Abstract 3588: Multiplex proteomics profiling with the imaging mass cytometry (IMC) identifies immune cell phenotypes associated with high NOS2/COX2 expression in ER- negative and TNBC patients
Authors: Pore Milind , Ridnour Lisa , Mallory Paul , Cardamone Ashley , Lockett Stephen , Bocik William , Wink David 
Published: June 2023

Abstract
Elevated expression of Inducible Nitric oxide Synthase-2 (NOS2) occurs during acute and chronic inflammation and wound healing processes however, it is also a predictor of bad prognosis in aggressive ER-negative tumors. Overexpression of NOS2 correlates with other prognostic biomarkers making it a unique multifunctional oncoprotein. Cyclooxygenase-2 (COX2) is another enzyme associated with cancer inflammation and is overexpressed in &gt;50% of breast cancers. In our previous studies, we have shown that NOS2/COX2 co-expression is a strong predictor of poor clinical outcome and promotes CD8+ T cell exclusion from tumor epithelia in Triple-negative breast cancers (TNBC). This observation was further explored using multiplexing proteomics technologies. Imaging Mass Cytometry (IMC) has revolutionized tumor microenvironment research by allowing the interrogation of over 35 protein markers simultaneously for the elucidation of the spatial distribution of immune and tumor cells at sub-cellular resolution. Herein, we used multiplex capability IMC to explore the interactions of immune and tumor cell counterparts in ER-negative and TNBC tumors. We employed Formalin-fixed paraffin-embedded (FFPE) tissue from 26 ER-negative and TNBC samples from a clinical cohort of African American breast cancer patients in the Baltimore MD area. We developed a panel of 15 protein metal-labeled antibody markers including antibodies targeting NOS2, COX2, T cells (CD45, CD3, CD4, CD8,) endothelium (CD31), monocytes (CD14), macrophages (CD68), basal markers (α-SMA), cancer stem cells (CD44v6), cancer cells (Pan-CK, B7H4, Cadherin), and serine proteases (granzyme B) secreted by T cells or Natural Killer (NK) cells. Single section slides of 4 µm size were stained with the cocktail of metal-labeled antibodies. Multiple regions of interest (ROIs) representing the tumor and immune cells compartments and tumor-facing stromal cell areas were ablated with the Hyperion Imaging system. The data analysis was performed by the Bodenmiller lab pipeline and VisioPharm software packages. Our preliminary data suggest that patient tumors with high or low NOS2/COX2 expression showed variable levels of CD68 positive macrophage population and T cells. We are currently analyzing the remaining data to understand the cell phenotypes associated with the immunosuppressive and immune-rich clinical samples and performing neighborhood analysis of tumor cells to immune cells and vessels.
Citation Format: Milind Pore, Lisa Ridnour, Paul Mallory, Ashley Cardamone, Stephen Lockett, William Bocik, David Wink. Multiplex proteomics profiling with the imaging mass cytometry (IMC) identifies immune cell phenotypes associated with high NOS2/COX2 expression in ER- negative and TNBC patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3588.