The ability of cancer cells to invade into surrounding tissues requires degradation of the extracellular matrix (ECM). Invasive structures, such as invadopodia, form on the plasma membrane of cancer cells and secrete ECM-degrading proteases that play crucial roles in cancer cell invasion. We have previously shown that protein tyrosine phosphatase alpha (PTPα) regulates focal adhesion formation and migration of normal cells. Here we report a novel role for PTPα in promoting triple-negative breast cancer cell invasion in vitro and in vivo. We show that PTPα knockdown reduces ECM degradation and cellular invasion of MDA-MB-231 cells through Matrigel. PTPα is not a component of TKS5-positive structures resembling invadopodia; rather, PTPα localizes with endosomal structures positive for MMP14, caveolin-1, and early endosome antigen 1. Furthermore, PTPα regulates MMP14 localization to plasma membrane protrusions, suggesting a role for PTPα in intracellular trafficking of MMP14. Importantly, we show that orthotopic MDA-MB-231 tumours depleted of PTPα exhibit reduced invasion into the surrounding mammary fat pad. These findings suggest a novel role for PTPα in regulating the invasion of triple-negative breast cancer cells.

Metastatic breast cancer is challenging to effectively treat, highlighting the need for an improved understanding of host factors that influence metastatic tumor cell colonization and growth in distant tissues. The lungs are a common site of breast cancer metastasis and are host to a population of tissue-resident eosinophils. Eosinophils are granulocytic innate immune cells known for their prominent roles in allergy and Th2 immunity. Though their presence in solid tumors and metastases have been reported for decades, the influence of eosinophils on metastatic tumor growth in the lungs is unclear. We used transgenic mouse models characterized by elevated pulmonary eosinophils (IL5Tg mice) and eosinophil-deficiency (ΔdblGATA mice), as well as antibody-mediated depletion of eosinophils, to study the role of eosinophils in EO771 mammary tumor growth in the lungs. We found that IL5Tg mice exhibit reduced pulmonary metastatic colonization and decreased metastatic tumor burden compared to wild-type (WT) mice or eosinophil-deficient mice. Eosinophils co-cultured with tumor cells ex vivo produced peroxidase activity and induced tumor cell death, indicating that eosinophils are capable of releasing eosinophil peroxidase (EPX) and killing EO771 tumor cells. We found that lung eosinophils expressed phenotypic markers of activation during EO771 tumor growth in the lungs, and that metastatic growth was accelerated in eosinophil-deficient mice and in WT mice after immunological depletion of eosinophils. Our results highlight an important role for eosinophils in restricting mammary tumor cell growth in the lungs and support further work to determine whether strategies to trigger local eosinophil degranulation may decrease pulmonary metastatic growth.

Hypoxia develops in germinal centers (GCs) induced by model antigens; however, it is unknown whether tumor-reactive GCs are also hypoxic. We identified GC hypoxia in lymph nodes (LNs) draining murine mammary tumors and lethally irradiated tumor cells, and found that hypoxia is associated with the levels of antibody-secreting B cells. Hypoxic culture conditions impaired the proliferation of activated B cells, and inhibited class-switching to IgG1 and IgA immunoglobulin isotypes in vitro . To assess the role of the hypoxic response in tumor-reactive GCs in vivo , we deleted von Hippel-Lindau factor (VHL) in class-switched B cells and found decreased GC B cells in tumor-draining LNs, reduced class-switched and tumor-specific antibodies in the circulation, and modified phenotypes of tumor-infiltrating T cells and macrophages. We also detected the hypoxia marker carbonic anhydrase IX in the GCs of LNs from breast cancer patients, providing evidence that GC hypoxia develops in humans. We conclude that GC hypoxia develops in TDLNs, and that the hypoxic response negatively regulates tumor-induced humoral immune responses in preclinical models.