The effects of nitric oxide on metastatic breast cancer cells: linking NOS2 expression and immune modulation to the anti-tumor response

Nitric oxide (NO) has dual roles which include pro- and anti-tumorigenic properties that are fundamental to metastatic breast cancer, Triple Negative Breast Cancer (TNBC). Recently, NO has surfaced as a key protein that drives TNBC. Under a normal physiological state, the production of NO is synthesized by the nitric oxide synthase (NOS); which forms three isoforms. Although, there are three isoforms of nitric oxide, NOS2 the inducible form generates higher amounts of NO and is overexpressed in TNBC. In TNBC, inappropriate NOS2 expression enhances tumor growth, metastasis, and production of tumor-derived cytokines and chemo-attractants. However, the effects of NO in TNBC, linking NOS2 expression to the anti-tumor response have been inconclusive. Further, the relationship between NO with immune cells in the tumor microenvironment is not well studied. The studies detailed in the thesis investigate the correlation involved in immune modulation of NOS2 in metastatic breast cancer to the antitumor response. Moreover, we used 4T1 murine metastatic breast cancer cells and CRISP/CAS9 mediated NOS2 knockouts (KO) E3 and A40 cell lines to further study the role of NOS2 in the tumor microenvironment. We were able to induce NOS2 expression in the 4T1 wild-type (parental) cells with inflammatory cytokines in serum deprivation as well as confirm that the NOS2 knockouts E3 and A40 lack the NOS2 gene. Additionally, we show that NOS2 expression does not depend on cytokine concentration in the 4T1 wild-type cells. Several surface receptors that may be overexpressed in the TNBC cells such as PD-L1, MHC-I, FAS, and CD47 were studied in the 4T1 cells. Interestingly, we saw upregulation of PD-L1 and MHC-I, and FAS during cytokine stimuli in nutrient starvation in the NOS2 knockout cell lines when compared to the parental cell line. Further, we used DETA/Nononate, a slow-releasing NO donor in the 4T1 wild-type and NOS2 KO cell lines. We observed, that in high concentrations the NOS2 knockout cell lines decreased when compared to the untreated cytokine serum-free media cells