Tumor and Peritoneum-Associated Macrophage Gene Signature as a Novel Molecular Biomarker in Gastric Cancer

Abstract

A spectrum of immune states resulting from tumor resident macrophages and T-lymphocytes in the solid tumor microenvironment correlates with patient outcomes. We hypothesized that in gastric cancer (GC), macrophages in a polarized immunosuppressive transcriptional state would be prognostic of poor survival. We derived transcriptomic signatures for M2 (M2_TS, MRC1; MS4A4A; CD36; CCL13; CCL18; CCL23; SLC38A6; FGL2; FN1; MAF) and M1 (M1_TS, CCR7; IL2RA; CXCL11; CCL19; CXCL10; PLA1A; PTX3) macrophages, and cytolytic T-lymphocytes (CTL_TS, GZMA; GZMB; GZMH; GZMM; PRF1). Primary GC in a TCGA stomach cancer dataset was evaluated for signature expressions, and a log-rank test determined overall survival (OS) and the disease-free interval (DFI). In 341 TCGA GC entries, high M2_TS expression was associated with histological types and later stages. Low M2_TS expression was associated with significantly better 5-year OS and DFI. We validated M2_TS in prospectively collected peritoneal fluid of a GC patient cohort (n = 28). Single-cell RNA sequencing was used for signature expression in CD68⁺CD163⁺ cells and the log-rank test compared OS. GC patients with high M2_TS in CD68⁺CD163⁺ cells in their peritoneal fluid had significantly worse OS than those with low expression. Multivariate analyses confirmed M2_TS was significantly and independently associated with survival. As an independent predictor of poor survival, M2_TS may be prognostic in primary tumors and peritoneal fluid of GC patients.

Keywords: biomarker; gastric cancer; liquid biopsy; macrophage; peritoneal metastases.

Figure 1

Association of M2-defining macrophage gene expression with histologic subtype and pTMN stage. (A) Histologic subtypes for intestinal adenocarcinomas, diffuse adenocarcinoma, mucinous adenocarcinoma, and signet ring cell adenocarcinoma are associated with M2-defining signatures. One-way ANOVA, p < 0.001. (B) Within each histologic subtype, M2-defining macrophage signature expression is significantly greater than both M1-defining and T-cell cytolytic signature expression. t-test for each comparison, * p < 0.05. (C) Greater M2-defining macrophage expression is associated with more advanced stages. t-test, p = 0.02. M2_TS, M2 transcriptomic signature. NOS type, not otherwise specified type. TNM, tumor node metastasis. M1_TS, M1 transcriptomic signature. CTL_TS, cytotoxic T-lymphocyte transcriptomic signature.

Figure 2

M2-defining macrophage, but not M1-defining or T-cell cytolytic, signature is associated with survival. The highest quintile of M2-defining macrophage signature expression is associated with worse OS (p = 0.03) and DFI (p = 0.05). High levels of M1-macrophage and T-cell cytolytic signature expression were not associated with OS or DFI (p > 0.05). OS, overall survival. DFI, disease free interval. M2_TS, M2 transcriptomic signature. M1_TS, M1 transcriptomic signature. CTL_TS, cytotoxic T-lymphocyte transcriptomic signature.

Figure 3

M2-defining signature is expressed in the M2 macrophage cell cluster on single-cell analysis of peritoneal fluid samples and is associated with survival in peritoneal fluid samples. (A) UMAP plot displays the cells with higher M2-defining signature expression as purple dot, which is primarily seen in the M2 macrophage cells (CD68⁺CD163⁺). (B) The expression profile of the M2-defining macrophage signature is plotted on the y-axis (log scale) using the single-cell RNA seq expression per a single sample. Each dot represents one single-cell M2-defining signature expression level. Cells tend to cluster in subtypes of M2 macrophages of varying expression of the M2-defining macrophage signature. (C) High (n = 13) and low (n = 15) cohorts can be defined from our entire cohort. (D) High expression of M2-defining macrophage gene signature in M2 defined cells within peritoneal samples is associated with worse overall survival (p = 0.018). UMAP, unform manifold approximation and projection.