Abstract B90: Improved mice survival by reducing pheochromocytoma burden through activation of innate immunity using mannan and toll-like receptors
Authors: Caisova Veronika , Gupta Garima , Jochmanova Ivana , Jha Abhishek , Li Liping , Huynh Thanh Truc , Pang Ying , Ghayee Hans Kumar , Taïeb David , Zenka Jan , Pacak Karel 
Published: June 2021

Abstract
Background and Purpose: Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are rare neuroendocrine/neural crest cell tumors. Therapeutic options for PHEOs/PGLs are limited, particularly in metastatic patients. Therefore, efforts to find new and more effective therapies are crucial in PHEO/PGL research. Recently, immunotherapy based on activation of innate immunity via pathogen-associated molecular patterns (PAMPs) has been tested in melanoma. PAMPs-based immunotherapy uses mannan, a simple polysaccharide from Saccharomyces cerevisiae, as a ligand stimulating phagocytosis in combination with toll-like receptor (TLR) ligands. Thus, in the present study we evaluated (1) the effect of intratumorally administered mannan-BAM + TLR ligands in a PHEO mouse model, (2) the participation of innate immunity on the PHEO growth reduction, (3) the leukocyte infiltration in treated PHEO, and (4) in vitro interactions of PHEO cells (with or without mannan) with neutrophils.
Methods: PHEO cells were subcutaneously transplanted into mice and mannan-BAM + TLR ligands were intratumorally administered. CD45+ infiltration in tumors was measured using flow cytometry. In vitro experiments were performed using mouse/human PHEO cell lines incubated with mouse/human neutrophils.
Results: The intratumoral administration of mannan-BAM and TLR ligands into PHEO resulted in 90% tumor growth reduction. The survival median increased from 16 days in the control group to 50 days in the group treated with mannan-BAM + TLR ligands. Subsequently, mice lacking functional T and B cells were used. Intratumoral administration of mannan-BAM + TLR ligands resulted in 86% reduction of tumor growth, which proved the key role of innate immunity in PHEO tumor elimination. The flow cytometry analysis of tumor-infiltrating CD45+ cells revealed higher levels of CD45+ cells in the group treated by mannan-BAM + TLR ligands. Significant increase in the levels of granulocytes was observed on days 3 and 18 of the treatment. Cytotoxic experiments using PHEO cell lines revealed increased cytotoxic effect of neutrophils toward PHEO cells labeled with mannan-BAM compared to the cells without mannan-BAM. Microscopic evaluation of neutrophils and PHEO cells labeled with mannan-BAM revealed enhanced frustrated phagocytosis and neutrophils rosette formation dependent on the presence of mannan-BAM.
Conclusion: We demonstrate excellent therapeutic effects of enhanced innate immunity using intratumorally administered PAMPs and TLR ligands in a subcutaneous PHEO mouse model.
Citation Format: Veronika Caisova, Garima Gupta, Ivana Jochmanova, Abhishek Jha, Liping Li, Thanh Truc Huynh, Ying Pang, Hans Kumar Ghayee, David Taïeb, Jan Zenka, Karel Pacak. Improved mice survival by reducing pheochromocytoma burden through activation of innate immunity using mannan and toll-like receptors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B90.