*4.19. Statistical Analysis*

Statistical analysis was performed applying GraphPad Prism software (GraphPad Inc., San Diego, CA, USA, version 8.2.1). Variable distribution was tested for normality using the Kolmogorov-Smirnov test. Respectively, Gaussian distributed, and non-Gaussian distributed patient samples were statistical analyzed using two-tailed paired student's *t*-test or Wilcoxon matched-pairs signed-ranks test. In vitro experiments were analyzed using one-way ANOVA. Cell culture experiments were performed at least three times (independent experiments using technical replicates). Patient samples were used in experiments upon availability. P values were considered significant at \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

#### **5. Conclusions**

This study provides new insights of a significantly altered iron metabolism in renal cell carcinoma. Most of the studied iron-regulated genes are associated with tumor grade and tumor pT-stage. Moreover, our results suggest that tumor-associated macrophages adopt a pro-tumorigenic iron-releasing phenotype through increased expression of FPN. These tumor-associated macrophages are then able to fuel the increased iron demands of tumor cells by secreting iron in the tumor microenvironment.

EC1, a novel iron chelator, specifically scavenges iron in the extracellular space and was able to reverse pro-tumorigenic effects of macrophage-conditioned media on proliferation and migration of cancer cells, including primary patient-derived renal cancer cells. These results might pave the way towards further in vivo studies addressing the possibility to interfere with iron availability in the tumor microenvironment by targeted chelation strategies.

#### **6. Patents**

Issued patent: Tomat E.; Chang, T. M. "Redox-Directed Chelators Targeting Intracellular Metal Ions" U.S. Patent No. 9,486,423, November 8th, 2016.

Pending patent application: Tomat, E.; Chang, T.; Akam, E. A. "Redox-activated Pro-chelators" U.S. Patent Appl. No.: 16/200,286, November 26th, 2018.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6694/12/3/530/s1, Figure S1: Primary human renal tissue verification, Figure S2: Perl's staining of histopathological renal cell carcinoma subtypes. Figure S3: Characterization of the extracellular iron chelator EC1.

**Author Contributions:** Conceptualization: M.J.; Methodology: M.S., C.R., C.M., E.T., E.A.A., M.J.; Software: M.S.; Validation: M.S., C.R., C.M., J.K.M., M.J.; Formal analysis: M.S., C.R., A.U., C.M., E.T., E.A.A., M.J.; Investigation: M.S., C.R., A.U., C.M., J.K.M., E.T., E.A.A., P.B., F.C.R., M.J.; Resources: A.U., P.B., F.R., B.B., M.J.; Data curation: M.J.; Writing—original draft preparation: M.S., E.T., B.B., M.J.; Writing—review and editing, M.S., E.T., B.B., M.J.; Visualization: M.S., M.J.; Supervision: M.J.; Project administration: M.J.; Funding acquisition: E.T., B.B., M.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** MS—PhD scholarship from the Faculty of Medicine, Goethe-University Frankfurt; CR—PhD scholarship from EKFS (Else Kröner-Fresenius-Graduiertenkolleg); BB—Else Kröner-Fresenius Foundation (EKFS), Research Training Group Translational Research Innovation–Pharma (TRIP); MJ—Wilhelm Sander-Stiftung (2017.130.1); ET—U.S. National Institutes of Health (GM127646).

**Acknowledgments:** The authors thank Aline Kraus and Yu-Shien Sung for assistance on the chemical characterization of EC1.

**Conflicts of Interest:** The authors declare no conflict of interest.
