Exploration of Dynamic Elastic Modulus Changes on Glioblastoma Cell Populations with Aberrant EGFR Expression as a Potential Therapeutic Intervention Using a Tunable Hyaluronic Acid Hydrogel Platform
"> Figure 1
<p>Tuning of hydrogel formulations by cross-linker manipulation. The three elastic moduli (100, 1000 and 2000 Pa) employed in S1, S2, and S3 hydrogels are obtained by modulating ratios of linear versus four-arm PEG-based cross-linkers. The elastic modulus corresponding to an environment less than normal brain elastic modulus (S1), normal brain elastic modulus (S2), and the brain tumor microenvironment (S3). Statistical significance: * <span class="html-italic">p</span> < 0.01 between hydrogel groups.</p> "> Figure 2
<p>Cell proliferation rates as an effect of elastic moduli. U373 cells (<b>A</b>), A172 cells (<b>B</b>), U87 (<b>C</b>), and U87 EGFRvIII cells (<b>D</b>). * denotes <span class="html-italic">p</span> value is less than 0.05 when S1 and S3 values were compared. & denotes <span class="html-italic">p</span> value is less than 0.05 when values of S2 and S3 were compared and # denotes when <span class="html-italic">p</span> value is less than 0.05 when S1 and S2 values where compared.</p> "> Figure 3
<p>Focal adhesion kinase (FAK) expression increases with elastic modulus. (<b>A</b>) Immunofluorescent staining for phosphorylated FAK in U373 cells, A172 cells and U87EGFRvIII cells at S1, S2, and S3. Scale bars—100 μm; (<b>B</b>) Increased resolution imaging of FAK expression at S3. Scale bars—50 μm. (<b>C</b>–<b>F</b>) Proliferation of glioblastoma (GBM) cell types on varying elastic moduli under inhibition of FAK phosphorylation with defactinib: U373 cells (<b>C</b>), A172 cells (<b>D</b>), U87 (<b>E</b>) and U87 EGFRvIII cells (<b>F</b>). * denotes <span class="html-italic">p</span>-value was less than 0.05 when S1 and S3 values were compared, & denotes <span class="html-italic">p</span>-value was less than 0.05 when values of S2 and S3 were compared, and # denotes when the <span class="html-italic">p</span>-value was less than 0.05 when S1 and S2 values were compared.</p> "> Figure 4
<p>Proliferation of GBM cell types on varying elastic moduli under inhibition of epidermal growth factor receptor (EGFR) inhibitors: U373 cells with erlotinib (<b>A</b>), A172 cells with dacomitinib (<b>B</b>), U87 with erlotinib (<b>C</b>) and U87 EGFRvIII cells with dacomitinib (<b>D</b>). * denotes <span class="html-italic">p</span>-value was less than 0.05 when S1 and S3 values were compared, & denotes <span class="html-italic">p</span>-value was less than 0.05 when values of S2 and S3 were compared, and # denotes when <span class="html-italic">p</span>-value was less than 0.05 when S1 and S2 values were compared.</p> "> Figure 5
<p>(<b>A</b>) The integration of a double sulfide bond in the cross-linker, which can then be broken on demand and thereby decrease the elastic modulus of the hydrogels on demand; (<b>B</b>) The elastic modulus reduction experiment modified to suit in vivo conditions, in which hydrogels without the breakable cross-linker are partially digested using collagenase/hyaluronidase to decrease the elastic modulus; (<b>C</b>,<b>D</b>) Rheological data demonstrating NAC treatment reduction PEGSSDA cross-linked hydrogels and collagenase/hyaluronidase treatment reduction of S3 hydrogels, respectively. Statistical significance: * <span class="html-italic">p</span> < 0.01.</p> "> Figure 6
<p>Targeting elastic modulus by using <span class="html-italic">N</span>-Acetyl-<span class="html-small-caps">l</span>-Cysteine to break disulfide bonds in PEGSSDA cross-linked hydrogels, and resulting proliferation rates: (<b>A</b>) U373 cells; (<b>B</b>) A172 cells; (<b>C</b>) U87 cells and (<b>D</b>) U87 EGFRvIII cells. Targeting elastic modulus with collagenase/hyaluronidase to degrade the hyaluronic acid (HA) and gelatin components in S3 hydrogels: (<b>E</b>) U373 cells; (<b>F</b>) A172 cells; (<b>G</b>) U87 cells and (<b>H</b>) U87 EGFR viii cells. * Denotes <span class="html-italic">p</span> value is less than 0.05.</p> "> Figure 7
<p>Biocompatibility assays of cells with <span class="html-italic">N</span>-Acetyl-<span class="html-small-caps">l</span>-cysteine (<b>A</b>) U373 cells; (<b>B</b>) A172 cells; (<b>C</b>) U87 cells and (<b>D</b>) U87 EGFRvIII cells. Biocompatibility assays of cells with collagenase/hyaluronidase; (<b>E</b>) U373 cells; (<b>F</b>) A172 cells; (<b>G</b>) U87 cells and (<b>H</b>) U87 EGFRvIII cells. Other than the case of U373 cells under collagenase treatment (* <span class="html-italic">p</span> < 0.05), control and treated conditions are not significantly different, indicating that the NAC and collagenase/hyaluronidase treatments do not contribute to a reduction in cell proliferation in the <b><span class="html-italic">E’</span></b> reduction studies.</p> ">
Abstract
:1. Introduction
2. Results and Discussion
2.1. Elastic Modulus-Dependent Proliferation Assays
2.2. The Role of FAK in Elastic Modulus-Dependent Cell Proliferation
2.3. The Effect of EGFR Inhibition on Elastic Modulus-Dependent Cell Proliferation
2.4. Dynamic Elastic Modulus Reduction as a Therapeutic Application
3. Conclusions
4. Materials and Methods
4.1. Cells Employed in Elastic Modulus Experiments
4.2. Hydrogels
4.3. Proliferation Assays
4.4. Immunofluorescence Staining for FAK Phosphorylation
4.5. FAK and EGFR Inhibition
4.7. Hydrogel Reduction Through a Breakable Cross-linker
4.8. Hydrogel Reduction through Enzymatic Degradation
4.9. NAC and Collagenase/Hyaluronidase Biocompatibility Verification
4.10. Statistical Analysis
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Stiffness Sensitivity Summary | |||
---|---|---|---|
Cell Line | Proliferation Rate Highest in Normal Condition | Proliferation Rate Highest in Defactinib | Proliferation Rate Highest in EGFR Inhibition |
U373-MG | S3 *,& | S1 * | No distinct preference |
A172 | S2 & and S3 * (DAY 4) | S1 * | S1 * and S2 & |
U87-MG | S2 & | S2 | No distinct preference |
U87-EGFRvIII | S3 | S1 | S1 |
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Sivakumar, H.; Strowd, R.; Skardal, A. Exploration of Dynamic Elastic Modulus Changes on Glioblastoma Cell Populations with Aberrant EGFR Expression as a Potential Therapeutic Intervention Using a Tunable Hyaluronic Acid Hydrogel Platform. Gels 2017, 3, 28. https://doi.org/10.3390/gels3030028
Sivakumar H, Strowd R, Skardal A. Exploration of Dynamic Elastic Modulus Changes on Glioblastoma Cell Populations with Aberrant EGFR Expression as a Potential Therapeutic Intervention Using a Tunable Hyaluronic Acid Hydrogel Platform. Gels. 2017; 3(3):28. https://doi.org/10.3390/gels3030028
Chicago/Turabian StyleSivakumar, Hemamylammal, Roy Strowd, and Aleksander Skardal. 2017. "Exploration of Dynamic Elastic Modulus Changes on Glioblastoma Cell Populations with Aberrant EGFR Expression as a Potential Therapeutic Intervention Using a Tunable Hyaluronic Acid Hydrogel Platform" Gels 3, no. 3: 28. https://doi.org/10.3390/gels3030028
APA StyleSivakumar, H., Strowd, R., & Skardal, A. (2017). Exploration of Dynamic Elastic Modulus Changes on Glioblastoma Cell Populations with Aberrant EGFR Expression as a Potential Therapeutic Intervention Using a Tunable Hyaluronic Acid Hydrogel Platform. Gels, 3(3), 28. https://doi.org/10.3390/gels3030028