ESTRO 2024 - Abstract Book

S5248

Radiobiology - Normal tissue radiobiology

ESTRO 2024

Keywords: integrin antagonism; pulmonary fibrosis; SHG

879

Poster Discussion

Impact of in vivo lung irradiation on endothelial and immune cells in mice

Claire LAGO 1 , Georges TARLET 1 , Loïc LECOMTE 1 , Agnès FRANCOIS 1 , Michele MONDINI 2 , Mohamedamine BENADJAOUD 1 , Fabien MILLIAT 1 , Olivier GUIPAUD 1 1 Institut de Radioprotection et de Sûreté Nucléaire (IRSN), PSE-SANTE/SERAMED/LRMed, Fontenay-aux-roses, France. 2 Université Paris-Saclay, Institut Gustave Roussy, INSERM, Radiothérapie Moléculaire et Innovation Thérapeutique, Villejuif, France

Purpose/Objective:

Radiotherapy causes vascular endothelial cell (EC) dysfunction, which plays a major role in the initiation and development of radiation-induced lesions [1]. The endothelium can modulate inflammation and immune response through its ability to interact with immune cells (ICs) and control their extravasation by producing adhesion and activation molecules [2]. However, chronic recruitment of ICs in response to radiotherapy could have a deleterious effect on healthy tissues. While the molecular response of ECs to radiation in vitro is well described, we are only just beginning to understand their response in vivo and their role in immune response. Using a single-cell RNA sequencing (scRNA-seq) approach, this work aims to uncover the molecular response of irradiated mouse lung ECs and determine their ability to interact with lung ICs to better understand their role in the immune response.

Material/Methods:

Mice were exposed to whole thorax irradiation at 18 Gy (10 MV, 2.5 Gy/min) with a LINAC to induce pulmonary fibrosis and were studied at 7 days, 3 months and 6 months post-irradiation. For each mouse, two distinct fractions enriched in ICs (CD45+ cells) and ECs (CD45-CD31+ cells) were prepared from lung dissociation using magnetic cell separation (Miltenyi Biotec). After cell encapsulation and creation of cDNA libraries (10X Genomics), cDNAs from the two enriched fractions were sequenced at the single-cell level using a NovaSeq 6000 system (Illumina). Cell purities, viabilities and proportions were evaluated by flow cytometry (CyTek Aurora). ScRNA-seq data were analyzed using the Seurat package to study cell landscapes and gene expression within each cell type. The scRNA-seq dataset was also used to investigate interactions between ICs and ECs using ICELLNET [3] and CellChat [4] tools, which reveal likely interactions between cell types based on the expression of receptor-ligand pairs. In parallel, characterization of the radiation-induced pulmonary fibrosis model was carried out by histological and immunohistochemical studies and micro-CT imaging, while assessment of the functional status of ECs isolated from the lungs was carried out by cell culture and immunostaining.

Results:

Histological analysis and micro-CT imaging allowed the characterization of lung fibrosis six months post irradiation. Flow cytometry analyses of EC and IC suspensions were performed at each time post-irradiation. EC enrichment was around 95%, enabling to perform EC cultures to assess the functional status and ability of the