APPLICATION OF IMMUNOMAGNETIC CELL SEPARATION IN CANCER CELL DETECTION: DEVELOPMENT AND OPTIMIZATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Liying Yang, B.S. ***** The Ohio State University 2008 Dissertation Committee: Approved by Prof. Jeffrey J. Chalmers, Adviser Prof. James C. Lang Prof. Jim F. Rathman Adviser Graduate Program in Chemical and Biomolecular Engineering ABSTRACT Detection of rare, circulating tumor cells (CTCs) in peripheral blood is a potential prognostic/diagnostic tool in oncology. The use of immunomagnetic cell separation has been shown to improve the target cell purity and thus detection sensitivity. In this dissertation, a repeatable enrichment process including a flow through immunomagnetic cell separation system, the quadrupole magnetic cell sorter (QMS), was continuously developed and optimized. Molecular analysis technologies such as immunocytochemical assay and the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) were combined with the enrichment process to reliably and accurately detect the presence of CTCs in peripheral blood. The novel technique was applied involving samples from head and neck patients undergoing surgery. Immunochemical staining and RT-PCR analysis of the same, enriched sample result in congruent outcome in all but one cases. Furthermore, the data with respect to the quantitative detection of CTCs is generally consistent with the pathological report on these patients. Data suggested that if a sample had 10 or more CTCs per ml of blood, a metastatic disease was present in the corresponding patient. ii In order to further improve the final purity of cancer cells, to be eligible for studies including cDNA microarray, continuous development and optimization of the novel system was desired. A kinetics model was used to describe the process of ligand binding to cell surface receptors, which demonstrated that the ratio between the initial free antibody concentration and its dissociation constant (L0/KD) is the limiting factor for a given magnetic labeling system. Based on the theory, an optimal labeling scheme is identified, including the use of a tetrameric antibody complex, resulting in significantly better separation performance with order of magnitude higher log depletion. In the second part of the study, a reaction-diffusion model was constructed to describe the in vitro tissue dissociation process. It is not only useful in recognizing the rate-limiting factor in the tissue dissociation process, but also provides quantitative guidelines to establish an optimal tissue dissociation technology. A rapid tissue dissociation process is established and characterization followed by a positive selection of EGFR targeted cancer cells. Evidence presented in this dissertation proofed the concept that it is feasible to isolate pure cancer cells from solid tumor biopsies within 2 hrs by applying the newly established rapid tissue dissociation method followed by a positive selection of cancer cells by immunomagnetic labeling. iii Dedicated to my parents and husband iv ACKNOWLEDGMENTS This work would have not been possible without the guidance, support, and encouragement by my advisor Dr. Chalmers, Jeffrey J. His great personality and understanding of cultural differences have made my staying here much smooth. I would like to thank Dr. Lang, James C. for his valuable insights and tremendous knowledge in the molecular biology area, which makes my research much easier. I also wish to thank Dr. Rathman, Jim, Dr. Yang, ST, and Dr. Palmer, Andre F. for having been on my committee and providing criticisms on my work. I would like to thank Dr. Schuller, David, Dr. Agrawal, Amit, and Dr. Jatana, Kris R. in the Department of Otolaryngology, Head and Neck Surgery for their effort and enthusiasm in this work, which makes the preclinical investigation possible. I would like to thank Dr. Zborowski and Mr. Lee R Moore in the Cleveland Clinic Foundation for their continuous help and comments during the development of work. I would like to thank Dr. Tong, Xiaodong and Dr. Metha, Bhavya for their guidance and help at the time I started this research. They are both mentors and friends to me. I wish to express my appreciation to every group member in Chalmers’s research group, especially Priya Balasubramanian for her contribution, Dr. Zhao, v Yang for his technical support, Ying Xiong, Rustin Shenkman for their suggestions to the completion of this work. I would like to thank Mr. Bryan McElwain at the Ohio State University Analytical Cytometry Laboratory for his help on flow cytometry analysis. Special thanks to those blood donors including Dr. Tong, Xiaodong, Dr. Chalmers, Jeffrey J. and Priya Balasubramanian. Finally, I would like to thank my parents, who love me and have faith in me, which had make me stronger and be able to come to this far. My husband, He Yuesheng, has always been standing by my side during these years. His encouragement and love has been the source of my spiritual power. vi VITA March 22, 1981……………………………….Born – Suzhou, Jiangsu, China July, 2003……………………………………..B.S. Bioengineering, Zhejiang University, China September, 2003-December, 2007……………Graduate Research Associate, Chemical & Biomolecular Engineering The Ohio State University PUBLICATIONS Xiaodong Tong, Liying Yang , James C Lang , Maciej Zborowski , Jeffrey J Chalmers. Application of immunomagnetic cell enrichment in combination with RT- PCR for the detection of rare circulating head and neck tumor cells in human peripheral blood. Cytometry Part B (Clinical Cytometry). 2007. 72B:310–323. Chunhui Zhao, Jianping Lin, Liying Yang, Jianghua Mu, Peilin Cen. Effect of Glycine and Succinate on 5-Aminolevulinic Acid Production by Photobacteria. Chemical reaction engineering and technology. 2004. 20(3): 275-279. FIELD OF STUDY Major Field: Chemical Engineering Specialization: Biochemical engineering vii TABLE OF CONTENTS Page ABSTRACT.................................................................................................................. ii DEDICATION............................................................................................................. iv ACKNOWLEDGMENTS .............................................................................................v VITA........................................................................................................................... vii LIST OF TABLES..................................................................................................... xiii LIST OF FIGURES .....................................................................................................xv Chapters: 1. INTRODUCTION ....................................................................................................1 1.1 Immunomagnetic cell separation ....................................................................1 1.2 Magnetophoretic mobility and CTV...............................................................3 1.3 Commercial magnetic cell sorters...................................................................8 1.4 Novel magnetic cell separation systems .........................................................9 1.4.1 Quadrupole magnetic cell sorters (QMS) .................................................9 1.4.2 Magnetic deposition system....................................................................12 1.5 Detection of Circulating Tumor Cells (CTCs)...............................................14 1.5.1 Biology of CTCs.....................................................................................14 1.5.2 Current CTC detection status..................................................................16 1.6 QMS enrichment of rare cancer cells............................................................18 1.7 Molecular analysis technologies in cancer research .....................................19 1.7.1 ImmunoCytochemical Staining (ICCS)...................................................19 1.7.2 RT-PCR....................................................................................................20 1.7.3 cDNA microarray.....................................................................................27 1.8 Technologies to obtain pure cancer cells from solid tumors ........................28 1.8.1 Importance of obtaining pure cell samples in cancer research ...............28 1.8.2 In vitro degradation of tissue ..................................................................29 1.8.3 Histology of the ECM.............................................................................30 1.9 Research objectives.......................................................................................32 1.10 Dissertation organization ............................................................................33 viii 2. APPLICATION OF IMMUNOMAGNETIC CELL SEPARATION IN COMBINATION WITH RT-PCR FOR THE DETECTION OF RARE CIRCULATING HEAD AND NECK TUMOR CELLS IN HUMAN PERIPHERAL BLOOD........................................................................................................................35 2.1 Motivation.....................................................................................................35 2.2 Material and Methods ...................................................................................38 2.2.1 Cell sources......................................................................................38 2.2.2 Immunomagnetic