Verbanac, KathrynBalestrieri, Kassondra2024-01-162023-122024-01-04December 2http://hdl.handle.net/10342/13286Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Patients with TNBC have a high rate of metastasis and mortality due to the lack of targeted therapies and resistance to chemotherapy. This research aimed to develop reliable models of metastatic TNBC to investigate targetable proteins and pathways. The 2225L and T11 murine tumor lines, which are highly analogous to human basal-like and claudin-low TNBC subtypes, were serial passaged heterotopically and orthotopically in syngeneic mice to generate lines that would consistently metastasize to the lung. Immunochemical and transcriptomic methods were used to define the immune microenvironment of the primary and metastatic tumors and to compare tumors with variable metastatic potential. Both resulting 2225LM and T11 tumor lines displayed an immunosuppressive tumor microenvironment characterized by high myeloid-to-lymphocyte ratio (CD11b+:CD3+) and elevated expression of protumor cytokines compared to control tissues. Subsequent analysis of T11 primary and metastatic claudin-low breast tumors employed a discovery-based proteomic approach that detected significant differential galectin-1 expression compared to normal control tissues. Label-free quantitation, Western immunoblot, and ELISA confirmed galectin-1 identity and elevated expression in primary and metastatic tumors compared to normal control tissues. Mass spectrometry spatial mapping and immunohistochemistry revealed high localization of galectin-1 in T11 metastatic lung foci. Transcriptomic analysis of mouse and human breast cancer subtypes showed significant and differential galectin-1 expression in claudin- low primary tumors compared to other breast cancer subtypes. Galectin-1 is an N-acetyllactosamine (LacNAc)-binding protein that promotes anti-inflammatory actions and has a wide range of reported intracellular and extracellular activities, but its role in breast cancer has not been well studied. We next evaluated galectin-1 effects on CD11b+ myeloid cells, based on their high proportion in T11 tumors, using in vitro, ex vivo, and in vivo models. The CD11b+ murine macrophage cell line RAW 264.7 was susceptible to galectin-1-induced apoptosis in vitro in a dose-dependent, carbohydrate-specific manner. However, ex vivo assays with murine CD11b+ peritoneal exudate cells showed no effect on apoptosis. In vivo systemic administration of galectin-1 allosteric inhibitor OTX008 in T11-tumor-bearing mice slowed tumor growth but did not affect the percentage of infiltrating myeloid populations. Fluorescence microscopy evaluation of overall tumor apoptosis and CD3+ and CD11b+ cell apoptosis in primary tumors at sacrifice showed no statistical difference between the OTX008 treatment and the vehicle control groups. These findings suggest that galectin-1 does not affect CD3+ or CD11b+ immune cell infiltration or apoptosis in claudin-low primary tumors. Larger in vivo studies and analysis of different drug regimens, smaller tumors and metastatic models are indicated. Future studies focusing on alternative actions of galectin-1 in claudin-low breast cancer, including its potential effects on tumor proliferation and angiogenesis, should be conducted to explore its viability as a therapeutic target.application/pdfenclaudin-lowbreast cancertriple negative breast cancergalectin-1proteomicsgenomicscarbohydrate-binding proteinDoctoral Dissertation2024-01-11