Purpose: We had previously identified Nw-hydroxy L-Arginine (NOHA) as a blood-based biomarker to distinguish estrogen receptor negative (ER–) breast cancer from estrogen receptor positive (ER+) breast tumor; based on disease burden, progression and molecular phenotype (U.S. Utility Patent 10,073,099). In this study, we assessed the distinction in NOHA response between ER– tumor subtypes of ovarian versus breast cancer.
Methods: Plasma samples from ER–/ER+ ovarian and breast cancer patients, categorized as low grade tumor, were assessed for NOHA levels. Three-dimensional (3D) spheroids of ER–/ER+ primary cells from breast cancer and ovarian cancer patients, along with control/healthy cells, were cultured in medium between weeks 1 and 9, and tested for cell proliferation and cell cycle by Guave–8HT® flow cytometry (Millipore, MA) using kit assays. Inducible nitric-oxide synthase (NOS2) expression was determined by ELISA kit assay (Bioassay systems, CA); and cellular nitric-oxide as total nitrite was determined by calorimetric assay (Cayman Chemicals, IL). Extracellular concentrations of Nw-hydroxy-L-Arginine (NOHA) in the culture medium, and cellular L-Arginine were determined by in-house developed custom ELISA assay. Statistical difference was set at p< 0.01.
Results: Among ER–/ER+ ovarian and breast cancer patients, a plasma NOHA reduction of ≥ 33.9 % was seen in only ER– cancer patient plasma samples (Fig. 1). This ER– tumor selective plasma NOHA reduction maintained its distinction between ER– ovarian cancer versus ER– breast cancer subtypes, with a ≥ 48.8% greater NOHA reduction in ER– Ovarian tumor than seen with ER– breast cancer (Fig. 1). In 3D-spheroids, our custom ELISA assay showed a progressive reduction of ≥1-fold for NOHA in extracellular medium of ER– 3D-spheroids (during 9 week culture), along with a progressive increase in cellular NOS2 and nitric-oxide levels by ≥1-fold was observed, compared to those in ER+ and control 3D-spheroids (p < 0.01, n=6; Fig 2). ER– ovarian cancer 3D-spheroids had reduced NOHA level, of ≥38.9%, than in ER– breast cancer 3D-spheroids, over 9-weeks. The distinction in extracellular NOHA reduction, between ER– ovarian and ER– breast tumor 3D spheroids, correlated with their respective 3D spheroid tumor grades. The cellular NOS2 expression and nitric-oxide levels in ER– ovarian cancer 3D-spheroids were also elevated by ≥ 17.4 % and ≥ 18.8%, respectively than those seen in ER– breast cancer 3D-spheroids during the 9-week incubation period.
Conclusion: The present study offers the first evidence for NOHA as a sensitive and selective indicator that is capable of distinguishing ER– tumor subtypes (i.e. ER– breast cancer vs ER– ovarian cancer), based on tumor grade. The present results also suggest the potential for our custom ELISA protocol to be developed as a point-of-care assay.