A growing body of evidence suggests that macrophage polarization dictates the expression of iron-regulated genes. Polarization towards iron sequestration depletes the microenvironment, whereby extracellular pathogen growth is limited and inflammation is fostered. In contrast, iron release contributes to cell proliferation, which is important for tissue regeneration. Moreover, macrophages constitute a major component of the infiltrates in most solid tumors. Considering the pivotal role of macrophages for iron homeostasis and their presence in association with poor clinical prognosis in tumors, we approached the possibility to target macrophages with intracellular iron chelators. Analyzing the expression of iron-regulated genes at mRNA and protein level in primary human macrophages, we found that the iron-release phenotype is a characteristic of polarized macrophages that, in turn, stimulate tumor cell growth and progression. The application of the intracellular iron chelator (TC3-S)2 shifted the macrophage phenotype from iron release towards sequestration, as determined by the iron-gene profile and atomic absorption spectroscopy (AAS). Moreover, whereas the addition of macrophage supernatants to tumor cells induced tumor growth and metastatic behavior, the supernatant of chelator-treated macrophages reversed this effect. Iron chelators demonstrated potent anti-neoplastic properties in a number of cancers, both in cell culture and in clinical trials. Our results suggest that iron chelation could affect not only cancer cells but also the tumor microenvironment by altering the iron-release phenotype of tumor-associated macrophages (TAMs). The study of iron chelators in conjunction with the effect of TAMs on tumor growth could lead to an improved understanding of the role of iron in cancer biology and to novel therapeutic avenues for iron chelation approaches.

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Background We aimed at clarifying the role of lipocalin-2 (LCN-2) in clear-cell renal cell carcinoma (ccRCC). Since LCN-2 was recently identified as a novel iron transporter, we explored its iron load as a decisive factor in conferring its biological function. Methods LCN-2 expression was analysed at the mRNA and protein level by using immunohistochemistry, RNAscope® and qRT-PCR in patients diagnosed with clear-cell renal cell carcinoma compared with adjacent healthy tissue. We measured LCN-2-bound iron by atomic absorption spectrometry from patient-derived samples and applied functional assays by using ccRCC cell lines, primary cells, and 3D tumour spheroids to verify the role of the LCN-2 iron load in tumour progression. Results LCN-2 was associated with poor patient survival and LCN-2 mRNA clustered in high- and low-expressing ccRCC patients. LCN-2 protein was found overexpressed in tumour compared with adjacent healthy tissue, whereby LCN-2 was iron loaded. In vitro, the iron load determines the biological function of LCN-2. Iron-loaded LCN-2 showed pro-tumour functions, whereas iron-free LCN-2 produced adverse effects. Conclusions We provide new insights into the pro-tumour function of LCN-2. LCN-2 donates iron to cells to promote migration and matrix adhesion. Since the iron load of LCN-2 determines its pro-tumour characteristics, targeting either its iron load or its receptor interaction might represent new therapeutic options.

Carcinogenesis is a multistep process. Besides somatic mutations in tumor cells, stroma-associated immunity is a major regulator of tumor growth. Tumor cells produce and secrete diverse mediators to create a local microenvironment that supports their own survival and growth. It is becoming apparent that iron acquisition, storage, and release in tumor cells is different from healthy counterparts. It is also appreciated that macrophages in the tumor microenvironment acquire a tumor-supportive, anti-inflammatory phenotype that promotes tumor cell proliferation, angiogenesis, and metastasis. Apparently, this behavior is attributed, at least in part, to the ability of macrophages to support tumor cells with iron. Polarization of macrophages by apoptotic tumor cells shifts the profile of genes involved in iron metabolism from an iron sequestering to an iron-release phenotype. Iron release from macrophages is supposed to be facilitated by ferroportin. However, lipid mediators such as sphingosine-1-phosphate, released form apoptotic tumor cells, upregulate lipocalin-2 (Lcn-2) in macrophages. This protein is known to bind siderophore-complexed iron and thus, may participate in iron transport in the tumor microenvironment. We describe how macrophages handle iron in the tumor microenvironment, discuss the relevance of an iron-release macrophage phenotype for tumor progression, and propose a new role for Lcn-2 in tumor-associated macrophages.

Accumulating evidence suggests that iron homeostasis is disturbed in tumors. We aimed at clarifying the distribution of iron in renal cell carcinoma (RCC). Considering the pivotal role of macrophages for iron homeostasis and their association with poor clinical outcome, we investigated the role of macrophage-secreted iron for tumor progression by applying a novel chelation approach. We applied flow cytometry and multiplex-immunohistochemistry to detect iron-dependent markers and analyzed iron distribution with atomic absorption spectrometry in patients diagnosed with RCC. We further analyzed the functional significance of iron by applying a novel extracellular chelator using RCC cell lines as well as patient-derived primary cells. The expression of iron-regulated genes was significantly elevated in tumors compared to adjacent healthy tissue. Iron retention was detected in tumor cells, whereas tumor-associated macrophages showed an iron-release phenotype accompanied by enhanced expression of ferroportin. We found increased iron amounts in extracellular fluids, which in turn stimulated tumor cell proliferation and migration. In vitro, macrophage-derived iron showed pro-tumor functions, whereas application of an extracellular chelator blocked these effects. Our study provides new insights in iron distribution and iron-handling in RCC. Chelators that specifically scavenge iron in the extracellular space confirmed the importance of macrophage-secreted iron in promoting tumor growth.