Abstract 2092: Tumor-infiltrating myeloid cells: Using MultiOmyx to distinguish between TAMs, TANs, and MDSCs in the pancreatic tumor microenvironment

Abstract

Abstract Myeloid cells are the primary recruited effector cells during inflammation. A subset of these, consisting primarily of tumor-associated M1/M2 macrophages (TAMs), tumor-associated neutrophils (TANs), and myeloid-derived suppressor cells (MDSCs), accumulate in tumors where they establish an inflammatory tumor microenvironment (TME) that is favorable for tumor progression (1-3). While these tumor-infiltrating myeloid cells (TIMs) are known to be major regulators of tumor-associated immune-suppression, angiogenesis, metastasis, and resistance to anti-cancer therapy much still remains to be understood about how they function within the TME and how they co-operate with tumor-resident lymphocytes to regulate antitumor immunity. Targeting TIMs to either eliminate them or to convert them from their immune-suppressive to an immune-stimulatory state has emerged as a new strategy complementing current cancer immunotherapy strategies. However, a major impediment to understanding the complexity of the distinct functions of subsets of TIMs and their spatial distribution within the TME is the ability to phenotypically characterize TIMs in FFPE tissues by standard immunohistochemistry, as most IHC studies do not utilize a panel of antibodies broad enough to characterize multiple myeloid cell subsets in the same sample. We will be using MultiOmyx, a proprietary, multiplexing assay with similar staining characteristics as standard IHC stains but with the significant advantage that up to 60 protein biomarkers can be interrogated from a single FFPE section (4). MultiOmyx protein immunofluorescence (IF) assays utilize a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining and deactivation. Using this MultiOmyx multiplexing assay in combination with a proprietary algorithm that takes into account the staining pattern for each specific biomarker, we will seek to identify different subsets of TIMs and their spatial relationship in tumor tissue from patients with pancreatic ductal adenocarcinoma (PDAC), characterized by an excessive amount of desmoplastic stroma seeded with inflammatory cells. TIM Panel: CD11b, CD14, CD15, CD16, CD33, CD68, CD163, HLA-DR, Arginase, PANCK. Human Myeloid Cell Subset Markers: M1 TAMs (CD68+HLA-DR+CD163-), M2 TAMs (CD68+CD163+HLA-DR+), TANs (CD11b+CD15+CD16+HLA-DR+Arginase+), M-MDSCs (CD11b+CD14+CD33+HLA-DRLoCD15-), G-MDSC (CD11b+CD15+CD33+Arginase+HLA-DRLoCD14-).