Modeling the Lymph Node Microenvironment to Uncover Metabolic Vulnerabilities in Breast Cancer

Abstract

Metastatic dissemination is responsible for a vast majority of cancer-related deaths, yet the biological mechanisms that enable tumor cells to survive during early metastatic spread remain incompletely understood. Lymph nodes represent a common early site of metastasis in breast cancer, among other cancer types (e.g., melanoma, pancreatic cancers) and provide a distinct metabolic microenvironment that may influence tumor cell survival and metastatic fitness. Emerging evidence suggests that ferroptosis, a regulated form of cell death driven by iron-dependent lipid peroxidation, acts as a critical barrier to metastatic progression in the bloodstream, but not lymphatic metastasis. However, the extent to which the metabolic microenvironment of the lymph node microenvironment influences tumor cell survival remains poorly defined. This dissertation investigates how the lymph node metabolic environment influences breast cancer survival. First, using murine and human breast cancer models, including spontaneous metastasis models and lymph node-derived tumor cell isolations, this work characterizes the metabolic features of the lymph node microenvironment that promotes breast cancer cell survival. To experimentally model lymph node conditions ex vivo, we designed LyMe (lymph-like medium), a physiologically informed culture system that recapitulates the key metabolic features of the lymph node microenvironment. LyMe enabled the interrogation of metabolic dependencies that influence cancer cell survival in in vitro cell culture contexts, thus facilitating an opportunity to more closely model the metabolic features of the lymph node microenvironment in standard cell culture. To assess the extent to which metabolic features observed in our in vitro and in vivo experimental models are reflected in human disease, a clinical study of human breast cancer fine-needle aspiration biopsy samples was initiated and completed as part of this dissertation work. Metabolomic and lipidomic profiling of lymph node-positive and lymph node-negative patient plasma and lymph node aspirates revealed distinct metabolic signatures associated with lymph node metastasis. These analyses identified enrichment of pathways related to lipid metabolism, purine metabolism, and immune-associated scavenger receptor signaling, including involvement of scavenger class F receptors. Together, these findings support the translational relevance of the experimental systems and suggest that lymph node metastases undergo coordinated metabolic adaptations that may promote tumor cell survival during metastatic progression. Furthermore, through targeted metabolic perturbation experiments, we demonstrate that specific nutrient pools contribute differentially to tumor cell survival in lymph-like conditions. In particular, cystine availability emerged as a critical determinant of tumor cell survivability, consistent with its central role in regulating ferroptosis through glutathione metabolism and redox homeostasis. These findings expose the metabolic features of lymph node microenvironments that influence breast cancer survival. Finally, complementary collaborative work examined metabolic regulation of ferroptosis in metastatic cancer, including the role of lysosomal iron handling and dietary lipid metabolism in shaping ferroptotic susceptibility. Collectively, these studies highlight ferroptosis and lipid peroxidation as metabolically regulated vulnerabilities during metastatic progression. Overall, this dissertation demonstrates that microenvironmental metabolic conditions within lymph nodes can influence tumor cell survival and ferroptosis susceptibility and introduces a metabolic media matching lymph conditions as a platform to study cancer biology of cancer cells residing in lymph nodes under physiologically relevant conditions in vitro. Together, these findings provide a foundation for understanding how tissue-specific metabolic environments support metastatic adaptation and reveal metabolic vulnerabilities that may be therapeutically leveraged to limit cancer cell survival within lymph nodes.