*2.3. Tumor Proliferation by Macrophage-Secreted Iron Is Suppressed by EC1*

According to our previous observation that tumor-associated MΦ adopt an iron releasing phenotype (Figure 3D), we further established an in vitro setting to analyze the role of macrophage-secreted iron in conferring renal tumor cell growth (Figure 5A).

**Figure 5.** Macrophage-secreted iron induces proliferation and migration of tumor cells in vitro. (**A**) Schematic overview of how to generate conditioned medium from iron-releasing human MΦ. (**B**) Iron amount measured by AAS relative to the total protein amount in the supernatant of primary human MΦ, either left untreated (ctrl) or stimulated with IL-10 (20 ng/mL; 24 h) (*n* = 5). Proliferation of (**C**) CAKI 1 (*n* = 4) and (**D**) 786-O (*n* = 4) cells as well as migration of **(E)** CAKI 1 (*n* = 4) and (**F**) 786-O cells (*n* = 4) upon stimulation with the supernatant of IL-10-stimulated MΦ in the presence or absence of an extracellular chelator (EC1, 100 μM) 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.

As previously published [41,42], IL-10 stimulation of primary human MΦ induced the release of iron into the supernatant measured by AAS (Figure 5B). We next applied macrophage-conditioned supernatants to renal tumor cells CAKI-1 (Figure 5C,E) and 786-O (Figure 5D,F) and observed enhanced proliferation (Figure 5C,D) as well as tumor cell migration (Figure 5E,F) measured by xCELLigence in real-time upon stimulation with IL-10-conditioned media.

To further verify the effect of EC1 on tumor proliferation and migration in the presence of macrophage-secreted iron, we stimulated tumor cells with MΦ-conditioned media supplemented by EC1 (100 μM). In line with our previous observations using EC fluids (Figure 4F–I), EC1 was able to significantly inhibit cellular proliferation and migrations of both CAKI-1 (Figure 5C,E) and 786-O cells (Figure 5D,F).
