WEANING-INDUCED LIVER INVOLUTION: A NOVEL MECHANISM OF METASTASIS IN POSTPARTUM BREAST CANCER By Erica T. Goddard A THESIS/DISSERTATION Presented to the Cancer Biology Program & the Oregon Health & Science University School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy January 2017 School of Medicine Oregon Health & Science University CERTIFICATE OF APPROVAL ______________________________ This is to certify that the PhD dissertation of Erica Thornton Goddard has been approved _________________________________ Pepper J. Schedin, PhD, Mentor/Advisor _________________________________ Virginia F. Borges, MD, MMSc, Clinical Mentor/Advisor _________________________________ Melissa H. Wong, PhD, Oral Exam Committee Chair _________________________________ Lisa Coussens, PhD, Member _________________________________ Amanda W. Lund, PhD, Member _________________________________ Caroline Enns, PhD, Member _________________________________ Willscott E. Naugler, MD, Member Table of Contents List of Figures ........................................................................................................................... iv List of Tables ............................................................................................................................ vii List of abbreviations ............................................................................................................ viii Acknowledgements .............................................................................................................. xiv Abstract ..................................................................................................................................... xv Chapter I: Background and introduction ......................................................................... 1 Postpartum breast cancer is associated with a high rate of metastasis ......................... 2 Mammary gland involution and the ‘involution hypothesis’ .............................................. 6 The metastatic cascade .................................................................................................................... 7 Early steps in the metastatic cascade: from primary tumor to circulation ................ 10 Primary tumor growth .............................................................................................................................. 10 Acquisition of tumor cell invasive phenotypes and local dissemination ............................. 11 Immune evasion .......................................................................................................................................... 13 Intravasation ................................................................................................................................................. 17 Survival in the circulation ........................................................................................................................ 19 Tumor cell arrest in vasculature ........................................................................................................... 20 Extravasation ................................................................................................................................................ 21 Preclinical models of postpartum breast cancer implicate the involuting mammary gland in metastasis ......................................................................................................................... 22 The microenvironment at secondary sites represents a major barrier to metastatic efficiency ............................................................................................................................................. 25 What determines site-specific metastatic patterns in cancer?................................................. 25 Metastatic efficiency is dictated by the microenvironment at the secondary site ........... 27 Establishment of the pre-metastatic niche ............................................................................ 28 Identifying the pre-metastatic niche ................................................................................................... 28 Secreted factors from the primary tumor establish pre-metastatic niches ........................ 30 Pre-metastatic and metastatic niches impact early cell fate decisions and outgrowth ........................................................................................................................................... 32 The perivascular niche is the first point of interaction upon entry into the secondary site ..................................................................................................................................................................... 32 Establishment of pro-metastatic microenvironments in the absence of tumor- education ........................................................................................................................................................ 34 The fate of tumor cells at metastatic sites in the postpartum setting .......................... 36 Limitations to studying site-specific metastasis and the metastatic niche ......................... 37 Chapter II: A portal vein injection model to study liver metastasis of breast cancer ......................................................................................................................................... 39 Short abstract ................................................................................................................................... 40 Long abstract ..................................................................................................................................... 40 Introduction ...................................................................................................................................... 41 Breast Cancer Metastasis to the Liver ................................................................................................ 41 Established Models to Study Breast Cancer Metastasis to the Liver ..................................... 42 Portal Vein Injection Model of Liver Metastasis ............................................................................. 45 i Protocol: .............................................................................................................................................. 47 Representative results .................................................................................................................. 60 Discussion .......................................................................................................................................... 67 Acknowledgements......................................................................................................................... 70 Chapter III: The Rodent Liver Undergoes Weaning-Induced Involution and Supports Breast Cancer Metastasis ................................................................................. 71 Abstract ............................................................................................................................................... 72 Statement of Significance ............................................................................................................. 72 Introduction ...................................................................................................................................... 73 Materials and Methods .................................................................................................................. 74 Supplementary Methods ............................................................................................................... 81 Results ................................................................................................................................................. 87 Dynamic regulation of the rodent liver during pregnancy, lactation, and weaning ........ 87 Weaning-induced liver involution is accompanied by stromal remodeling ....................... 92 Immune cell accumulation during weaning-induced liver involution .................................. 96 Weaning-induced liver involution establishes a pro-metastatic microenvironment ..... 99 Evidence for elevated risk for liver metastasis in postpartum breast cancer patients .......................................................................................................................................................................... 101 Discussion ........................................................................................................................................ 105 Supplementary Figures ............................................................................................................... 109 Supplementary Tables ...................................................................................................... 119 Acknowledgments ......................................................................................................................... 126 Grant Support ................................................................................................................................. 126 Chapter IV: Low-dose ibuprofen intervention has minimal impact on postpartum breast cancer metastasis to the liver ................................................... 128 Abstract ............................................................................................................................................