PhD Oral Exam - Elahe Memari, Physics

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

Immunotherapy has shown great promise for treating hematological malignancies. However, its potential for solid cancer, such as brain cancer, remains limited due to the presence of restrictive blood-brain barrier and suppressive tumor microenvironment. Therefore, developing strategies to improve the efficacy of molecular and cellular immunotherapy is in urgent need. In this thesis, I explore the potential of therapeutic ultrasound under fluid flow conditions to enhance endothelial cell permeabilization and immunobiology for improving immunotherapy. I hypothesized that ultrasound-stimulated microbubbles could enhance CAR NK-92 cell recruitment and extravasation, thus improving the efficacy of cellular immunotherapy. In this thesis, an in vitro setup was established using a custom-designed acoustically coupled inverted microscope to visualize the acoustic focus within the microscope field of view. Given that microbubble flow pattern varies across different vascular regions, understanding the influence of flow dynamics on ultrasound-mediated cell permeabilization provides valuable insights for localized drug delivery. Thus, fluidic systems were employed to cultivate and treat two human endothelial cell types, either umbilical vein (HUVEC) or brain endothelial cells (HBEC-5i), under fluid flow conditions. The findings demonstrated a direct correlation between microbubble flow velocity and ultrasound-assisted cell permeabilization. The velocity of microbubble perfusion also substantially influenced the dynamics of Ca2+ influx in both directly and indirectly perforated cells. Besides flow velocity, the impact of varied flow patterns was investigated on endothelial cell physiology and response to ultrasound treatment. Sustained shear-flow preconditioning for two days influenced endothelial cells morphology and cytokine profile, which significantly enhanced their susceptibility to ultrasound-mediated cell permeabilization. In addition, distinct microbubble flow patterns, either laminar, non-reversing pulsatile or reversing oscillatory flow profile, dramatically influenced the dynamics and efficiency of cell permeabilization. Building on these findings, I first explored the effects of shear flow as a baseline, followed by microbubble-induced shear stress on endothelial cell immunobiology. The results indicated that ultrasound-stimulated microbubbles led to a time-dependent upregulation of cell adhesion molecules involved in immune cell homing. Additionally, ultrasound treatment under identical conditions significantly increased the secretion of 20 cytokines and chemokines, most of which contribute to promoting anti-tumor immunity. At the end, the influence of ultrasound-modulated endothelial cell molecular profile was assessed on CAR NK-92 cell recruitment and trafficking in vitro. The findings illustrated that ultrasound-assisted treatment of brain endothelial cells significantly improved CAR NK-92 cells homing and trafficking, 4 hours post-sonication. Overall, this thesis highlights the potential of ultrasound-activated microbubbles to overcome the challenges of tumor microenvironment, thereby enhancing the efficacy of cellular immunotherapy.