Development of a Microfluidic Platform for Size- Based Enrichment and Immunomagnetic Isolation of Circulating Tumour Cells by Hadi Esmaeilsabzali M.A.Sc., Electrical Engineering, Iran University of Science and Technology, 2006 B. Eng., Biomedical Engineering, Shahid Beheshti University of Medical Sciences, 2003 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the School of Mechatronic Systems Engineering Faculty of Applied Sciences © Hadi Esmaeilsabzali SIMON FRASER UNIVERSITY Summer 2017 Copyright in this work rests with the author. Please ensure that any reproduction or re-use is done in accordance with the relevant national copyright legislation. Approval Name: Hadi Esmaeilsabzali Degree: Doctor of Philosophy Title: Development of a Microfluidic Platform for Size- Based Enrichment and Immunomagnetic Isolation of Circulating Tumour Cells Examining Committee: Chair: Dr. Behraad Bahreyni Associate Professor Dr. Edward J. Park Senior Supervisor Professor Dr. Timothy V. Beischlag Senior Co-Supervisor Professor Dr. Ash Parameswaran Supervisor Professor Dr. Bonnie L. Gray Internal Examiner Professor School of Engineering Science Dr. Yu Sun External Examiner Professor Department of Mechanical & Industrial Engineering University of Toronto Date Defended/Approved: August 16, 2017 ii Ethics Statement iii Abstract Cancer is a leading cause of death worldwide. Efforts to improve the longevity and quality of life of cancer patients are hindered by delays in diagnosis of tumours and treatment deficiency, as well as inaccurate prognosis that leads to unnecessary or inefficient treatments. More accurate biomarkers may address these issues and could facilitate the selection of effective treatment courses and development of new therapeutic regimens. Circulating tumour cells (CTCs), which are cancer cells that are shed from tumours and enter the vasculature, hold such a promise. Therefore, there is much interest in the isolation of CTCs from the blood. However, this is not a trivial task given the extreme scarcity of CTCs in the circulation. In this thesis, the development of a microfluidic immunomagnetic approach for isolation of CTCs is presented. First, the design, microfabrication, and experimental evaluation of a novel integrated microfluidic magnetic chip for sensitive and selective isolation of immunomagnetically labelled cancer cells from blood samples is reported. In general, to ensure the efficient immunomagnetic labelling of target cancer cells in a blood sample, an excessive number of magnetic beads should be added to the sample. When an immunomagnetically labelled sample is processed through the chip, not only cancer cells but also free magnetic beads that are not bonded to any target cells would be captured. The accumulation of these beads could disrupt the capture and visual detection of target cells. This is an inherent drawback associated with immunomagnetic cell separation systems and has rarely been addressed in the past. Therefore, the design, microfabrication, and characterization of a microfluidic filter for continuous size-based removal of free magnetic beads from immunomagnetically labelled blood samples is presented next. Connected in tandem, the two chips developed in this work form a microfluidic platform for size-based enrichment and immunomagnetic isolation of CTCs. Preclinical studies showed that the proposed approach can capture up to 75% of blood- borne prostate cancer cells at clinically-relevant low concentrations (as low as 5 cells/mL) at an acceptable throughput (200 μL/min). The retrieval and successful propagation of captured prostate cancer cells is also investigated and discussed in this thesis. Keywords: BioMEMS; Cancer; Circulating Tumour Cells; Immunomagnetic Cell Separation; Single-Cell Research; Microfluidics iv Dedication To My Parents, Esmat and Karim v Acknowledgements I am fortunate to have had Dr. Edward Park as my senior supervisor during my PhD studies. Without his broad interdisciplinary vision, continuous guidance, and capacity to form fruitful collaborations with other researchers, this work would have not been possible. I thank him for giving me the freedom to pursue my research ideas while providing me with constructive directions. I appreciate his encouragement, patience, and support throughout these years. Dr. Timothy Beischlag has been significantly influential in my PhD research. Working with him helped me in defining the scope of my research and learning the skills and concepts that I would not have known otherwise. He taught me scientific writing and the significance of self-criticism and perseverance in research. I am grateful to him for all the lessons. I am thankful to Dr. Ash Parameswaran for his original ideas and willingness to advise on microfabrication issues whenever I needed his valuable expertise. I would like to thank Dr. Nikolai Dechev for his guidance and support during the early stages of my PhD studies. I am also appreciative to Dr. Frank Lee, Dr. Gratien Prefontaine, and the Faculty of Health Sciences for their support and hospitality. I have been lucky to work with several amazing lab-mates over the past few years. Their accompany made this occasionally bumpy PhD journey enjoyable, and it would not be an overstatement to say I learned something new from each and every of them. Thank you, Kelly Sakaki, Shabnam Massah, Samaneh Khakshour, Jung Keun Lee, Mark Labrecque, Rebecca Moncur, Shazina Khan, Beryl Luk, Mandeep Takhar, Julienne Jagdeo, and Kevin Tam, for your friendship and fun memories. I cannot begin to express how grateful I am to my parents, Esmat and Karim, and my brothers, Bahram, Shahram, and Kourosh, for their love and support. Hearing their voice was all the motivation I needed to firmly get back to work when I was having a frustrating day in the lab. vi Table of Contents Approval .......................................................................................................................... ii Ethics Statement ............................................................................................................ iii Abstract .......................................................................................................................... iv Dedication ....................................................................................................................... v Acknowledgements ........................................................................................................ vi Table of Contents .......................................................................................................... vii List of Tables ................................................................................................................... x List of Figures................................................................................................................. xi List of Acronyms ............................................................................................................ xiii Introductory Image ........................................................................................................ xv Chapter 1. Introduction .............................................................................................. 1 1.1. Background ........................................................................................................... 1 1.2. Thesis Outline ....................................................................................................... 2 1.3. Motivation .............................................................................................................. 2 1.4. Literature Review ................................................................................................... 6 1.4.1. Carcinogenesis, Metastasis, and Circulating Tumour Cells............................ 6 1.4.2. CTC Detection and Isolation: Principles and Methods ................................. 10 Nucleic acid-based methods for CTC detection ...................................................... 11 Physical properties-based methods for CTC isolation ............................................ 13 Antibody-based methods for CTC detection and isolation ...................................... 19 1.5. Contributions ....................................................................................................... 31 Chapter 2. An Integrated Microfluidic Magnetic Chip for Immunomagnetic Isolation of Cancer Cells ................................................................................... 34 2.1. Microfluidic Immunomagnetic Cell Isolation ......................................................... 34 2.1.1. Background ................................................................................................. 34 2.2. Theory ................................................................................................................. 35 2.3. Materials and Methods ........................................................................................ 38 2.3.1. PSMA and Prostate Cancer ......................................................................... 38 2.3.2. Immunomagnetic Labelling .......................................................................... 41 Indirect Labelling Using Tetrameric Antibody Complex (TAC) Technology ............. 42 Direct Labelling Using Anti-mouse IgG Coated Magnetic Beads ............................ 42 Indirect Labelling Using Streptavidin-Biotin Interaction ..........................................