**4. Materials and Methods**

#### *4.1. Ethics*

Investigations were conducted in accordance with the ethical standards according to the Declaration of Helsinki and to national and international guidelines. Primary human tumor and adjacent healthy tissues were obtained from 64 patients with the approval of the ethics committees of the Goethe-University Hospital Frankfurt am Main (04/09 UGO 03/10) and the Philipps-University Hospital Marburg (122/14). Patients gave their written informed consent prior to surgery (UCT 122/14 and 04/09 UGO 03/10).

#### *4.2. Participants*

Patients included in this study underwent nephrectomy or partial nephrectomy for renal lesions histopathologically diagnosed with renal cancer between 2016 and 2019 at University Hospitals Frankfurt am Main and Marburg (see Table 1). Patients underwent preoperative staging either by computed tomography or Magnetic resonance imaging and surgery was performed before receiving other therapy. Tissue was collected immediately after surgery and processed for single cell suspensions, fixed in 4% paraformaldehyde (PFA) or stored at −80 ◦C. Pathological examination was performed by independent pathologists applying the UICC TNM classification of malignant tumors [51].

#### *4.3. RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)*

Total RNA was extracted from homogenized tissue samples using peqGold RNAPure (VWR, Darmstadt, Germany, 732-3312) and transcribed using Maxima First Strand cDNA synthesis kit (Thermo Fisher, Dreieich, Germany, K1642). Gene expression profiles were determined by qPCR using the SYBR Green Supermix (Bio-Rad, Munich, Germany, 1725006CUST) on a CFX-Connect real-time-PCR detection system (Bio-Rad). Results were quantified using the Bio-Rad CFX-Manager (Bio-Rad, version 3) with 18S mRNA expression as housekeeping control. All primers except TfR1 Primer (Qiagen, Hilden, Germany, QT00094850) are listed in the supplemental information file and were purchased from Biomers (Ulm, Germany).

#### *4.4. Data baSe Analysis*

To show mRNA expression of *FPN*, *FTL*, *FTH*, *TfR1* and *IRP2* in different renal cancer subtypes, gene expression data of the Cancer Genome Atlas were analyzed (https://portal.gdc.cancer.gov/). Expression data of TCGA files were used of the following data sets: "Tumor Kidney Renal Clear Cell Carcinoma" (KIRC, *n* = 533), "Tumor Kidney Renal Papillary Cell Carcinoma" (KIRP, *n* = 290), and "Tumor Kidney Chromophobe" (KICH, *n* = 66). Cases with tumor and adjacent renal healthy tissue data available were included in the analysis (KIRC: *n* = 70; KIRP: *n* = 31; KICH: *n* = 23).

Kaplan-Meier plots were generated using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). The dataset "Tumor Kidney Renal Clear Cell Carcinoma" (*n* = 533) was chosen. Default settings of the KaplanScan including a log rank comparison between the groups were used to determine an optimum survival cut-off as described in the portal. The resulting p-value as well as the Bonferroni correction of the log rank comparison are included in the plots.

#### *4.5. Atomic Absorption Spectroscopy (AAS)*

Iron measurements were performed as previously described [35]. Whole tissue homogenates where either measured as whole homogenates and normalized to total protein amount or underwent FACS sorting with the final cell suspension being analyzed for its iron content and normalized to the total number of sorted cells.
