ESTRO 2023 - Abstract Book

S2013

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ESTRO 2023

PO-2237 Role of protein glycosylation in radiation-induced immune cell recruitment

O. Guipaud 1 , F. Milliat 2 , S. Ladaigue 2

1 IRSN, Human Health Radiation Protection Unit, Fontenay aux Roses, France; 2 IRSN, Human Health Radiation Protection Unit, Fontenay-aux-Roses, France Purpose or Objective Radiation therapy results in the recruitment of immune cells into tumors and healthy tissue. As a key player in this process, the irradiated vascular endothelium captures immune cells and actively promotes their passage to the injured tissue by transendothelial migration. However, the regulation of immune cell recruitment is not well understood in the general context and after radiotherapy. The interactions between endothelial cells and monocytes involve binding between monocyte receptors and glycosylations carried by endothelial membrane proteins. Endothelial high-mannose N-glycans are structures that could play an important role in inflammation in different pathological contexts such as atherosclerosis. Trimmed by class I alpha-mannosidases, these glycans are quite rare under normal conditions but particularly abundant after irradiation of endothelial cells. They are, at least in part, responsible for the interaction between monocytes and irradiated endothelial cells. The objective of this work was to better understand how high-mannose N-glycans are regulated in endothelial cells exposed to radiation. Materials and Methods To better understand the role of high-mannose N-glycans in endothelium-monocyte interactions, we used primary human endothelial cells (HUVECs) and a human monocyte cell line (THP-1). HUVECs were irradiated at 20 Gy with a LINAC. Two days after irradiation, two crucial steps in monocyte recruitment via endothelial cells, namely monocyte adhesion and transendothelial migration, were assessed in vitro by flow adhesion experiments videomicroscopy and transmigration experiments using real-time imaging. The role of the classe-1 alpha-mannosidase MAN1C1 was explored by performing the functional assays after inhibiting (by siRNA) or overexpressing (by lentiviral vectors) the gene encoding the mannosidase. The N-glycan content was identified and quantified by liquid chromatography-tandem mass spectrometry under the different experimental conditions. Results We show that the expression of the endothelial MAN1C1 endogenous protein decreases after irradiation. Inhibition of MAN1C1 expression by siRNA gene silencing increases the abundance of high-mannose N-glycans, improves the adhesion rate of monocytes on endothelial cells in flow conditions and, in contrast, decreases radiation-induced transendothelial migration of monocytes. Consistently, overexpression of MAN1C1 in endothelial cells using lentiviral vectors decreases the abundance of high-mannose N-glycans and monocyte adhesion, and enhances transendothelial migration of monocytes. Conclusion Overall, this research supports the theory that radiation-induced downregulation of, at least, MAN1C1 is involved in monocyte adhesion to irradiated endothelial cells through the increased expression of high-mannose N-glycans. Hence, we propose a role for endothelial MAN1C1 in the recruitment of monocytes, particularly in the adhesion step to the endothelium. 1 Institute of Clinical Medicine, Experimental Clinical Oncology - Dept. Oncology , Aarhus, Denmark; 2 Institute of Clinical Medicine , Experimental Clinical Oncology - Dept. Oncology, Aarhus, Denmark; 3 Institute of Clinical Medicine, Experimental Clinical Oncology - Dept. Oncology, Aarhus, Denmark; 4 Institute of Clinical Medicine, Danish Centre of Particle Therapy, Aarhus , Denmark Purpose or Objective Introduction: There is great focus on establishing combinational therapies where radiotherapy or chemotherapy is given beforehand such that tumor becomes more immunogenic prior to administration of immunotherapy. This holds great importance for solid tumors, that are traditionally known to be poor candidates for immunotherapy. Our plan would be to investigate the combination of one such inhibitor (anti-CTLA-4; anti-cytotoxic T lymphocyte associated protein - 4) with treatments that have shown to have a bi-functional cell killing mechanisms. These include high dose proton radiation, and the vascular disrupting drug OXi4503, that not only have potential for tumor ablation, but also have shown to mediate tumor immunogenicity. Objectives: To investigate the critical factors influencing the tumor growth inhibition seen when we combine anti-CTLA-4 with radiation, and vascular disrupting agent OXi4503. Materials and Methods Materials & Methods: All experiments used C3H mammary carcinoma grown in the right rear foot of CDF1 mice. Treatments started when tumors were at specific s of 50, 100, 200, or 400 mm3. These included proton radiation (local tumor irradiation with 20 Gy on day 0), OXi4503 (50 mg/kg, injected i.p. on days 0, 3, 7, and 10), achieved by immersing the tumor bearing leg in a water-bath), and anti-CTLA-4 (injected i.p. on days 1, 4, 8, and 11). The endpoint was tumor growth delay (time to grow to 1000 mm3). PO-2238 Improving immunotherapy outcome in solid tumor by combining with other established cancer treatments P. Sinha 1 , F.C. Asonganyi 2 , S. Nygaard 3 , M.K. Sitarz 4 , P.B. Elming 1 , M.R. Horsman 1

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