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. 2019 Nov;116(11):3084-3097.
doi: 10.1002/bit.27119. Epub 2019 Aug 1.

Fluid shear stress stimulates breast cancer cells to display invasive and chemoresistant phenotypes while upregulating PLAU in a 3D bioreactor

Caymen M Novak  1 , Eric N Horst  1   2 , Charles C Taylor  1 , Catherine Z Liu  1 , Geeta Mehta  1   2   3
Affiliations

Fluid shear stress stimulates breast cancer cells to display invasive and chemoresistant phenotypes while upregulating PLAU in a 3D bioreactor

Caymen M Novak et al. Biotechnol Bioeng. 2019 Nov.

Abstract

Breast cancer cells experience a range of shear stresses in the tumor microenvironment (TME). However most current in vitro three-dimensional (3D) models fail to systematically probe the effects of this biophysical stimuli on cancer cell metastasis, proliferation, and chemoresistance. To investigate the roles of shear stress within the mammary and lung pleural effusion TME, a bioreactor capable of applying shear stress to cells within a 3D extracellular matrix was designed and characterized. Breast cancer cells were encapsulated within an interpenetrating network hydrogel and subjected to shear stress of 5.4 dynes cm-2 for 72 hr. Finite element modeling assessed shear stress profiles within the bioreactor. Cells exposed to shear stress had significantly higher cellular area and significantly lower circularity, indicating a motile phenotype. Stimulated cells were more proliferative than static controls and showed higher rates of chemoresistance to the anti-neoplastic drug paclitaxel. Fluid shear stress-induced significant upregulation of the PLAU gene and elevated urokinase activity was confirmed through zymography and activity assay. Overall, these results indicate that pulsatile shear stress promotes breast cancer cell proliferation, invasive potential, chemoresistance, and PLAU signaling.

Keywords: 3D bioreactor; PLAU; breast cancer; interpenetrating hydrogel; mechanotransduction; shear stress.

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Conflict of interest statement

Conflicts of Interest

The authors have no conflicts to declare.

Figures

Figure 1.
Figure 1.. Schematic of the 3D shear stress bioreactor.
A) Layout of the shear bioreactor. Cell culture medium is contained in IV bag, pumped via peristaltic pump into the bottom of the reactor, and recirculated into the cell culture medium reservoir, as indicated by the directional arrows in the schematic. B) Photo of the disassembled (I.) and assembled (II.) bioreactor. I.) Flow plate showing radial flow chambers connecting the inlet of media flow to the cell-laden hydrogel stacks. II.) Constructed bioreactor, composed of flow plate with PDMS seals and steal endplates bolted together. C) Schematic of interpenetrating (IPN) hydrogel stack composition and dimensions. Each stack was connected via a radial flow channel to both the inlet and outlet chamber. Hydrogel stack consisted of PE plug, PMMA fluid distribution net, PE plug sandwich surrounding cell laden hydrogel to both hold the hydrogel in place and evenly distribute fluid flow.
Figure 2.
Figure 2.. Material characterization of interpenetrating (IPN) hydrogel stack components.
A) SEM images of collagen/agarose IPN hydrogels in order of increasing magnification, where a reticulated network of collagen fibers was observed within a global agarose construct. B) Graphical representation of a frequency sweep from rheometric testing of collagen/agarose IPN hydrogels. Four trials were utilized to determine viscoelastic modulus of the IPN hydrogel, averaging 10358 ± 81.56 Pa. C) Table of material properties for each component of the hydrogel stack. Values were determined by experimental measure through SEM analysis, ImageJ quantification, hydrostatic water column flow rate, and mercury porosimetry.
Figure 3:
Figure 3:. Finite element analysis of the 3D shear bioreactor quantifies shear stress.
A) Open face bioreactor with dotted line denoting modeled section B) 3D COMSOL schematic of modeled section. IPN hydrogel highlighted in blue. C) Flow velocity of modeled section in m s−1. Velocity ranges from 0–0.3 m s−1 along surface of y-z plane. D) Flow velocity in x-z plane of midline cross section of hydrogel in mm s−1 E) Secondary model of shear stress field experienced by a single cell within the IPN hydrogel. Average fluid velocity within the hydrogel, determined from primary model (3C) was applied over an idealized spherical cell and resulting shear stress on the surface was determined. F) Resulting shear stress around perimeter of cell within the flow field demonstrated in Figure 3E. Shear stress experienced by the cell is reported as the maximum value of 5.41 dyne cm−2.
Figure 4:
Figure 4:. Shear stress increases area and decreases roundness in breast cancer cells.
A) Representative H&E images of shear and control studies for each cell type. Noticeable cellular elongation and increased cellular area are seen in all shear samples. All scale bars are 50 μm. B) Graph of cellular area quantification comparing control and sheared cells. Shear stress exposure shows significant increase in cellular area for all cell types (One-way ANOVA ****p< 0.0001, n≥3). C) Quantification of cell roundness comparing control and sheared cells. Shear stress exposure shows significant decrease in cell roundness for all cell types (One-way ANOVA ****p< 0.0001, n≥3).
Figure 5:
Figure 5:. Shear stress increases breast cancer proliferation.
A) Representative images of Ki67 IHC counterstained with hematoxylin. Deep brown coloring indicates a proliferating cell and was quantified manually. Counts were normalized to total cell count within each image. Scale bars are 200 μm in the main images and 20 μm in the corner frames for MCF7 cells. B) Graphical representation of the fraction of proliferating cells for each cell type under control and shear conditions (****p< 0.0001, n≥3). All cell types show significantly increased proliferation tendency under shear stress stimulus.
Figure 6:
Figure 6:. Shear stress induced chemoresistance to paclitaxel treatment.
A) Representative images of caspase 3 IHC staining for MDA-MB-231 cells. Arrows indicate examples of cells positively expressing caspase 3. B) Cells were treated with paclitaxel chemotherapy (25 μM) in either control or shear conditions and cellular death was quantified using caspase 3 IHC staining, counterstained with hematoxylin. Results were manually quantified and compared to total cell number within the image. The fraction of proliferating cells was then normalized to the non-stimulated control average of the respective cell type. All cell types showed significantly decreased death under sheared conditions (*p< 0.1, n≥3). Three or more images were quantified for each experiment and a minimum of three biological replicates were performed for each condition. C) Representative images of Ki67 IHC staining for MDA-MB-468 cells. Arrows indicate examples of cells positively expressing Ki67. D) Cellular proliferation was analyzed using Ki67 IHC staining on paclitaxel treated experiments. Results were manually quantified, and each cell type showed significantly enhanced proliferation in sheared samples (**p< 0.01, n≥3).
Figure 7:
Figure 7:. Activity assay and zymography confirm enhanced urokinase activity under shear stimulation.
A) qPCR analysis on PLAU expression showed significant upregulation for MDA-MB-231 (*p< 0.1, n≥3) and MDA-MB-468 (**p< 0.01, n≥3). MCF7 cells showed an increase in PLAU expression but was not significant (n≥3). B) Confirmation of PLAU upregulation was performed using a urokinase activity assay on MDA-MB-468 experimental medium. Results showed a significant increase in enzymatic activity for shear samples normalized to controls. C) Additional confirmation of urokinase protein activity was performed via plasminogen zymography. Band intensity for three MDA-MB-468 experimental sets is sown (32 kDa low molecular weight form uPA). D) Quantification of the zymography band intensity was normalized to their respective controls and statistically analyzed with a two tailed t-test. MDA-MB-468 sheared samples showed a significant increase in band brightness, indicating increased urokinase enzyme activity.
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