Graduation Semester and Year

2018

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Biomedical Engineering

Department

Bioengineering

First Advisor

Cheng-Jen Chuong

Abstract

Glioblastoma multiforme (GBM) is the most common and malignant form of primary brain tumor. One of the major challenges facing the treatment of GBM tumors is the detachment and subsequent migration of peripheral cells. Migration of GBM cells take place in mechanically distinct confined microenvironment along the borders of white matter tracts. Studies using microfluidic devices mimicking the in-vivo microenvironment have shown that the migration characteristics are significantly altered with the change in kinematic state of the migrating cell. Several studies on different types of cells have consistently shown that a cancerous cell is more compliant than a normal cell in open 2D environment. However, in the in-vivo microenvironment the migrating pathway of GBM cells are confined and the effect of kinematic states on the compliance of migrating cells are yet to be understood. In this work, we examined the differences in mechanical property of GBM cells when in two different kinematic states. Using a microfluidic platform, we studied the creep response of GBM cells in response to the sudden application of negative pressure (-20, -25, -30, -35, and -40 cm H2O). Cells studied are either actively migrating in a confined channel of 5 x 5 μm cross section or in a stationary state located at the entrance region to the confined channel from an open surface. Our results showed that, in response to the aspiration pressure load, GBM cells in actively migrating state exhibited higher stiffness than those in stationary state. Through the deformation process, cells in migrating state absorbed more energy elastically with relatively small dissipative energy loss compared with those in the stationary state. At elevated negative pressure loads, there was a linear increase in elastic stiffness and higher distribution in elastic storage than energy loss up to - 30 cm H2O. For further increase in negative pressure load, the response appeared to reach a plateau. To explore the underlying cause, we carried out immuno-histochemical studies of these cells immediately after creep study. Results showed polarized distribution in actin and myosin at both the front and posterior end of the cells in migrating state, whereas data from cells in stationary state show the normalized intensity oscillate around unity without specific regional differences. Cell adhesion plays a major role in migration of a cell and alteration of adhesion mechanism can significantly affect the dynamics of migration of a cell. Studies quantifying adhesion forces have developed our understanding of the migration process on 2D substrate. However, effect of geometric confinement on the adhesion strength between the cell and the extracellular matrix (ECM) have not been investigated. In this study, we quantified the adhesion strength of GBM cells migrating in microchannel of 5 x 5 μm cross section. Using a microfluidic platform, we applied negative pressure to detach migrating GBM cells from its surrounding extracellular matrix. Our results demonstrate detachment force of 685.78±68.65 nN for a total of eight individual experiments. Comparing our results with previous studies we have identified that although a migrating cell prefers adhesion-independent migration in 3D conditions, the overall attachment force is this state is comparable with migration on 2D substrate. Analysis of relative deformation of the cell prior to detachment also revealed that most of the resistance to applied negative pressure is generated by the nuclear region and the posterior end of the cell. Our study underscores the importance of transverse forces applied by the cell on the surrounding ECM for generating traction force during adhesion-independent migration mechanism. During adhesion independent migration, cells have demonstrated that the propelling force is generated partly by hydrostatic pressure. The rise of hydrostatic pressure is signified by the local detachment of plasma membrane from the actin cortex. The detachment of plasma membrane results in spherical protrusions known as blebs. Although it has been suggested in previous studies that the source of hydrostatic pressure is actomyosin contraction in the actin cortex, the mechanism of actomyosin contraction in the intracellular level have not been identified. In this study, we developed a numerical model of a migrating cell in confinement and applied the understanding of actomyosin contraction in muscle tissues in the model. Our study finds, that using the mechanism of muscle contraction, the migrating cell generates intracellular pressure of approximately 3 Pa which is sufficient to generate bleb protrusion comparable to experimentally observed cellular blebs. The findings of this study help broaden our understanding of the changes in mechanical property and adhesion mechanism the GBM cell undergoes while migrating through a confined space in the brain.

Keywords

Glioblastoma, Cell migration, Creep response, Adhesion

Disciplines

Biomedical Engineering and Bioengineering | Engineering

Comments

Degree granted by The University of Texas at Arlington

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