**2. Results**

#### *2.1. Iron Homeostasis Is Altered in RCC*

In order to determine whether renal iron homeostasis is altered in RCC, we first analyzed mRNA expression of several iron-dependent genes, including *FPN*, *FTL*, *FTH*, *IRP2,* and *TfR1* in whole tissue homogenates of our patient cohort (Table 1).

**Table 1.** Patient cohort. The patient cohort is composed of 64 patients, grouped into three major renal tumor types ccRCC, pRCC, and chRCC. Patient parameters age, sex, pT-stage and grade are depicted in the table.


We found a significantly increased mRNA expression in tumor tissue compared to adjacent healthy tissue for all genes (Figure 1A–E). We performed hematoxylin staining in both healthy adjacent tissue and RCC subtypes of clear cell RCC (ccRCC), papillary RCC (pRCC) as well as chromophobe RCC (chRCC) that were included in our patient cohort (Figure S1A), and analyzed the *CAIX* mRNA expression, which was shown to be upregulated in more than 90% of RCC cases [32] (Figure S1B). Accordingly, *CAIX* mRNA expression was significantly upregulated in ccRCC and pRCC tumor subtypes, whilst varying in chRCC compared to adjacent healthy tissue. We next analyzed the mRNA expression of iron-dependent genes in relation to tumor grade (G1-G2 vs. G3-G4) and tumor pT-stage (pT1 pT2 vs. pT3-pT4). *FPN* mRNA expression was significantly increased in all tumor pT-stages and tumor grades compared to adjacent

healthy tissue with the notion of enhanced expression in higher tumor pT-stage (Figure 1F). This expression pattern was also observed for mRNA expression of *TfR1* (Figure 1G).

**Figure 1.** Expression of iron-regulated genes in human renal cancer samples. mRNA expression normalized to the housekeeping gene *18S* in whole tissue homogenates of renal tumor tissue and adjacent healthy tissue of (**A**) *FPN* (*n* = 48), (**B**) *TfR1* (*n* = 47), (**C**) *FTL* (*n* = 48), (**D**) *FTH* (*n* = 48), and (**E**) *IRP2* (*n* = 46). (**F**–**J**) Left: mRNA expression of (**F**) *FPN*, (**G**) *TfR1*, (**H**) *FTL*, (**I**) *FTH,* and (**J**) *IRP2* correlated to low (G1-G2) and high (G3-G4) tumor grade. Right: mRNA expression of (**F**) *FPN*, (**G**) *TfR1*, (**H**) *FTL*, (**I**) *FTH,* and (**J**) *IRP2* correlated to low (pT1–pT2) and high (pT3–pT4) tumor pT-stage. Number of tested patients differ between genes due to patients with failed measurements of initially low sample RNA amount. No samples have been excluded as outliers. Graphs are displayed as means ± SEM with \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

For *FTL*, *FTH,* and *IRP2,* we found an increased mRNA expression in lower tumor grades (G1-G2) and lower tumor pT-stage (pT1–pT2), but either similar or lower expression within the group of higher tumor grades (G3-G4) and higher tumor pT-stage (pT3–pT4; Figure 1H–J).

Since RCC subtypes significantly differ regarding in the prognosis and treatment [33], we analyzed the mRNA expression of iron-dependent gene expression in patients with ccRCC, pRCC, or chRCC of our cohort (Figure 2A–E, left panel). While the defined iron-dependent genes were significantly upregulated within the ccRCC subgroup in comparison to adjacent healthy tissue, mRNA expression in the pRCC and the chRCC subtype varied, depending on the analyzed gene. Expression of *FPN*, *FTH*, and *IRP2* was higher in all RCC subtypes compared to adjacent healthy tissue, whereas *FTL* remained unaltered in the chRCC subtype and *TfR1* was lower in pRCC subtypes. In order to verify our data, especially regarding patients diagnosed with pRCC and chRCC, where less patients were included in our cohort, we analyzed publically available TCGA KIRC (ccRCC), KIRP (pRCC), and KICH (chRCC) data sets (Figure 2A–E, right panel). RNA expression in the TCGA data sets confirmed a significant upregulation of *FPN*, *FTL*, and *FTH* in ccRCC. In pRCC, *FPN*, *FTL,* and *FTH* are significantly higher expressed, while *IRP2* remained unaltered. Our data regarding reduced *TfR1* expression in pRCC and unaltered *FTL* expression in chRCC was corroborated using the TCGA data analysis.

As we showed an altered iron homeostasis in all histopathological subtypes, we next aimed at looking into the iron distribution in RCC tissue. We first analyzed the iron amount of tumor and adjacent healthy tissues by AAS measurements. Tumor tissue showed an overall significantly higher iron amount than adjacent healthy renal tissue (Figure 3A). When analyzing the histopathological subtypes, both ccRCC and chRCC showed a higher iron amount compared to adjacent healthy tissue, whereas in pRCC the total iron amount remained nearly unaltered (Figure 3B). To address the question of iron localization within the tissues, Perl's staining of tumor versus adjacent healthy tissue slides was used. In line with our AAS analysis, healthy renal tissue showed a low amount of iron deposits appearing in blue. Compared to the healthy adjacent tissue, a more intense staining in ccRCC was observed, whereas iron deposits in pRCC remained low (Figure 3C and Figure S2A–D). Intriguingly, the iron load in chRCC varies considerably between different patients (Figure S2D) with the notion of overall enhanced iron deposits in tumor tissue compared to adjacent healthy tissue. In ccRCC tissue, we hypothesize that the highly intense blue-colored cells might be tumor cells, whereas the diffuse positive staining around long-shaped cells in the stroma might be iron secreted by MΦ. There are also other positive-stained cells in the stroma that appear much smaller, which we believe might be lymphocytes that are also able to handle iron in the tumor stroma as previously described by Marques et al. in mammary carcinoma [34]. For pRCC we only detect low amounts of overall Perl's staining, with localized positive staining mostly in tumor cells, whereas we observed high amounts of iron deposits in chRCC, mostly within the tumor stroma. We and others previously showed that tumor cells are prone to adopt an iron retaining phenotype, whereas cells from the tumor stroma such as MΦ rather adopt an iron mobilization and iron releasing phenotype [34,35]. In order to verify the location of iron within different tumor compartments in RCC tissues, we sorted both tumor cells and tumor-associated MΦ from tumor tissue of all histopathological RCC subtypes and compared them to sorted epithelial cells and MΦ isolated from adjacent healthy tissue (Figure 3D,E). A significantly reduced intracellular iron amount in MΦ isolated from ccRCC and pRCC tissues was observed, whereas MΦ from chRCC tissues showed similar intracellular iron levels as cells from adjacent healthy tissue (Figure 3D). In contrast, tumor cells showed a significant increased iron amount in ccRCC and pRCC compared to adjacent renal epithelial cells. In chRCC, iron amount in tumor cells showed a larger variation resulting in a non-significant increase compared to renal epithelial cells isolated from adjacent healthy tissue (Figure 3E).

**Figure 2.** Profile of iron-regulated genes in histopathologically distinct RCC subtypes. mRNA expression of renal tumor and adjacent healthy samples in clear cell (ccRCC), papillary (pRCC), and chromophobe (chRCC) RCC of own patient cohort (left) compared to mRNA expression acquired from the TCGA database applying the ccRCC-KIRC (*n* = 70), pRCC-KIRP (*n* = 31), and chRCC-KICH (*n* = 23) datasets (right). Analyzed genes include (**A**) *FPN*, (**B**) *TfR1*, (**C**) *FTL*, (**D**) *FTH,* and (**E**) *IRP2*. Own cohort is normalized to housekeeping gene *18S* expression. Graphs are displayed as means ± SEM with \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

**Figure 3.** Iron homeostasis and distribution is altered in RCC. (**A**) Iron load normalized to protein amount in whole tissue homogenates of renal cancer tissue in comparison to adjacent healthy renal tissue measured by AAS (*n* = 31). (**B**) Iron load in whole tissue homogenates of clear cell (ccRCC; *n* = 17), papillary (pRCC; *n* = 7), and chromophobe (chRCC; *n* = 7) RCC in comparison to corresponding healthy renal tissue measured by AAS. (**C**) Representative pictures of Perl's staining of RCC tissue and adjacent healthy renal tissue of ccRCC, pRCC, and chRCC. Representative pictures (scale bar: 200 μm) with corresponding detailed pictures (scale bar: 100 μm) are given. (**D,E**) Macrophages (MΦ) and CD326+ cells were isolated by FACS-sorting from RCC tissue and adjacent healthy tissue. Intracellular iron load of (**D**) MΦ and (**E**) either tumor cells (TC) or epithelial cells from adjacent healthy tissue of ccRCC (*n* = 7), pRCC (*n* = 13), and chRCC (*n* = 4) measured by AAS. Statistical analysis was performed comparing tumor to adjacent healthy tissue within the histopathological subtypes. Graphs are displayed as means ± SEM. \* *p* < 0.05, \*\* *p* < 0.01.
