*2.4. Macrophage-Derived Iron Is Exported Via the Iron Exporter FPN, Which Is Positively Associated with Poor Patient Outcome*

We next asked whether the iron exporter FPN was expressed in tumor-associated MΦ. Therefore, FPN protein expression in MΦ by flow cytometry (Figure 6A) was measured, showing higher FPN expression in both tumor stroma (Figure 6B) as well as tumor-associated MΦ (Figure 6C) compared to stroma and MΦ from adjacent healthy tissue. In order to localize FPN protein within the tissue, multiplex-immunohistochemistry was applied, combining CD163 as macrophage marker, FPN, and DAPI as nuclear stain (Figure 6D), showing enhanced co-localization of CD163 and FPN in tumor tissue compared to adjacent healthy tissue. Taking our previous *FPN* mRNA data into consideration that showed enhanced *FPN* expression in tumor tissue compared to adjacent healthy tissue (Figures 1A and 2A), we next questioned the association of *FPN* expression with tumor grade (Figure 6E) and tumor (Figure 6F) of our own cohort (upper part) compared to the TCGA data set (lower part). In line with the TCGA data set, we observed association of FPN mRNA expression only with lower tumor grade (Figure 6E). For tumor pT-stage (Figure 6F), a positive association with lower tumor pT-stages (pT1–pT2) was noticed, which was more pronounced in higher tumor pT-stages (pT3–pT4). However, TCGA data suggests higher FPN expression in patients with low tumor grade (G1–G2) and tumor pT-stage (pT1–pT2). Accordingly, low tissue FPN expression correlated with a lower overall survival probability analyzed by the R2: Genomics Analysis and Visualization Platform applying the 'Tumor Kidney Renal Clear Cell Carcinoma—TCGA-533data set (Figure 6G).

**Figure 6.** *Cont*.

**Figure 6.** Tumor-associated MΦ express enhanced FPN protein. (**A**) FACS panel how to gate CD45<sup>+</sup> immune cells and CD326<sup>+</sup> epithelial/tumor cells. Immune cells were further gated for CD33+/HLA-DR<sup>+</sup> cells of which CD64<sup>+</sup> and MerTK<sup>+</sup> MΦ were sub-selected. Cells were subsequently analyzed for their FPN protein expression, displayed as MFI (mean fluorescence intensity). (**B**) FPN protein expression as MFI of CD45- /CD326- stroma cells (*n* = 26). (**C**) FPN protein expression as MFI of MΦ in tumor tissue compared to adjacent healthy tissue (*n* = 26). (**D**) Representative pictures for the MΦ marker CD163 and FPN protein expression in tumor tissue compared to healthy adjacent tissue applying confocal laser scanning miscroscopy. DAPI was used as nuclear stain. Scale bar: 200 μm. *FPN* mRNA expression normalized to *18S* expression correlated to (**E**) low (G1–G2) and high (G3–G4) tumor grade and (**F**) low (pT1–pT2) and high (pT3–pT4) tumor pT-stage in our patient cohort (upper panels; *n* = 48) compared to the TCGA data base, applying the ccRCC-KIRC data set (lower panels; *n* = 70). **(G)** Kaplan-Meier curve of high or low *FPN* expression from the R2 bioinformatics platform using the ccRCC-KIRC data set. Graphs are displayed as means ± SEM with \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

#### **3. Discussion**

We present evidence that iron metabolism is significantly altered in renal cancer. We observed elevated iron deposits in renal tumor tissue compared to adjacent healthy tissue as well as enhanced expression of iron-regulated genes in tumor tissue isolated from patients with renal cancer compared to adjacent healthy tissue. As iron is of importance for essentially all tumor hallmarks, we further investigated the role of iron in determining tumor cell proliferation and migration. In this setting, we also described and characterized the use of a novel extracellular iron chelator that scavenges iron in the extracellular space, thereby providing a valuable tool to investigate its role in the tumor microenvironment.

Numerous studies support a positive association between increased iron levels and cancer development, whereby cancer cells evolved specialized mechanisms for iron acquisition, storage, and mobilization in order to ensure their enhanced metabolic turnover [43]. Despite the fact that both availability as well as distribution of iron is strictly regulated under healthy conditions, cancers exert a profoundly dysregulated iron-handling capacity with altered expression of iron-regulated genes [15–17,44,45]. These observations are corroborated by the present study, detecting enhanced expression of iron-regulated genes in renal tumor tissue as compared to adjacent healthy tissue, which was most prominent in ccRCC. This effect was further confirmed by TCGA data base analysis. In this regard, we also found that cancer cells isolated from patients with renal cancer showed enhanced iron sequestration compared to their healthy counterparts.

Taking the unique role of the kidney in iron physiology into consideration [4], several markers, including erythropoietin [46] have been tested in RCC. Regardless of the initial promising effects, they only showed low predictive value. There are still no specific and reliable tumor markers neither for RCC diagnosis nor for monitoring post-operative disease outcome. Despite the apparent association of RCC with the development of systemic anemia in RCC patients [47], the role of iron in human RCC carcinogenesis is largely unknown and was only scarcely investigated so far. Because of the growing evidence on their tumor-promoting effects, the expression of iron-regulated genes could become an important factor among the markers of tumorigenesis. Along these lines, Greene at al. recently showed an association of TfR1 expression and RCC progression [7], with TfR1 levels being highest in benign primary tumors, subsequently dropping during the course of disease progression. TfR1 levels were therefore inversely associated with worse survival, but independent of tumor pathology. In line, we observed overall enhanced iron amounts in tumor tissue as compared to adjacent healthy tissue for all investigated renal cancer subtypes. Interestingly, iron amounts in chRCC varied considerably between different patients and needs to be further addressed in follow-up studies including higher patient number. Intriguingly, we also found initial differences in iron levels in adjacent healthy tissue for each histopathological subtype. These observations might arise both from the original localization of the tissue for individual samples as well as result from different basal iron levels of each individual patient. Therefore, healthy control tissue has to be controlled carefully to avoid misleading interpretation.

Nonetheless, modulating the iron-retaining tumor phenotype reduced growth and progression of both human and mouse carcinomas [48]. The use of iron chelators in the treatment of cancer inhibited DNA synthesis and caused a G1-S-phase cell cycle arrest, attenuated epithelial-mesenchymal-transition, and promoted cancer cell apoptosis [39]. Furthermore, chemically-induced and oncogene-driven cancer models corroborated these findings and stressed the relevance of iron for tumor development [49,50]. Numerous studies investigated methods to interfere with iron-handling in cancer cells, either by directly modulating iron-regulated genes [43] or by the use of iron chelators [37]. Nevertheless, a detailed knowledge of the effects of chelators within the tumor microenvironment (and on potential iron sources thereof) is still lacking [43]. In this study, we identified enhanced iron levels in extracellular fluids of tumor tissue in comparison to healthy adjacent tissue, suggesting that cells of the tumor microenvironment secreted iron in extracellular fluids. Due to their important role in tumor development and iron handling, we proposed that MΦ might adopt a pro-tumorigenic iron-releasing phenotype, whereby tumor growth is favored. Since MΦ are central players in systemic iron homeostasis, they have evolved unique mechanisms to recycle, store, and release iron to their local microenvironment. However, our data suggest differences in total tissue iron levels versus iron amounts of both MΦ and tumor cells for histopathological renal cancer subtypes. We hypothesize that even if the overall amount of iron is not changed in tumor tissue compared to adjacent healthy tissue in pRCC patients, the different distribution of iron within the tissue and in cells of the tumor mass, i.e. MΦ or tumor cells might add to the characteristics of iron as a pro-tumoral factor.

Macrophage iron homeostasis is functionally coupled to their heterogeneity and plasticity, with their polarization status being reflected also by their expression profiles of iron-regulated genes. We previously showed that treatment of MΦ with LPS/IFNγ enhanced the retention of iron within the cell, whereas stimulation with anti-inflammatory cytokines such as IL-10 or IL-4 induced the

release of iron [41]. This observation falls in line with a typical cytokine/chemokine profile of differentially polarized MΦ. We used this setting also in the present study to generate supernatant from IL-10-polarized iron-releasing MΦ. We tested the effect of iron, which was released by MΦ, in combination with an iron chelator that specifically binds iron in the extracellular space. EC1, which was designed and synthesized specifically for this study, is a thiosemicarbazone chelator featuring a sulfonate group that is negatively charged near neutral pH. While the tridentate (*O,N,S*) binding unit (see Figure S3A) ensures high-affinity iron coordination, the negative charge on the scaffold was incorporated to limit or hamper cellular membrane permeability. This strategy is particularly advantageous for the study of iron with respect to the crosstalk between TAMs and cancer cells. We found that the addition of EC1 reversed the positive effect of macrophage-conditioned media on the proliferation and migration of cancer cells. Although the exact molecular speciation of iron released in the macrophage supernatant remains to be determined, these experiments indicated that this iron pool is accessible by small-molecule chelators and could represent a hitherto unrecognized effect of these antiproliferative compounds in the tumor microenvironment.

Our previous studies using intracellularly active pro-chelators underscore the importance of macrophage-released iron for tumor cell proliferation [41]. Intriguingly, current research focuses primarily on the role of iron and iron-chelation therapy in tumor cells, whereas detailed knowledge on the crosstalk between tumor cells and tumor-associated MΦ as a possible source of iron is lacking. Taking into account that the presence of MΦ in tumor tissue is closely linked to tumor progression, we further analyzed the expression of the iron exporter FPN as a determinant of the iron releasing capacity of MΦ. We found enhanced expression of FPN in tumor MΦ compared to MΦ of adjacent healthy tissues, which was significantly associated with tumor pT-stage. However, these data could not be corroborated by TCGA data base analysis regarding overall patient survival. This discrepancy might arise from low cohort size of analyzed patients. Furthermore, it might also be necessary to distinguish FPN expression in stromal cells versus tumor cells as compared to whole tissue analysis. Recently, it was shown that tumor-associated MΦ also secrete iron in form of FT, which, in turn, stimulated tumor cell proliferation [44]. FTL expression in MΦ was further described as an independent prognostic marker in node-negative breast cancer. However, in the present study, we did not observe significant changes in FT protein expression in tumor-associated MΦ compared to MΦ isolated from adjacent healthy tissue. Recently, Marques et al. observed that FT expression was elevated in tumor-infiltrating lymphocytes, whereas no changes were detected for FT expression in tumor-associated MΦ [34]. The crucial implication of MΦ in tumor development and their role in iron distribution within the tumor microenvironment represents an important area of investigation in contemporary cancer biology.

Collectively, the results of the present study indicate that iron homeostasis is significantly disturbed in renal cancer with most of the investigated iron-regulated genes being associated with tumor grade and tumor pT-stage. Moreover, we observed that iron availability in the tumor microenvironment might be controlled by tumor-associated MΦ, which adopt an iron-release phenotype through increased expression of FPN. Application of chelators that are able to specifically scavenge iron in the extracellular space confirmed the importance of macrophage-secreted iron in promoting tumor cell proliferation and migration.

Future experimental in vivo studies should address the possibility to either interfere with iron availability in the tumor microenvironment or use macrophage-targeted chelation strategies. Moreover, more research is needed with regard to the questions of: i) the molecular networks that allow tumor cells to actually take up, store, and utilize iron and ii) the release of tumor cell-derived mediators that re-program stromal cells, i.e., MΦ, to serve as an iron source in order to maintain their enhanced metabolism and growth.
