(W0930-03-19) Assessing the Relevance of Nw-Hydroxy L-Arginine to Inflammatory and Immune Checkpoint Molecules in Breast Cancer Based on Estrogen Receptor Status and Disease Progression

Purpose: We have previously delineated N^w-hydroxy L-Arginine, (NOHA) as a predictive biomarker in distinguishing breast cancer based on estrogen-hormone-receptor status (USPTO 10,073,099), and on its utility with disease screening, as well as with high-risk breast cancer treatment response and therapy monitoring. In this study, we compare NOHA response with estrogen-receptor status selective breast cancer associated immune check point molecules (i.e., PD-L1) and inflammation indicators (i.e., IL8), based on disease progression.

Methods: Three-dimensional (3D) spheroids of estrogen receptor negative (ER–) and estrogen receptor positive (ER+) primary cells from Caucasian breast cancer patients, along with control/healthy cells (ATCC, MD), were cultured in medium between weeks 1 and 5, and tested weekly for cell proliferation and cell cycle status by Guave–8HT^® flow cytometry (Millipore, MA) using kit assays. Each week 3D spheroids were lysed, and cell lysate assayed for cellular inducible nitric-oxide synthase (NOS2) expression, PD-L1, and IL8 using respective ELISA kit assays (abcam, MA); and their nitric-oxide production as total nitrite was determined by calorimetric assay (Cayman Chemicals, IL). NOHA in each week 3D spheroid cell lysate and cultured medium were determined by our proprietary ELISA assay, with a lower limit of quantification set at 0.06nM. Statistical difference for this study was set at p< 0.01.

Results: Our in vitro studies show a progressive increase of ≥1-fold in cellular NOS2, total nitrites, PD-L1 and IL8, along with progressive decrease in cellular and medium NOHA by ≥ 1-fold only with ER– 3D spheroid group (p < 0.01, Figure. 1). ER+ or control 3D spheroid groups remained unchanged for all 5-weeks of the study period and their tested conditions. In ER– 3D spheroid group, a weekly decrease in cellular and medium NOHA by ≥ 5.2 % and ≥ 7.3 % respectively, correlated with cellular increases in PD-L1 by ≥18.8%, and IL8 by ≥23.4%. ER– 3D spheroid group also showed weekly cellular gain of ≥ 6.1% in NOS2 expression and ≥16.7% for total nitrite.

Conclusion: The present study provides first evidence for the strict association of NOHA with inflammation and immune-checkpoint molecules only under ER– distinctive breast cancer condition, and their disease progression. This work offers vital foundational knowledge for future delineation of NOHA metabolic impact in ER– breast cancer and associated phenotypic cellular modifications.