Abstract
BACKGROUND: Estrogen receptor-negative (ER-) breast cancer remains clinically challenging due to its aggressive behavior, lack of targeted therapies, and limited biomarker tools for diagnosis and disease monitoring. While immune checkpoint markers (such as PD-L1) and inflammatory cytokines (viz., IL8) offer insight into tumor immune dynamics, their clinical application is hampered by tissue-dependence and/or biological variability. We have previously delineated N(w)-hydroxy l-Arginine, (NOHA) as a simple, yet sensitive biomarker in distinguishing breast cancer based on estrogen-hormone-receptor status (USPTO 10,073,099). This study investigates whether NOHA functions as key biomarker linking immune checkpoint activation and inflammatory cytokine signaling in ER- breast cancer. METHODS: Three-dimensional (3D) spheroid cultures were generated from ER- and ER + breast cancer cell lines of African American (AA) and Caucasian (CA) origin, as well as healthy control cells. 3D spheroids were maintained for 7 weeks, with weekly measurements of their medium NOHA as well as lysates for cellular NOHA, NOS2 activity, total nitrites, PD-L1, and IL-8. Quantification was performed using validated ELISA and fluorometric assays. Statistical comparisons were performed using ANOVA with post-hoc testing. RESULTS: ER- spheroids exhibited a progressive ≥1-fold increase in cellular NOS2, total nitrites, PD-L1, and IL-8, accompanied by a ≥1-fold decrease in both cellular and medium NOHA over 7 weeks (p < 0.01). No significant changes were observed in ER + or control spheroids. The ratio means of cellular PD-L1/medium NOHA and cellular IL-8/medium NOHA increased significantly in ER- groups, with AA-derived ER- 3D spheroids demonstrating ≥43.8 % and ≥48.4 % higher mean ratios, respectively, compared to ER- CA-derived counterparts (p < 0.01). A corresponding analysis comparing ratio means of cellular PD-L1/cellular NOHA and cellular IL-8/cellular NOHA showed a similar pattern, with ≥52.4 % and ≥55.9 % higher mean ratios in ER- groups, with AA-derived ER- 3D spheroids, than in ER- CA group (p < 0.01). DISCUSSION: These findings identify NOHA as a potential non-invasive biomarker that reflects dynamic immuno-inflammatory changes in ER- breast cancer. The inverse relationship between NOHA and both PD-L1 and IL-8 suggests an inflammatory loop driven by NOS2 activity. The pronounced effects in AA-derived ER- spheroids align with known disparities in tumor aggressiveness and suggest future NOHA clinical utility potential in racially diverse populations. Further in vivo validation is warranted to support and confirm such clinical translation.