Multiplexed analysis of macrophage polarisation in pulmonary metastases of microsatellite stable colorectal cancer

Abstract

Tumour-associated macrophages (TAMs) express a continuum of phenotypes ranging from an anti-tumoural M1-like phenotype to a pro-tumoural M2-like phenotype. During cancer progression, TAMs may shift to a more M2-like polarisation state, but the role of TAMs in CRC metastases is unclear. We conducted a comprehensive spatial and prognostic analysis of TAMs in CRC pulmonary metastases and corresponding primary tumours using multiplexed immunohistochemistry and machine learning-based image analysis. We obtained data from 106 resected pulmonary metastases and 74 corresponding primary tumours. TAMs in the resected pulmonary metastases were located closer to the cancer cells and presented a more M2-like polarised state in comparison to the primary tumours. Higher stromal M2-like macrophage densities in the invasive margin of pulmonary metastases were associated with worse 5-year overall survival (HR 3.19, 95% CI 1.35-7.55, p = 0.008). The results of this study highlight the value of multiplexed analysis of macrophage polarisation in cancer metastases and might have clinical implications in future cancer therapy.

Keywords: Cancer immunology; Colorectal cancer; Macrophage polarisation; Pulmonary metastases; Tumour-associated macrophages.

Fig. 1

A haematoxylin–eosin-stained section of a pulmonary metastasis illustrating the selection of tissue microarray core locations. The red circles indicate tumour cores from the invasive margin and yellow circles indicate tumour cores from the tumour centre

Fig. 2

Multiplex immunohistochemistry visualisation of macrophage phenotypes and image analysis. A, 6-plex immunohistochemistry image, in which each marker is represented with a unique colour. B, Detection and phenotyping cells into macrophages, tumour cells, and other cells in QuPath bioimage software using machine-learning based algorithms. C, Examples of three macrophages with an M1-like, M2-like, and mixed-like polarisation phenotype. D, The segmentation of tissue compartments into tumour epithelium and stroma. The length of the scale bar is 2.5 µm

Fig. 3

Macrophage polarisation in primary colorectal tumours and the corresponding first resected pulmonary metastases. Comparison of M1- (A) and M2-like macrophage densities (B) and M1:M2 ratios (C) between the primary tumours and pulmonary metastases. Rounds indicate outliers, and crosses indicate extreme outliers. The third and fourth quartile values for epithelial M1:M2 ratios were 203,503,770 and 20,083,912,500 in the tumour centre (TC) and 692,847,301 and 11,470,838,740 in the invasive margin of the primary tumours, respectively. ***p < 0.001, **p < 0.01, *p < 0.05. The statistical significance was tested with the Wilcoxon signed-rank test. Strom = stromal

Fig. 4

Spatial analysis of macrophages in primary tumours and in pulmonary metastases using the nearest neighbour distance (NND) function. A, Pseudocoloured multiplex immunohistochemistry image from an example tissue microarray core and from a smaller tumour region. B, Cell phenotyping maps with nearest neighbour distance analysis from each M1-like and M2-like macrophage to the closest tumour cell. C, Boxplots visualising the nearest neighbour distances (NNDs) from M1-like macrophages and M2-like macrophages to the closest tumour cell across all tumour images of primary tumours and pulmonary metastases. D, Boxplots visualise the distribution of nearest neighbour distances from all macrophages, M1-like macrophages, and M2-like macrophages to the closest tumour cell in primary tumours and pulmonary metastases. The results are based on 27,775 M1-like and 39,386 M2-like macrophages in the pulmonary metastases, and 146,438 M1-like and 83,159 M2-like macrophages in the primary tumours. The statistical significance was tested with the Wilcoxon rank-sum test. ****P < 0.0001

Fig. 5

Kaplan–Meier survival analysis of macrophage densities. The plots present 5-year overall survival curves stratified by epithelial and stromal M1-like and M2-like macrophage densities and M1:M2 ratios in the tumour centre (TC) and the invasive margin (IM) of the first resected pulmonary metastases of CRC. Log-rank tests were applied

Fig. 6

Kaplan–Meier survival analysis of intratumoural heterogeneity (ITH) of macrophage infiltrates. The plots present 5-year overall survival curves stratified by low versus high standard deviations of M1-like and M2-like macrophage densities in the tumour epithelial and stromal compartments of the first resected pulmonary metastases. Log-rank tests were applied