Comprehensive single-cell analysis demonstrates radiotherapy-induced infiltration of macrophages expressing immunosuppressive genes into tumor in esophageal squamous cell carcinoma

Abstract

Radiotherapy (RT) combined with immunotherapy is promising; however, the immune response signature in the clinical setting after RT remains unclear. Here, by integrative spatial and single-cell analyses using multiplex immunostaining (CODEX), spatial transcriptome (VISIUM), and single-cell RNA sequencing, we substantiated the infiltration of immune cells into tumors with dynamic changes in immunostimulatory and immunosuppressive gene expression after RT. In addition, our comprehensive analysis uncovered time- and cell type-dependent alterations in the gene expression profile after RT. Furthermore, myeloid cells showed prominent up-regulation of immune response-associated genes after RT. Notably, a subset of infiltrating tumor-associated myeloid cells showing PD-L1 positivity exhibited significant up-regulation of immunostimulatory (HMGB1 and ISG15), immunosuppressive (SIRPA and IDO1), and protumor genes (CXCL8, CCL3, IL-6, and IL-1AB), which can be targets of immunotherapy in combination with PD-L1. These datasets will provide information on the RT-induced gene signature to seek an appropriate target for personalized immunotherapy combined with RT and guide the timing of combination therapy.

Fig. 1.. Spatial identification of immune cell infiltration in the tumor microenvironment after radiotherapy.

(A) Workflow of this research. See also figs. S1 to S3. For the specialty analyses (CODEX and VISIUM), the data were obtained from a single patient for each pre-radiotherapy (RT) and post-RT sample. (B) Hematoxylin and eosin (H&E) observation and Uniform Manifold Approximation (UMAP) visualization of esophageal squamous cell carcinoma (ESCC) cell, stromal cell, and immune cell clusters in resected ESCC tissue. (C) Expression patterns of signature genes in distinct ESCC clusters. Clusters 5 to 10 showed the ESCC part in resection. A numerical heatmap is shown in fig. S4A. (D) H&E observation and UMAP visualization of ESCC cell, stromal cell, and immune cell clusters in resected tissue after RT. (E) Expression patterns of signature genes in distinct ESCC clusters. Clusters 5 to 10 indicated the resected ESCC tumor after RT. A numerical heatmap is shown in fig. S4B. (F and G) RT increased immune cell infiltration into ESCC. Multicolor immunohistochemistry (IHC) staining in ESCC tissue after resection (F) and resection after RT (G). Scale bars, 20 μm. The low-power field is shown in fig. S4C. (H) Ratio of immune cells in the tumor/stroma. The absolute number of each cell type is shown in fig. S4 (E and F).

Fig. 2.. Characterization of gene expression profiles in the tumor microenvironment (TME) of esophageal squamous cell carcinoma (ESCC) after radiotherapy.

(A) Identification of the microenvironment surrounding ESCCs after radiotherapy (RT). Multicolor immunohistochemistry (IHC) staining showed immune cell infiltration, major histocompatibility complex (MHC) class I expression [human leukocyte antigen A (HLA-A)], DNA damage (γH2AX), and cell proliferation (Ki67). (B) Workflow of integrated spatial analysis. The TME was identified by IHC, and TME transcriptome data were extracted from the IHC-merged field in VISIUM. H&E, hematoxylin and eosin. (C) Gene expression of MHC class I (HLA-A), MHC class II (HLA-DPB1), PD-L1 (CD274), and STAT1 according to 10x VISIUM (****P < 1 × 10⁻¹⁶, *P < 0.05, Bonferroni-adjusted Wilcoxon test; ns, no significance). Figure S5A shows gene expression in the total and IHC fields. (D) Differentially expressed gene (DEG) analysis between the Re and RT groups. Volcano plot showing gene expression in each contoured field in Fig. 2B. (E) Gene enrichment analysis showed up-regulated biological pathways in the viable ESCC field after RT. (F) Pathways enriched in non-RT and after RT tissue (adjusted P < 0.01). (G) Transcription factors that were significantly enriched in non-RT and post-RT tissues (P < 1 × 10⁻⁶).

Fig. 3.. Single-cell RNA sequencing (scRNA-seq) uncovers the up-regulation of immune response genes in myeloid cells by radiotherapy.

(A) Workflow of scRNA-seq analysis. (B) Uniform Manifold Approximation (UMAP) of major cell clusters based on scRNA-seq data after integration. (C) Proportions of the cell clusters annotated in (B). (D to G) Expression of representative markers of the indicated biological class as assessed by scRNA-seq. The grayscale bar indicates the time point during radiotherapy (RT): white, pre-RT; light gray, during RT; dark gray, immediately post-RT; black, post-RT. The red arrowhead shows immune-inhibitory genes. The blue arrowhead shows tumor progression–related genes. IFNs, interferons; ILs, interleukins.

Fig. 4.. Characterization of the immune response gene expression profile in myeloid cells with time after radiotherapy.

(A) Uniform Manifold Approximation (UMAP) of subclusters in the myeloid cell cluster. (B) Bubble charts of the percentage of cells within the indicated myeloid cell subclusters that express immune coinhibitory, interleukin (IL) and interferon (IFN), chemokine, and human leukocyte antigen (HLA) genes and the corresponding average gene expression level. A larger dot indicates a higher percentage of cells expressing a particular gene; a darker color dot indicates a higher average gene expression level. DCs, dendritic cells.

Fig. 5.. Identification of PD-L1–positive myeloid cells showing high expression of multiple immune-inhibitory genes after radiotherapy.

(A) Gene expression signature of myeloid cells from non-radiotherapy (RT) and post-RT patients. Myeloid cells from patients after RT were classified as PD-L1–negative or PD-L1–positive. Bubble charts of total myeloid cells and non-RT (1,1493 cells), PD-L1–negative (15,294 cells), and PD-L1–positive (5,266 cells) myeloid cells after RT; PD-L1–positive myeloid cells showed high expression of immune checkpoint inhibitor (ICI) target genes (red arrowhead) and other protumor genes (blue arrowhead). (B) Differentially expressed genes (DEGs) between non-RT total myeloid cells and PD-L1–positive myeloid cells after RT. DEGs were defined by P < 0.05 (Bonferroni-adjusted Wilcoxon test) and fold change > 2. (C and D) GSEA of DEGs in PD-L1–positive myeloid cells after RT and total myeloid cells. (****P < 1 × 10⁻¹⁶, Bonferroni-adjusted Wilcoxon test; ns, no significance). (E) Identification of PD-L1–expressing cells after RT by multicolor immunohistochemistry (IHC) staining. In addition to PD-L1, the upper line shows CD68, CD163, PanKRT, CD4, CD8, CD20, FOXP3, CD56, and CD11c expression for cell orientation. The lower line shows the ICI target genes PD-1, IDO1, LAG3, and TIGIT. Figures S7 and S8 show the low-power field for each antibody staining experiment. (F) Graphical summary of tumor microenvironment (TME) alterations after RT in esophageal squamous cell carcinoma (ESCC) tissue.