*2.2. Iron Promotes Renal Tumor Cell Growth*

In order to test the role of iron released into the tumor stroma, we generated extracellular fluids (EC fluids) from both tumor tissue as well as adjacent healthy tissue (Figure 4A). First, we analyzed the iron amount in EC fluids by AAS and observed significantly higher iron amounts in EC fluids isolated from tumor tissue as compared to EC fluids from adjacent healthy tissue (Figure 4B). We then stimulated renal tumor cells CAKI-1 (Figure 4C) and 786-O (Figure 4D) as well as primary patient-derived tumor tubular epithelial cells (TTEC; Figure 4E) with tumor EC fluids. Cellular proliferation was analyzed applying xCELLigence real-time measurements. Results showed that all tested cell lines as well as primary tumor cells positively responded to treatments with tumor EC fluids and augmented cellular proliferation upon stimulation.

**Figure 4.** Extracellular iron induces proliferation and migration of tumor cells in vitro. (**A**) Schematic overview of how to generate extracellular (EC) fluids (ECF) from primary human renal tumor and adjacent healthy tissue. (**B**) Iron load measured by AAS relative to the total protein amount of EC fluids of ccRCC tissue compared to adjacent healthy renal tissue (*n* = 8). Proliferation of (**C**) CAKI-1 (*n* = 7), (**D**) 786-O (*n* = 8), and (**E**) primary human tumor tubular epithelial cells (TTEC) upon stimulation with EC fluids in vitro measured with the xCELLigence system (*n* = 8). Proliferation of **(F)** CAKI 1 (*n* = 4) and (**G**) 786-O (*n* = 4) cells as well as migration of (**H**) CAKI 1 (*n* = 4) and (**I**) 786-O cells (*n* = 4) upon stimulation with EC fluids in the presence or absence of an extracellular chelator (EC1, 100μM) or dimethyl sulfoxide (DMSO) as negative control measured with the xCELLigence system. Graphs are displayed as means ± SEM with \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

To further verify the role of extracellular iron on tumor proliferation and migration, we stimulated tumor cells with EC fluids in the presence of a specific extracellular chelator (EC1). This novel compound was designed for extracellular chelation as it features an established iron-binding unit as well as a negatively charged group to hinder cell membrane permeation (Figure S3A). In particular, the tridentate chelating unit of EC1 includes a thiosemicarbazone moiety that is common to many anti-proliferative iron chelators [36,37]; however, the incorporation of a negatively charged sulfonate

group significantly limits the ability of EC1 to cross cellular membranes. As a result, EC1 is expected to chelate iron only in the extracellular space without affecting intracellular iron levels. The iron binding abilities were validated using optical absorption spectroscopy (Figure S3B). The effects of EC1 on cellular viability and proliferation were tested in vitro in CAKI-1 and 786-O cells in comparison to the unspecific chelator 2,2- -dipyridine (2- 2-DPD) and the intracellularly activated prochelator (TC3-S)2 [38–40]. Whereas EC1 showed no effect at concentrations up to 100 μM with regard to both, viability (Figure S3C,D) and cellular proliferation (Figure S3E,F) under basal growth conditions, both 2- 2-DPD and (TC3-S)2 showed increasingly adverse effects at higher concentrations regarding cellular viability and anti-proliferative capacity due to the fact that both are able to chelate intracellular iron. In contrast, EC1 showed toxicity effects only at very high concentrations (500 μM), which might be due to non-specific side effects. Supplementation of EC fluids with EC1 (100 μM) in order to specifically block iron secreted to the supernatant resulted in a significant inhibition of cellular proliferation and migration of both CAKI-1 (Figure 4F,H) and 786-O cells (Figure 4G,I).
