Infiltration Characteristics of Tumor-associated Macrophage Subsets in Colorectal Cancer Liver Metastases and Their Association with Postoperative Recurrence

Abstract

Background: Tumor-associated macrophages (TAMs) are a dominant myeloid population within the colorectal cancer liver metastasis (CRLM) microenvironment, yet whether specific TAM subsets are spatially organized in a manner relevant to postoperative recurrence remains unclear. This study aimed to characterize the spatial infiltration of TAM subsets in CRLM and to assess their association with early postoperative recurrence. Methods: In this retrospective cohort study, 142 patients with CRLM who underwent hepatectomy at a single center between January 2024 and August 2025 were included (follow-up cutoff: 31 August 2026). Multiplex immunofluorescence was performed for CD68, CD163, CD80, SPP1, HLA-DRB5, and TREM2. Regions were defined relative to the tumor–stroma boundary as the tumor center (TC), invasive margin (IM), and peritumoral region (PT), and region-specific spatial quantification was performed. The primary endpoint was early recurrence (≤6 months). Kaplan–Meier analysis, log-rank tests, and Cox regression were used; the multivariable model adhered to an events-per-variable (EPV) threshold of ≥10. Results: Early recurrence occurred in 38 patients (26.8%). TAM subsets displayed substantial spatial heterogeneity: SPP1⁺ TAM were enriched at the invasive margin (156 ± 54 cells/mm²), approximately 2.4-fold higher than in the tumor center (64 ± 28 cells/mm²) (P<0.001). In multivariable Cox regression, invasive-margin SPP1⁺ TAM density (per 1-SD increase) was independently associated with shorter recurrence-free survival (RFS) (HR=1.78, 95% CI 1.16–2.73, P=0.004), whereas the CD80/CD163 ratio (PT region) was independently associated with longer RFS (HR=0.62, 95% CI 0.43–0.89, P=0.011). Early recurrence was associated with a microenvironment characterized by SPP1⁺ TAM enrichment and lower HLA-DRB5⁺ TAM density. Conclusions: TAM subsets show marked spatial heterogeneity in CRLM, and SPP1⁺ TAM enrichment at the invasive margin is associated with an increased risk of early postoperative recurrence, which may inform recurrence risk stratification, pending prospective validation. Given the retrospective design and limited follow-up, these findings remain exploratory.