Glioblastoma multiforme (GBM) is an aggressive, Grade IV astrocytoma with a poor survival rate, primarily due to the GBM tumor cells migrating away from the primary tumor site along the nanotopography of white matter tracts and blood vessels. It is unclear whether this nanotopography influences the biomechanical properties (i.e. cytoskeletal stiffness) of GBM tumor cells. Although GBM tumor cells have an innate propensity to migrate, we believe this capability is enhanced due to the influence of nanotopography on the tumor cells’ biomechanical properties. In this study, we used an aligned nanofiber film that mimics the nanotopography in the tumor microenvironment to investigate the mechanical properties of GBM tumor cells in vitro. The data demonstrate that the cytoskeletal stiffness, cell traction stress, and focal adhesion area were significantly lower in the GBM tumor cells compared to healthy astrocytes. Moreover, the cytoskeletal stiffness was significantly reduced when cultured on aligned nanofiber films compared to smooth and randomly aligned nanofiber films. Gene expression analysis showed that tumor cells cultured on the aligned nanotopography upregulated key migratory genes and downregulated key proliferative genes. Therefore, our data suggest that the migratory potential is elevated when GBM tumor cells are migrating along aligned nanotopographical substrates.
Sewing cuffs incorporated within tissue-engineered blood vessels (TEBVs) enable graft anastomosis in vivo, and secure TEBVs to bioreactors in vitro. Alternative approaches to cuff design are required to achieve cuff integration with scaffold-free TEBVs during tissue maturation. To create porous materials that promote tissue integration, we used electrospinning to fabricate cuffs from polycaprolactone (PCL), PCL blended with gelatin, and PCL coated with gelatin, and evaluated cuff mechanical properties, porosity, and cellular attachment and infiltration. Gelatin blending significantly decreased cuff ultimate tensile stress and failure strain over PCL alone, but no significant differences were observed in elastic modulus or failure load. Interestingly, gelatin incorporation by blending or coating did not produce significant differences in cellular attachment or pore size. We then created tissue tubes by fusing self-assembled smooth muscle cell rings together with electrospun cuffs on either end. After 7 days, rings and cuffs fused seamlessly, and the resulting tubes were harvested for pull-to-failure tests to measure the strength of cuff-tissue integration. Tubes with gelatin-coated PCL cuffs failed more frequently at the cuff-tissue interface compared to PCL and PCL:gelatin blended groups. This work demonstrates that electrospun cuffs integrated successfully with scaffold-free TEBVs, and that the addition of gelatin did not significantly improve cuff integration over PCL alone for this application. Electrospun cuffs may aid cannulation for dynamic culture and testing of tubular constructs during engineered tissue maturation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 817-826, 2018.
Glioblastoma multiforme (GBM) is an aggressive and invasive brain tumor primarily found in adults. Survival outcomes after standard clinical treatment of GBM are poor due to the sub-population of GBMs, the cancer initiating cells (CICs). CICs are highly invasive and resistant to multimodal therapeutic treatment strategies. It has been shown that microRNAs (miRs) that modulate apoptotic signaling pathways are dysregulated in GBMs, particularly miRs 34a and 21. Our study investigated the therapeutic potential of modulating levels of the pro-apoptotic miR34a and anti-apoptotic miR21 in the CICs. To accomplish this, we complexed both miR34a and the anti-sense oligonucleotide inhibitor for miR21 (ASO21) to targeted cationic liposomes, as these can be systemically administered in vivo and extravasate through the leaky vasculature of the blood-brain barrier to reach the tumor. The isolated CICs overexpress epidermal growth factor receptor (EGFR). An EGFR peptide was custom synthesized that has a high binding affinity to the receptor and was used as the targeting ligand on the cationic liposomes. We analyzed the effect of miR34a and/or ASO21 on the CIC viability, senescence, and invasion. Our results demonstrate that after treatment with miR34a, ASO21, and combination of the two, the tumor viability decreased to 65%, 70%, and 45%, respectively as compared to the media control (100%). In addition, using β-galactosidase as an indicator for senescent cells, it was observed that the number of cells that were senescent increased. For the miR34a, ASO21, and the combination of the two, 40%, 23%, and 70%, respectively, of the cells were senescent, compared to the 4% of untreated cancer initiating cells. The number of CIC spheres formed was also significantly reduced, as cancer initiating cells treated with the combination had an average of 1125 spheres formed versus the media (untreated) of 2860 spheres formed, suggesting a decreased potential to form bulk tumors. Preliminary protein and gene profiling has demonstrated fold changes in pathway and migration markers Stat3, Reck, and Notch, as well as apoptosis markers Bax, PDCD4, and Caspase 3 for the treatment conditions compared to the medial control. Furthermore, our data demonstrates that the enhanced effect of the miR34a and ASO21 combination treatment had a synergistic effect compared to the individual treatments rather than additive. Therefore, this study demonstrates that modulating miR34a and 21 is a potential therapeutic strategy to induce apoptosis and senescence, and decrease migration in the difficult to treat GBM cancer initiating cells. Citation Format: Kimberly Ornell, Yuan Yin, Alexander Alexander Beliveau, Anjana Jain. Synergistic modulation of microRNAs for treatment of glioblastoma cancer initiating cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3124. doi:10.1158/1538-7445.AM2015-3124
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