Assessing N ^w-hydroxy-L-arginine applicability as a novel ethnic specific estrogen-negative breast cancer marker

In our prior study we identified N ^w-hydroxy-L-arginine (NOHA) as a simple, yet sensitive indicator for estrogen negative (ER^-) breast cancer early-prognosis, but not estrogen positive (ER⁺), and to offer ethnic selectivity for ER^- detection. However, the ability of NOHA to assess ER^- breast tumor based on disease progression, and tumor severity needs further delineation. Also, the overall NOHA storage stability needs to be validated. To assess the NOHA predictive capability based on disease progression, ER^-/ER⁺ 3D-spheroids (from breast tumor cell lines of human origin) were cultured for 10 weeks. We found only ER^- 3D-spheroid cultured for 10 weeks to show a gradual reduction in NOHA (both in culture medium and 3D-spheroid lysates) that correlated with a progressive increase in cellular NOS2 expression and NOS2 activity (measured as total nitrites). We additionally identified the NOHA-NOS2 correlation to be ethnically selective between ER^- African American versus ER^- Caucasian groups. Interestingly, such NOHA reduction was observed earlier in ER^- culture medium (viz., after week 1) than from ER^- 3D-spheroids lysates (viz., at the end of 3 weeks). When categorized based on 3D-spheroid grade, we found a ≥ 68% NOHA reduction in ER^- spheroids that were ≤ 3 weeks old, that was categorized as "low-grade" (based on tumor size ≤ 250 µm, and with cellular characteristics identical to healthy cells). A substantial reduction in NOHA of ≥ 87% occurred with ER^- 3D-spheroids grown for 6 weeks, which were categorized as "intermediate-grade" (with tumor size of ≥ 400 µm, and with less characteristic similarity to control spheroids). These in vitro findings thus suggest a distinct correlation between NOHA reduction and ER^- tumor grade. Such distinctive correlation between NOHA and ER^- tumor grade was additionally observed in de-identified clinical samples where a onefold higher reduction in NOHA occurred in grade-2 than with grade-1 de-identified patient plasma (when compared with control), and such correlation offered ethnic selectivity between ER^- African American and ER^- Caucasian groups. Of additional interest, when NOHA overall storage stability was assessed by incubating patient plasma and culture medium spiked with 75 pg/ml NOHA at multiple incubation temperatures and time-points, we found NOHA to maintain its stability for up to 6 weeks in culture medium and for 7 days in plasma at 4 °C and below. These results thus provide the first evidence of NOHA as a stable indicator to monitor ER^- disease progression and tumor severity in ethnically distinctive populations.