**The Disturbed Iron Phenotype of Tumor Cells and Macrophages in Renal Cell Carcinoma Influences Tumor Growth**

**Matthias Schnetz 1, Julia K. Meier 1, Claudia Rehwald 1, Christina Mertens 1, Anja Urbschat 2, Elisa Tomat 3, Eman A. Akam 3, Patrick Baer 4, Frederik C. Roos 5, Bernhard Brüne 1,6,7,8 and Michaela Jung 1,\***


Received: 11 December 2019; Accepted: 23 February 2020; Published: 25 February 2020

**Abstract:** 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.

**Keywords:** renal cell carcinoma; iron; macrophages; chelation therapy

#### **1. Introduction**

Iron is the most abundant transition metal in the human body and drives a variety of mechanisms considered as hallmarks of cancer. Due to its role as critical cofactor for the rate-limiting step of DNA synthesis, iron controls cell division, DNA repair, and chromatin remodeling [1]. Iron is essential for basic cellular processes such as mitochondrial respiration and the enhanced metabolic turnover under cancerous conditions is controlled by iron-sulfur cluster proteins [2]. Considering the poor bioavailability of iron and its potent role in tumorigenesis, the interplay of different proteins important for iron import, storage, and export has to be tightly regulated through the interplay of various proteins, including the major iron storage protein ferritin with its subunits ferritin light chain (FTL) and ferritin heavy chain (FTH), the iron exporter ferroportin (FPN), transferrin receptor 1 (TfR1) for iron uptake, and iron-regulatory proteins 1 and 2 (IRP1/2) [3].

The kidney plays a unique role in systemic iron homeostasis by filtering and reabsorbing iron as well as providing the main body source of erythropoietin, which promotes hemoglobin synthesis [4]. It was previously shown that renal iron overload in anemic patients requiring chronic transfusions enhanced the incidence of renal cell carcinoma (RCC) development [5]. Repeated injections of iron led to RCC development with increased metastasis to the lungs and lymph nodes in experimental models [6]. Recently, the expression of TfR1 was associated with progression and mortality in clear cell RCC (ccRCC), identifying TfR1 as a novel RCC biomarker and potential therapeutic target [7]. Despite these compelling observations and the fact that RCC is one of the 15 most common cancers in humans as well as the third most common cause of death among urological cancers in 2018 [8], the role of iron for renal cancer was not investigated in detail so far. As RCC is considered to be resistant against conventional chemo- and radiation therapy, medical therapeutic options are currently still limited, thus making nephrectomy the first treatment approach in localized disease [9]. For metastatic disease state, treatment options include systemic therapy with multitarget tyrosine kinase inhibitors (TKIs), including sunitinib, cabozantinib, and pazopanib as well as mammalian target of rapamycin (mTOR) inhibitors such as everolimus or temsirolimus, offering only modest benefits [10]. Novel promising approaches for the treatment of metastatic RCC include immunotherapy and immune checkpoint inhibitors (ICI) targeting the cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death-1 (PD-1) with monoclonal antibodies [11]. Herein, besides to monotherapy a combinatory immunotherapy with checkpoint inhibitors has recently been approved on the base of a clinical phase-3 trial [12]. However, it is clear that there is still an urgent need for a deeper understanding of the molecular processes underlying RCC, which could provide new strategies to interfere during cancer therapy or might help to better determine patient prognosis.

Based on its concentration-dependent toxicity under physiological conditions, cellular iron homeostasis has to be strictly regulated [13]. This balance is shown to be compromised in the tumor microenvironment [14]. The malignant state of cancer cells is associated with a deregulation in cellular iron homeostasis, particularly in the expression of iron-regulated genes to fuel their higher metabolic iron demand needed for division, growth, and survival [14]. Cancer cells of various tumor entities develop an iron retaining phenotype by upregulating FTL, FTH [15,16], TfR1 [7,17], and IRP1/2 [18], while downregulating the iron exporter FPN [17]. These alterations result in increased tumor growth, aggressiveness and a poor patient outcome [14,19]. However, it still remains partly unclear how cancer cells acquire iron from the tumor microenvironment. One of the key players of iron homeostasis are macrophages (MΦ), which play a dual, activation-dependent role in iron homeostasis [20]. While classical, pro-inflammatory MΦ sequester iron to restrict iron availability for bacterial growth [21], alternatively activated anti-inflammatory MΦ recycle iron from dying cells by enhanced phagocytic activity [22]. Due to their physiological function, alternatively activated MΦ promote tissue repair, cell proliferation, and angiogenesis [23]. In the context of carcinogenesis, tumor associated MΦ (TAM) are major players when looking at abundance [24] and pro-tumoral function [25]. TAMs show characteristics of both pro-inflammatory MΦ that create an inflammatory environment during early stages of tumor development as well as anti-inflammatory MΦ [25,26]

during later stages that suppress anti-tumor immunity and stimulate tumor neovascularization as well as metastasis [27,28]. Accordingly, TAMs were shown to positively associate with tumor progression and worse patient prognosis [29–31].

Although the control of iron availability in the tumor microenvironment seems to be crucial for tumor development, the distribution of iron within cellular compartments of the tumor, in particular tumor cells and TAMs, as well their association with tumor outcome have not been investigated so far in renal cancer. In the present study, we provide evidence that iron-dependent genes are highly expressed in renal cancer and are associated with tumor pT-stage (tumor size and invasion as defined by UICC) and tumor grade. We further show that TAMs adopt an iron-release phenotype with increased expression of the iron exporter FPN, whereas tumor cells retain intracellular iron. *In vitro* assays with patient-derived extracellular fluids as well as novel extracellular iron chelators showed the iron-dependence of renal tumor growth and metastasis.
