X. Huang1, Y. Ma2, B. Tian1, J. Yu1, and D. Chen1; 1Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, 2Shandong Cancer Hospital and Institute, Jinan, Shandong, China
Purpose/Objective(s): Radiation induced esophageal injury (RIEI) is one of the major complications of radiotherapy (RT),imposing a severe restriction on the application of radiotherapy. The immune microenvironment knowledge about RIEI remains unknown and needs to be further characterized. This study aims to explore the immune network at the single-cell level how radiation esophagitis transformed to radiation esophageal fibrosis, and to identify predictive markers and therapeutic targets for RIEI. Materials/
Methods: An RIEI model was established using a high-dose single 40 Gy RT to the cervical esophagus of C57BL/6J mice, and the critical stages of RIEI development were determined by body weight and food intake, magnetic resonance imaging (MRI), ELISA, flow cytometry and hematoxylin-eosin (HE) staining. We performed single-cell RNA sequencing (scRNA-seq) analysis of mouse esophageal tissues from acute inflammatory stage (day 7), reparative stage (day 28), and normal mice (day 0). High throughput discoveries were verified using multiplex immunofluorescence (mIF) staining. Results: Our study found that 7 days after radiation, mice presented a significant decrease in body weight and food intake, and an increased T2 signal intensity of esophagus was shown on MRI. The levels of inflammatory cytokines in serum, including TNF-a, IL-1ß, and IL-6, increased at day 7, and the proportion of immune cell infiltration was significantly increased. The HE staining further validated the above results. Although the acute manifestations of RIEI were alleviated at day 28, it remained higher than those at day 0. ScRNA-seq analysis of 28744 cells revealed a population of 13 cell subsets. Myeloid cells and fibroblasts constituted the main cell population at all stages. We identified a subpopulation of fibroblasts with activation of proinflammatory transcriptome at the acute inflammatory stage, in which the expression of Saa3 gene was upregulated. In the reparative stage, S100a4high fibroblast was mainly identified and exhibited a mesenchymal phenotype. Also, we found that the proportion of macrophages and neutrophils in myeloid subsets increased significantly during the acute inflammatory phase, and cytokines, such as Cxcr2, Ccr2 and so on, were released to participate in RIEI process. Meanwhile, myeloid cells facilitate leukocyte recruitment and infiltration in the inflammatory esophagus by interacting with activated endothelial cells. Meanwhile, Atf4, as a hallmark transcription factor in the process of RIEI, could orchestrate immune and inflammatory responses by regulating expression of its target genes in multiple cell types. Conclusion: Here, we present a comprehensive single-cell landscape of different RIEI phases. Collectively, Atf4 may serve as a potential clinical therapeutic strategy for RIEI.
