Tumor-associated macrophages and risk of recurrence in stage III colorectal cancer

Abstract

Tumor-associated macrophages (TAMs) have a unique favorable effect on the prognosis of colorectal cancer (CRC), although their association with stage-specific outcomes remains unclear. We assessed the densities of CD68⁺ and CD163⁺ TAMs at the invasive front of resected CRC stage III CRC from 236 patients, 165 of whom received post-surgical FOLFOX treatment, and their relationship with disease-free survival (DFS). Associations between macrophage mRNAs and clinical outcome were investigated in silico in 59 stage III CRC and FOLFOX-treated patients from The Cancer Genome Atlas (TCGA). Biological interactions of SW480 and HT29 cells and macrophages with FOLFOX were tested in co-culture models. Low TAM densities were associated with shorter DFS among patients receiving FOLFOX (CD68⁺ , p = 0.0001; CD163⁺ , p = 0.0008) but not among those who were untreated. By multivariate Cox analysis, only low TAM (CD68⁺ , p = 0.001; CD163⁺ , p = 0.002) and nodal status (CD68⁺ , p = 0.009; CD163⁺ , p = 0.007) maintained an independent predictive value. In the TCGA cohort, high CD68 mRNA levels were associated with better outcome (p = 0.02). Macrophages enhanced FOLFOX cytotoxicity on CRC cells (p < 0.01), and drugs oriented macrophage polarization from M2- to M1-phenotype. Low TAM densities identify stage III CRC patients at higher risk of recurrence after adjuvant therapy, and macrophages can augment the chemo-sensitivity of micro-metastases.

Keywords: adjuvant therapy; cancer immunology; colorectal cancer; macrophage polarization; tumor-associated macrophages.

Figure 1

DFS by TAM densities in patients resected for stage III CRC and treated with FOLFOX. High versus low densities of both CD68+ and CD163+ TAM were determined by median of percent immunoreactivity values.

Figure 2

Tumor CD68 mRNA levels and postoperative recurrence in an in silico cohort of patients resected for stage III CRC and treated with FOLFOX. Upper panel: mRNA levels were significantly higher in patients with no recurrence (Fisher's exact, p = 0.02). Line bar: mean of CD68 mRNA. CD68 mRNA expression (high versus low expression) was reported as FPKM. Lower panel: at CART analysis, higher mRNA levels were associated with no recurrence. **p value < 0.01.

Figure 3

Effects of FOLFOX exposure in co‐cultures of CRC cells and macrophages. (A) Viability of colon cancer cells after exposure to FOLFOX agents. Loss of SW480 and HT29 cells viability, as measured in relative luminescence units (RLUs), was significantly enhanced by macrophage co‐culture. (B) When co‐cultured with cancer cells, macrophages exposed to FOLFOX had higher expression of surface markers typical of the M1‐like phenotype and lower expression of M2‐like markers. The median of fluorescence intensity (MFI) for each marker was measured by fluorescence‐activated cell sorting (FACS) in triplicate‐run experiments.