New Insights into Macrophage Polarization and its Prognostic Role in Patients with Colorectal Cancer Liver Metastasis

Abstract

Background: As liver metastasis is the most common cause of mortality in patients with colorectal cancer, studying colorectal cancer liver metastasis (CLM) microenvironment is essential for improved understanding of tumor biology and to identify novel therapeutic targets.

Methods: We used multiplex immunofluorescence platform to study tumor associated macrophage (TAM) polarization and adaptive T cell subtypes in tumor samples from 105 CLM patients (49 without and 56 with preoperative chemotherapy).

Results: CLM exhibited M2 macrophage polarization, and helper T cells were the prevalent adaptive T cell subtype. The density of total, M2 and TGFβ-expressing macrophages, and regulatory T cells was lower in CLM treated with preoperative chemotherapy. CLM with right-sided primary demonstrated enrichment of TGFβ-expressing macrophages, and with left-sided primary had higher densities of helper and cytotoxic T cells. In multivariate analysis, high density of M2 macrophages correlated with longer recurrence-free survival (RFS) in the entire cohort [hazard ratio (HR) 0.425, 95% CI 0.219-0.825, p=0.011) and in patients without preoperative chemotherapy (HR 0.45, 95% CI 0.221-0.932, p=0.032). High pSMAD3-expressing macrophages were associated with shorter RFS in CLM after preoperative chemotherapy.

Conclusions: Our results highlight the significance of a multi-marker approach to define the macrophage subtypes and identify M2 macrophages as a predictor of favorable prognosis in CLM.

Keywords: colorectal; immunofluorescence; liver metastasis; macrophage; multiplex; polarization; survival.

Figure 1. Workflow of multiplex immunofluorescence digital image analysis.

A, Multiplex immunofluorescence image of a representative tumor core. B, Tissue segmentation was performed by training the software using representative examples from each compartment (red-tumor, green-stroma). C, Cell limits were defined, and cells were individually identified. D, Cells were phenotyped based on expression of surface proteins. E-J, Representative examples of macrophages (E) and their specific phenotypes (F-J) based on co-expression of markers. K-P, Representative examples of adaptive T cells (K) and their specific phenotypes (L-P) based on co-expression of markers.

Figure 2. Density of different immune cell types.

A, Density of cells expressing individual markers in the multiplex immunofluorescence panel. B, Density of macrophage and adaptive T cell subtypes defined by marker co-expression. The horizontal bars indicate median values.

Figure 3. Recurrence-free survival (RFS) according to density of macrophage subtypes.

A-D, RFS according to density of (A) total macrophages (CD68+), (B) TGFβ-expressing macrophages (CD68+TGFβ+), (C) M2 macrophages (CD68+CD163+), and (D) M2 macrophages and TGFβ-expressing macrophages. Numbers before cell phonotype descriptions are numbers of cells per mm². E, Univariate, and multivariate Cox regression analysis for RFS (n=105). Median value used as cutoff.

Figure 4. Recurrence-free survival (RFS) according to density of macrophage subtypes in patients treated with and without preoperative chemotherapy.

A-B, RFS in patients treated without preoperative chemotherapy (n=49) according to density of (A) M2 macrophages (CD68+CD163+) and (B) TGFβ-expressing macrophages (CD68+TGFβ+). Numbers before cell phonotype descriptions are numbers of cells per mm². C, Univariate, and multivariate Cox regression analysis for RFS in patients treated without preoperative chemotherapy (n=49). D-E, RFS in patients treated with preoperative chemotherapy (n=56) according to density of (A) M2 macrophages and (B) pSMAD3-expressing macrophages (CD68+pSMAD3+). Numbers before cell phonotype descriptions are numbers of cells per mm². F, Univariate, and multivariate Cox regression analysis for RFS in patients treated with preoperative chemotherapy (n=56).