................................................................................................................................................ 2 RÉSUMÉ ..................................................................................................................................................... 3 ABBREVIATIONS ..................................................................................................................................... 5 TABLE OF CONTENTS ........................................................................................................................... 8 LIST OF FIGURES .................................................................................................................................. 10 LIST OF TABLES .................................................................................................................................... 12 ACKNOWLEDGEMENTS ..................................................................................................................... 13 CONTRIBUTION OF AUTHORS.......................................................................................................... 15 CHAPTER 1: REVIEW OF LITERATURE ......................................................................................... 16 1.1. Classification .................................................................................................................................... 18 1.1.1. Histopathology ....................................................................................................................... 18 1.1.2. Staging ................................................................................................................................... 19 1.1.3. Grading................................................................................................................................... 20 1.1.4. Molecular subtype .................................................................................................................. 20 1.2. Breast tumorigenesis ........................................................................................................................ 23 1.3. Breast cancer metastasis ................................................................................................................... 26 1.3.1. The pre-metastatic niche ........................................................................................................ 31 1.3.2. Liver metastasis ...................................................................................................................... 33 1.4. Metastatic microenvironment ........................................................................................................... 36 1.4.1. Tumor-stroma interactions in the liver microenvironment .................................................... 37 1.5. Rationale and approach .................................................................................................................... 38 CHAPTER 2: MATERIALS AND METHODS .................................................................................... 41 2.1. Cell lines and culture conditions ...................................................................................................... 42 2.2. Antibodies ........................................................................................................................................ 42 2.3. Generation of primary tumors and liver metastases ......................................................................... 42 2.4. Hematoxylin and Eosin staining ...................................................................................................... 43 2.5. Laser capture microdissection of frozen sections ............................................................................ 43 2.6. RNA isolation, amplification, labeling, and hybridization to microarrays ...................................... 44 2.7. Data processing and statistical analysis ........................................................................................... 45 2.8. Immunohistochemistry ..................................................................................................................... 45 2.9. Protein extraction ............................................................................................................................. 46 2.10. Immunoblotting .............................................................................................................................. 46 9 CHAPTER 3: RESULTS ......................................................................................................................... 48 3.1. Generation of murine primary mammary tumors and liver metastases ........................................... 49 3.2. Capturing regions of interest using LCM ......................................................................................... 50 3.3. Quality control and gene expression analysis of microarray data .................................................... 52 3.4. Class comparisons to find differentially expressed genes ................................................................ 53 3.5. Target gene validation: Metastasis core vs. margin comparison ..................................................... 55 3.5.1. Foxm1 ....................................................................................................................................... 56 3.5.2. Dnmt1 ........................................................................................................................................ 57 3.5.3. Brms1 ........................................................................................................................................ 58 3.5.4. Ndrg1 ........................................................................................................................................ 60 3.5.5. Tm4sf4 ...................................................................................................................................... 60 3.6. Gene target validations: Primary tumor vs. liver metastases .......................................................... 60 3.6.1. Lcn2 and S100a8/S100a9 as possible markers of myeloid/granulocytic cell recruitment ........ 61 CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS .............................................................. 63 4.1. Validation of genes differentially expressed between the core and margin of liver metastases ...... 65 4.2. Class comparisons between primary tumors and liver metastases revealed an enrichment of liver function genes in the metastases ............................................................................................................. 67 4.3. Lcn2 and S100a8 are putative myeloid/granulocytic cell markers .................................................. 70 4.4. Future directions emanating from this work .................................................................................... 74 REFERENCES .......................................................................................................................................... 77 APPENDIX ................................................................................................................................................ 83