Introduction

Colorectal cancer (CRC) is strongly associated with iron due to the role of gut epithelium in iron uptake and the exposure of luminal epithelial cells to iron, iron trafficking within the tumour microenvironment (TM), and the role of iron in cell function including that of immune cells and tumour cells (1,2).
Tumours do not grow in isolation; they are reliant on the function of other cells within the TM (3). These cells interact with the tumour, providing tumour cells with nutrients including iron (4,5). Within the TM, monocytes are polarised into proinflammatory (M1) macrophages and alternatively activated (M2) macrophages. M1 macrophages are characterised by production of pro-inflammatory cytokines such as IL-12, production of reactive oxygen species, and activation of Th1 responses. These cells are involved in immune surveillance and anti-cancer functions. In contrast, M2 macrophages have an anti-inflammatory phenotype and tumour promotion (6). Tumour associated macrophages that resemble M2 macrophages have been shown to promote angiogenesis, matrix remodelling, and immune suppression (7,8) and have been associated with worse patient outcomes (9).
Iron may affect the tumour cells within the TM by influencing macrophage polarisation. Mice fed an iron-enriched diet increased M2 polarization of macrophages with increase in M2 markers (10). However, the situation is more complex as extravasation of red blood cells during neoangiogenesis resulting in a haemorrhagic environment repolarised the macrophages to an M1 phenotype with the ability to kill cancer cells (8). These results suggest that haemolytic RBCs and iron influence macrophage polarisation and are important players within the TM and an important focus for research in CRC.

Aims & Methods

The study aims were to determine the effects of iron treatments on the differentiation of macrophages within the TM in anaemic patients with CRC.
Normal and tumour tissues were obtained from patients in the IVICA trial (REC reference 11/EM/0237). Patients with CRC and iron deficiency anaemia were randomised to receive oral ferrous sulphate (n=20) or IV ferric carboxymaltose (n=20). Using immunofluorescence microscopy, we identified macrophages as being CD68 positive and demonstrated the expression of TLR2, suggesting that these are M1 macrophages. The pattern of M1 macrophages were confirmed with further experiments as cells that were CD68 and MHC-II (HLA-DR) positive. M2 macrophages were identified as CD68 and CD163 positive cells.

Results

Macrophages expression was significantly higher on tumour tissues from patients who received IV ferric carboxymaltose compared with oral ferrous sulphate (p<0.02). In CRC tissues, macrophages were localised as clusters mainly in the stromal tissue.
The number of M1 polarised macrophages was significantly higher in the TM of patients treated with IV ferric carboxymaltose than in those colorectal cancer patients treated with oral ferrous sulphate (p=0.01).
There was a trend towards an increase in M2 polarised macrophages in the tumour samples of those patients treated with oral iron, however, this was not significant.

Conclusion

This is the first study in humans to investigate the effect of iron treatment on the phenotype of macrophages within the TM. Data demonstrates that IV ferric carboxymaltose may increase the recruitment and / or polarisation of macrophages to an M1 phenotype within the TM in anaemic CRC patients. These results have the potential to influence patients’ management as our work suggests that IV ferric carboxymaltose may be preferable to oral ferrous sulphate.

References

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