Date of Completion

5-4-2017

Embargo Period

5-2-2027

Keywords

cancer, spheroid, nanoparticles, stiffness analysis, microcantilever, single cell analysis, immunomagnetic capture

Major Advisor

Dr. Kazunori Hoshino

Associate Advisor

Dr. Mu-Ping Nieh

Associate Advisor

Dr. Guoan Zheng

Field of Study

Biomedical Engineering

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

The main purpose of this dissertation is to conduct mechanical characterization of in vitro 3D tumor models and study diffusion with respect to Young’s modulus of tumors.

According to American Institute of Cancer Research, cancer causes approximately 7.6 million deaths worldwide per year. The goals of cancer research include early detection as well as better drug delivery to reduce cancer mortality rate. For decades, researchers have developed cancer models in laboratory to study cancer biology and drug delivery. Most commonly used models for research are monolayer cultures, cells grown on a flat petri dish which fails to mimic the physiological conditions, and in vivo models, which are time-consuming and more expensive. Since last decade, growing interest has been on 3D multicellular models known as spheroids. Spheroids closely mimic tumor characteristics found in natural physiological conditions such as effect of gradient nutrient supply, necrotic core, extracellular buildup and hypoxia. Various drug studies have been associated with the above factors which may affect penetration of drugs. These factors can also change the mechanical properties of tumorous tissue compared to normal tissue. Mechanical properties have been considered as an early biomarker to distinguish between cancerous and normal cell. In this dissertation, a novel micro-tweezer device developed for mechanical characterization of spheroids is discussed. The chop-stick like action of the arm facilitated easy sample handling and microscopic observation for mechanical characterization. Cantilever bending was tracked using a custom build optical tracking software. The cantilevers were calibrated and the efficacy of the method was demonstrated by using agarose pillars of known concentration. The method was also evaluated by confirming the agarose Young’s modulus with the established micro-indentation technique. After the initial evaluation, three cancerous (MCF7, T47D and BT474) and one normal epithelial (MCF10A) breast cell lines were used to conduct multi-cellular spheroid measurements to find Young’s moduli of 130, 250, 460 and 1350 Pa for MCF7, BT474, T47D, and MCF10A, respectively. The results showed that MCF7, BT474 and T47D spheroids are ten, six and three times softer than epithelial MCF10A spheroids, respectively. Since micro-cantilevers are replaceable, this method successfully characterized samples with wide range of Young’s modulus including agarose (25-100 kPa), spheroids of cancerous and non-malignant cells (190-200 µm, 250- 1350 Pa), collagenase-treated spheroids (215 µm, 180 Pa) and overgrown spheroids (410µm, day 20, 820 Pa). The device was also built for confocal microscope which enabled localized strain mapping of tumors. Von Mises strain was mapped for section of spheroids showing softer and stiffer regions of spheroids which are not shown in bulk analysis.

Correlation between the factors that affect mechanical properties and the penetration of drugs carrying molecules to the spheroid has been also studied. Diffusion of liposomes (50nm) was measured for day 5 (200 µm), day 20 (420µm) and day 5 (size matched with day 20, 400 µm) spheroids made from BT474 and T474D cell lines. The diffusion study showed ~1.5 times more total fluorescence for day 5 (size matched) spheroids compared to day 20 spheroids for both the cell lines. Similarly, 3 hour collagenase (0.1%) treated spheroids showed 1.4 times more total fluorescence than control spheroids. The diffusion results were related to mechanical characteristics of the spheroids, measured using micro-cantilevers, which showed comparatively more liposome uptake for softer spheroids than stiffer spheroids.

In conclusion, a novel method was developed for mechanical characterization of cancer spheroids and the results were correlated with nanoparticle diffusion. As future application, the devices developed during the course of this dissertation can be applied for studying complex heterogeneous tumors and other non-cancerous multicellular in vitro models such as organoids.

Download

Available for download on Sunday, May 02, 2027

Share

COinS