells were treated with different concentrations of FVIIa (0–100nM) prior to perfusion with TFPI-immobilized channels. Increasing the concentration of FVIIa to 100nM significantly increased adhesion of MDA-MB-231 to immobilized TFPI in microfluidic channels (* p < 0.05, n = 3).</p
TF surface expression and density on breast cancer cells.
<p>Representative fluorescence histograms of TF expression on MDA-MB-231 and MCF-7 cells. Cells (5x10<sup>5</sup>) were incubated with a monoclonal antibody against TF (TF9-5B7, 80μg/mL), followed by an Alexa-488-conjugated secondary antibody (10μg/mL). Fluorescence was detected (bold line) using flow cytometry with isotype IgG as a control (dotted line). Tissue factor was strongly expressed on MDA-MB-231, but little expression was found on MCF-7 (n = 3). The surface ligand density is also shown for each cell line.</p
Microfluidic-Based Cell-Embedded Microgels Using Nonfluorinated Oil as a Model for the Gastrointestinal Niche
Microfluidic-based
cell encapsulation has promising potential in therapeutic applications.
It also provides a unique approach for studying cellular dynamics
and interactions, though this concept has not yet been fully explored.
No in vitro model currently exists that allows us to study the interaction
between crypt cells and Peyer’s patch immune cells because
of the difficulty in recreating, with sufficient control, the two
different microenvironments in the intestine in which these cell types
belong. However, we demonstrate that a microfluidic technique is able
to provide such precise control and that these cells can proliferate
inside microgels. Current microfluidic-based cell microencapsulation
techniques primarily use fluorinated oils. Herein, we study the feasibility
and biocompatibility of different nonfluorinated oils for application
in gastrointestinal cell encapsulation and further introduce a model
for studying intercellular chemical interactions with this approach.
Our results demonstrate that cell viability is more affected by the
solidification and purification processes that occur after droplet
formation rather than the oil type used for the carrier phase. Specifically,
a shorter polymer cross-linking time and consequently lower cell exposure
to the harsh environment (e.g., acidic pH) results in a high cell
viability of over 90% within the protected microgels. Using nonfluorinated
oils, we propose a model system demonstrating the interplay between
crypt and Peyer’s patch cells using this microfluidic approach
to separately encapsulate the cells inside distinct alginate/gelatin
microgels, which allow for intercellular chemical communication. We
observed that the coculture of crypt cells alongside Peyer’s
patch immune cells improves the growth of healthy organoids inside
these microgels, which contain both differentiated and undifferentiated
cells over 21 days of coculture. These results indicate the possibility
of using droplet-based microfluidics for culturing organoids to expand
their applicability in clinical research
Microfluidic-Based Cell-Embedded Microgels Using Nonfluorinated Oil as a Model for the Gastrointestinal Niche
Microfluidic-based
cell encapsulation has promising potential in therapeutic applications.
It also provides a unique approach for studying cellular dynamics
and interactions, though this concept has not yet been fully explored.
No in vitro model currently exists that allows us to study the interaction
between crypt cells and Peyer’s patch immune cells because
of the difficulty in recreating, with sufficient control, the two
different microenvironments in the intestine in which these cell types
belong. However, we demonstrate that a microfluidic technique is able
to provide such precise control and that these cells can proliferate
inside microgels. Current microfluidic-based cell microencapsulation
techniques primarily use fluorinated oils. Herein, we study the feasibility
and biocompatibility of different nonfluorinated oils for application
in gastrointestinal cell encapsulation and further introduce a model
for studying intercellular chemical interactions with this approach.
Our results demonstrate that cell viability is more affected by the
solidification and purification processes that occur after droplet
formation rather than the oil type used for the carrier phase. Specifically,
a shorter polymer cross-linking time and consequently lower cell exposure
to the harsh environment (e.g., acidic pH) results in a high cell
viability of over 90% within the protected microgels. Using nonfluorinated
oils, we propose a model system demonstrating the interplay between
crypt and Peyer’s patch cells using this microfluidic approach
to separately encapsulate the cells inside distinct alginate/gelatin
microgels, which allow for intercellular chemical communication. We
observed that the coculture of crypt cells alongside Peyer’s
patch immune cells improves the growth of healthy organoids inside
these microgels, which contain both differentiated and undifferentiated
cells over 21 days of coculture. These results indicate the possibility
of using droplet-based microfluidics for culturing organoids to expand
their applicability in clinical research
Microfluidic-Based Cell-Embedded Microgels Using Nonfluorinated Oil as a Model for the Gastrointestinal Niche
Microfluidic-based
cell encapsulation has promising potential in therapeutic applications.
It also provides a unique approach for studying cellular dynamics
and interactions, though this concept has not yet been fully explored.
No in vitro model currently exists that allows us to study the interaction
between crypt cells and Peyer’s patch immune cells because
of the difficulty in recreating, with sufficient control, the two
different microenvironments in the intestine in which these cell types
belong. However, we demonstrate that a microfluidic technique is able
to provide such precise control and that these cells can proliferate
inside microgels. Current microfluidic-based cell microencapsulation
techniques primarily use fluorinated oils. Herein, we study the feasibility
and biocompatibility of different nonfluorinated oils for application
in gastrointestinal cell encapsulation and further introduce a model
for studying intercellular chemical interactions with this approach.
Our results demonstrate that cell viability is more affected by the
solidification and purification processes that occur after droplet
formation rather than the oil type used for the carrier phase. Specifically,
a shorter polymer cross-linking time and consequently lower cell exposure
to the harsh environment (e.g., acidic pH) results in a high cell
viability of over 90% within the protected microgels. Using nonfluorinated
oils, we propose a model system demonstrating the interplay between
crypt and Peyer’s patch cells using this microfluidic approach
to separately encapsulate the cells inside distinct alginate/gelatin
microgels, which allow for intercellular chemical communication. We
observed that the coculture of crypt cells alongside Peyer’s
patch immune cells improves the growth of healthy organoids inside
these microgels, which contain both differentiated and undifferentiated
cells over 21 days of coculture. These results indicate the possibility
of using droplet-based microfluidics for culturing organoids to expand
their applicability in clinical research
Microfluidic-Based Cell-Embedded Microgels Using Nonfluorinated Oil as a Model for the Gastrointestinal Niche
Microfluidic-based
cell encapsulation has promising potential in therapeutic applications.
It also provides a unique approach for studying cellular dynamics
and interactions, though this concept has not yet been fully explored.
No in vitro model currently exists that allows us to study the interaction
between crypt cells and Peyer’s patch immune cells because
of the difficulty in recreating, with sufficient control, the two
different microenvironments in the intestine in which these cell types
belong. However, we demonstrate that a microfluidic technique is able
to provide such precise control and that these cells can proliferate
inside microgels. Current microfluidic-based cell microencapsulation
techniques primarily use fluorinated oils. Herein, we study the feasibility
and biocompatibility of different nonfluorinated oils for application
in gastrointestinal cell encapsulation and further introduce a model
for studying intercellular chemical interactions with this approach.
Our results demonstrate that cell viability is more affected by the
solidification and purification processes that occur after droplet
formation rather than the oil type used for the carrier phase. Specifically,
a shorter polymer cross-linking time and consequently lower cell exposure
to the harsh environment (e.g., acidic pH) results in a high cell
viability of over 90% within the protected microgels. Using nonfluorinated
oils, we propose a model system demonstrating the interplay between
crypt and Peyer’s patch cells using this microfluidic approach
to separately encapsulate the cells inside distinct alginate/gelatin
microgels, which allow for intercellular chemical communication. We
observed that the coculture of crypt cells alongside Peyer’s
patch immune cells improves the growth of healthy organoids inside
these microgels, which contain both differentiated and undifferentiated
cells over 21 days of coculture. These results indicate the possibility
of using droplet-based microfluidics for culturing organoids to expand
their applicability in clinical research