Single Cell Analysis of Macrophage Heterogeneity and NK-Cell Exhaustion in Lewis Lung Cancer Xenograft Tumor

Abstract

Stereotactic body radiation therapy (SBRT) has attracted much attention because of its ability to stimulate anti-tumor immune response. However, the mechanism of SBRT reprogramming the tumor microenvironment remains to be elucidated. Using Lewis lung carcinoma (LLC) xenograft mice model treated with SBRT (8Gy x 3F), multiplex assay was performed to measure serum chemokine levels, and single-cell RNA sequencing was performed to assess tumor microenvironment. The differential expression genes of each cell subcluster were identified by the "Find-All markers" function with default parameters provided by Seurat. Intercellular communication analysis was explored by using CellPhone DB package. The majority of serum chemokines involved macrophage recruitment, including CCL3, CCL4, CCL8, and CCL20, were highly secreted at 7 days after SBRT. Single-cell RNA sequencing of 108,741 cells were contained from 6 mouse Lewis lung carcinoma samples (n = 3 tumors for SBRT, n = 3 tumors pooled for SHAM). Besides Lewis cancer cells, myeloid cells were 57.61% ,70.82% in Sham-irradiation (SHAM) and SBRT while NT and T cells were 20.50%, 7.81% in SHAM and SBRT, respectively. When compared with SHAM group, upregulation of Ccl3, Ccl4, Ccl8 chemokine genes were observed in cancer cells of SBRT group. Differential expression genes analysis showed high expression level of Ccl8 (Log2FC 2.54, p<0.01) in cluster of Mrc1+macrophage. The SBRT group consisted of more Ccl8+Mrc1+macrophages (proportion 36.28% for SBRT, 27.44% for SHAM) and exhausted NK cells (proportion 22.56% for SBRT, 13.70% for SHAM). More importantly, intercellular communication analysis revealed a potential communication network between Ccl8+Mrc1+macrophages and exhausted NK cells. Our results provide a potential therapeutic strategy by disrupting Ccl8+ Mrc1+macrophages and NK-cell interaction to facilitate the stimulation of the anti-tumor immune response by SBRT.