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UNIVERSITY of CALIFORNIA, IRVINE Molecular and Metabolic UNIVERSITY OF CALIFORNIA, IRVINE Molecular and metabolic hallmarks of metastasis in triple negative breast cancer DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biomedical Sciences by Ryan Tevia Davis Dissertation Committee: Assistant Professor Devon A. Lawson, Chair Associate Professor Francesco Tombola Assistant Professor Sergio Armando Villalta Professor David A. Fruman Associate Professor Mei Kong 2020 Portion of Chapter 1 © 2018 Springer Nature Portion Chapter 2 and 3 © 2020 Springer Nature All other materials © 2020 Ryan Tevia Davis Dedication To my wife, the one who make me smile, my parents, the ones who sacrificed so much, my friends the ones who helped me hit level two, in recognition of their worth Perseverance When asked how it felt to have failed 10,000 times in the process of inventing the light bulb, Thomas Edison responded: “I have not failed. I’ve just found 10,000 ways that won’t work” ii Table of Contents Table of Contents ........................................................................................................................ iii List of Figures ............................................................................................................................... v List of Tables ................................................................................................................................ vi List of Acronyms and Symbols ................................................................................................. vii Acknowledgements ..................................................................................................................... ix Vita ................................................................................................................................................. xi Abstract of the Dissertation ..................................................................................................... xiv Chapter 1: Introduction ................................................................................................................ 1 1.1 Significance ............................................................................................................................. 1 1.2 Innovation ................................................................................................................................ 1 1.3 Background ............................................................................................................................. 2 1.3.1 Breast cancer prevalence and subtypes ........................................................................ 2 1.3.2 Breast cancer and the process of metastasis ................................................................ 3 1.3.3 Intratumoral heterogeneity and metastasis .................................................................... 4 1.3.4 Application of scRNA-seq technologies to cancer research .......................................... 5 1.3.5 Metabolism and cancer: Warburg and beyond............................................................... 9 1.4 Project Goal and Key Outcomes ..................................................................................... 10 Chapter 2: Defining heterogeneity in primary tumor and metastatic cells.......................... 12 2.1 Introduction ........................................................................................................................... 12 2.2 Materials and Methods ......................................................................................................... 12 2.2.1 Orthotopic transplantation of PDX tumors into immunocompromised mice ................ 12 2.2.2 Tissue harvest and dissociation.................................................................................... 13 2.2.3 Flow cytometry .............................................................................................................. 14 2.2.4 Generation of single-cell RNA sequencing data........................................................... 14 2.2.5 Processing of single-cell RNA sequencing data........................................................... 15 2.2.6 Dimensionality reduction, cell cluster identification, and differential gene expression analysis ................................................................................................................................... 15 2.2.7 Development of logistic regression model for identifying candidate biomarkers ......... 16 2.2.8 Gene scoring ................................................................................................................. 17 2.2.9 Relapse-free survival analysis ...................................................................................... 17 2.2.10 Breast cancer PAM50 subtyping ................................................................................ 17 2.2.11 Histology...................................................................................................................... 17 2.2.12 Fluorescent in situ hybridization (FISH) ..................................................................... 18 2.3 Results ................................................................................................................................... 18 2.3.1 Single-cell RNA sequencing of matched primary tumors and micrometastases ......... 18 2.3.2 Transcriptional diversity in micrometastatic and primary tumor cells........................... 20 2.3.3 Breast cancer subtype analysis of primary tumors and metastatic cells ..................... 25 2.3.4 Micrometastatic cells display a distinct transcriptome program ................................... 29 2.4 Discussion ............................................................................................................................. 36 Chapter 3: Metabolic phenotype ............................................................................................... 40 3.1 Introduction ........................................................................................................................... 40 3.2 Materials and Methods ......................................................................................................... 40 3.2.1 Seahorse Assay ............................................................................................................ 40 iii 3.2.2 Proliferation Assay ........................................................................................................ 41 3.2.3 Viability Assay ............................................................................................................... 42 3.2.4 FLIM Acquisition ............................................................................................................ 42 3.2.5 FLIM Data Analysis ....................................................................................................... 43 3.2.6 Orthotopic transplantation and intravenous injection of cell lines into mice ................ 43 3.2.7 Flow cytometry .............................................................................................................. 44 3.2.8 Quantitative real-time PCR ........................................................................................... 44 3.2.9 Generation of steady-state metabolomics data using LC-HRMS ................................ 45 3.3 Results ................................................................................................................................... 46 3.3.1 Micrometastatic cells upregulate mitochondrial oxidative phosphorylation ................. 46 3.3.2 Micrometastatic cells display a distinct metabolic profile ............................................. 52 3.3.3 Oxidative phosphorylation is critical for lung metastasis .............................................. 55 3.4 Discussion ............................................................................................................................. 59 Chapter 4: Conclusion and Final Remarks .............................................................................. 63 References ................................................................................................................................... 71 iv List of Figures Figure 1.1 Common types of intratumor heterogeneity and its regulation by intrinsic and extrinsic factors............................................................................................................................................... 6 Figure 1.2 Genetic and phenotypic properties of metastasis-initiating cells at the single-cell level. ................................................................................................................................................. 7 Figure 2.1 Single-cell RNA sequencing of micrometastatic and primary tumor cells. ................. 19 Figure 2.2 Quality control and exclusion criteria for single-cell RNA sequencing ....................... 21 Figure 2.3 Transcriptional diversity in micrometastatic and primary tumor cells ......................... 24 Figure 2.4 Marker and GO term analysis of cell clusters from each PDX model ........................ 26 Figure 2.5 Identification of an ECM remodeling and EMT co-correlated population in each PDX model.............................................................................................................................................. 28 Figure 2.6 Relevance of intratumor heterogeneity on subtype classification
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