Real-time nitric oxide detection in cytokine stimulated cancer cells and macrophages

Inflammation is increasingly linked to disease progression, particularly in cancer, where elevated levels of inducible nitric oxide synthase (iNOS or NOS2), driven by tumor inflammation, is correlated with aggressive tumors and poor outcomes. Measuring nitric oxide levels in tumor cells is hampered by the reactive nature of the molecule and generally inferred through indirect measurement of reaction products such as nitrate and nitrite. Here, we adapt the oxyhemoglobin detection method to tissue culture and examine nitric oxide production in tumor cells in response to inflammatory cytokines. Our assay provides real-time nitric oxide measurement, is highly sensitive, linear for at least an hour, inexpensive, and easy to implement. We show that triple negative breast and colorectal cancer cells respond to interferon gamma (IFNγ), interleukin 1-β (IL1-β) and tumor necrosis factor α (TNFα) to generate surprisingly high levels of NOS2 protein and nitric oxide, as high as seen in activated macrophages for fighting infection. NO detection levels reach 1.3 pmol NO/min/μg total cellular protein. The assay is readily adapted to assessing IC50 values for NOS2 inhibition, inhibition rates, and inhibition persistence. Using triple negative breast cancer cell line 4T1, a syngeneic murine tumor model, we estimate an IC₅₀ = 3.4 μM for NOS2-specific inhibitor 1400W, which displays a low nanomolar binding constant to isolated protein. Inhibition is rapid (<10 min) and persists for at least an hour. These results highlight the importance of nitric oxide production in the tumor and provide a means for developing new therapeutic strategies.