Purpose: Estrogen-negative (ER–) patients has been recognized as the more aggressive subtype, more difficult to treat, greater ethnic disparity concerns, worse prognosis, and twice the risk of mortality compared to estrogen-positive (ER+) tumors. We have delineated the predictive distinction of a novel biomarker (viz., Nw-hydroxy L-Arginine, NOHA; U.S. Utility Patent Application., 15/272,841; and International Cooperative Treaty Patent Application., PCT/US2016/053055) between ER– breast cancer and ER– ovarian cancer detection and disease progression measurement.
Methods: Three-dimensional (3D) spheroids of ER– and 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 flow cytometry using kit assays. Inducible nitric-oxide synthase (NOS2) expression in cells was determined by ELISA kit assay; and cell nitric-oxide as total nitrite was determined by calorimetric assay. Extracellular concentrations of Nw-hydroxy-L-Arginine (NOHA) in the culture medium were determined by LC–MS, with a lower limit of quantification set at 0.25nM. Statistical difference was set at p<0.01.
Results: Our in vitro studies in ER– and ER+ breast and ovarian tumor 3D-spheroids, cultured for up to 9-weeks show a progressive reduction of ≥1-fold for NOHA only in the extracellular medium of ER– 3D-spheroids, along with a progressive increase in cellular NOS2 and nitric-oxide levels by ≥1-fold, compared to those in ER+ and control 3D-spheroids (p < 0.01, n=6, Fig. 1). Additionally, the NOHA reduction in ER– ovarian cancer 3D-spheroids was ≥47% greater than those seen in ER– breast cancer 3D-spheroids, over 9-weeks. The distinction in extracellular NOHA reduction as seen between ER– ovarian and ER– breast tumor 3D spheroids, was also maintained based on their respective 3D spheroid tumor grades. The cellular NOS2 expression and nitric-oxide levels in ER– ovarian cancer 3D-spheroids were elevated by ≥43% and ≥36%, respectively than those seen in ER– breast cancer 3D-spheroids during the 9-week incubation period.
Conclusion: The present study provides the first evidence for NOHA as a sensitive and stable indicator capable of detecting different types of solid tumor (i.e. ER– breast cancer vs ER– ovarian cancer) and distinguishing them based on tumor grade (based on disease progression). Further in-depth studies are needed to validate NOHA as a new indicator that can monitor therapy outcome in responsive subgroups and prevent unnecessary exposure of unresponsive patients to ineffective therapy.