Metastasis continues to be the most dangerous stage of cancer, responsible for over 95% of breast cancer- related deaths for which we currently have no treatments. The NIH SEER program estimates over a quarter of a million new cases of breast cancer and over 40,000 deaths attributed to this disease for the US in 2017 alone; it is critical to understand metastatic progression at the molecular, cellular, and tissue levels to develop therapeutic strategies that target this threat to human health. An estimated 20% of metastatic breast cancer patients will not relapse within 10 years of completing treatment. This window of latency presents an opportunity for therapeutic intervention: it is thought that late-arising metastases emerge from disseminated tumor cells (DTC) lying dormant in the tissues until they are triggered to reawaken. DTC dormancy is regulated by external cues from tissue microenvironments that harbor these cells, such as the lymph node. The lymph node is the most common site of metastasis yet very little is known about how it sustains dormant DTCs and what changes to trigger DTC reawakening. This proposal will investigate the hypothesis that stable lymph node endothelia contribute to a pro-dormancy niche, which becomes pro-metastatic under physiological perturbation such as inflammation. To test this hypothesis, tumor dormancy will be modeled with in vivo spontaneous metastasis assays and a novel organotypic co-culture of the lymph node microvasculature. The localization of quiescent DTCs to lymph node stromal populations will be determined by immunofluorescence. The effect of the lymph node endothelia on tumor cell quiescence will be determined by quantifying mitotic markers and area outgrowth of human breast cancer lines on the microvascular niches. Co- culture conditions that suppress tumor proliferation relative to controls will be profiled by proteomics and RNAseq for candidate factors that relate to survival and proliferation which will be assessed by gain- and loss- of-function studies in the microvascular niches and verified in vivo using metastatic outgrowth as a readout. To test whether disruption of the stable microenvironment induces dormant DTC reawakening, the in vivo spontaneous metastasis model will be treated with inflammatory stimuli to determine changes to dormant DTC localization and growth. The effect of inflammation on lymph node endothelia will be directly assessed by translating ribosomal affinity purification and differentially expressed candidates will be functionally validated in the microvascular niches. Finally, the effect of inflammation on dormant tumor cell dynamics will be directly observed by intravital imaging through an inguinal lymph node imaging window. These studies will provide the first mechanistic understanding of how the lymph node microenvironment promotes tumor cell dormancy and demonstrate whether destabilizing the niche through inflammation can wake dormant tumor cells up. The proposed research has broad application to tissues that experience physiological perturbation, which may provide new therapeutic strategies to neutralize residual minimal disease in a tissue-specific manner. !
This proposal aims to identify mechanisms of lymph node-specific regulation of breast cancer dormancy and reawakening under normal and inflammatory conditions. Using novel animal and culture models, these studies will provide insight into how the lymph node microenvironment sustains disseminated tumor cell quiescence and whether destabilizing this niche through inflammatory perturbation triggers metastatic reawakening. The results of this proposal will contribute to our understanding of site-specific metastasis with the goal of identifying targets to clinically modulate tumor dormancy and prevent delayed relapse in the lymph node and elsewhere.
