Session Item

Radiotherapy (RT) is a cornerstone treatment in the battle against cancer. However, radioresistance, mainly caused by a hypoxic tumor environment, remains a major hurdle. Recently, ionizing radiation appeared to induce a specific type of programmed cell death, namely, ferroptosis. Ferroptosis is an iron-dependent form of cell death characterized by an accumulation of lipid peroxidation and dysregulated mitochondria. Cancer cells can develop resistance to ferroptosis via diverse defense mechanisms. A multitude of ferroptosis inducers (FINs) have been identified which primarily target the main ferroptosis defense axis: SLC7A11-GSH-GPX4. As RT on its own induces ferroptosis, combination with FINs could be a promising avenue in overcoming radioresistance. In this study we used FIN sulfasalazine (SSZ), a known SLC7A11 inhibitor, to explore the radiomodulatory potential.

Two human colorectal cancer cell lines DLD-1 and HCT116 were included in the study. Cells were treated with the FIN, SSZ, under normoxic and hypoxic conditions. The C11BODIPY dye was used to assess the levels of lipid peroxidation, an indicator for ferroptosis induction. The radiomodulatory properties of SSZ were evaluated by colony formation assay (2D) and multicellular tumor spheroids (3D). In vivo DLD-1 xenografts were treated wither either vehicle, SSZ 250mg/kg, 3*4Gy, or the combination, and tumor size was monitored. Examination of ferroptosis levels within DLD-1 tumors will be performed by IHC staining for 4-HNE.  

Treatment with SSZ did not radiosensitize human colorectal cancer cell lines under normoxic conditions. However, under hypoxic condition a pronounced effect was observed in DLD-1 cells (ER: 1.9) and in HCT116 cells (ER: 1.6). Irradiation alone induced increased levels of lipid peroxidation in both cell lines (up to 3-fold), and combination therapy with SSZ further increased the levels of lipid peroxidation (up to 7.6-fold) in DLD-1 cells, while the levels of lipid peroxidation in HCT116 cells remained unchanged. The radiosensitizing effect was partly reversed by the addition of ferrostatin-1, a ferroptosis inhibitor, in the DLD-1 cells. The enhanced levels of ferroptosis as well as the radiosensitizing effect of SSZ was confirmed in hypoxic 3D tumor spheroids. DLD-1 xenografts that were treated with combination therapy displayed a growth delay of 32 days compared to controls, indicating a synergistic effect between SSZ and RT. 4-HNE staining within isolated tumors is still in progress.

Our preliminary results suggest that the FIN SSZ significantly induced ferroptosis in the DLD-1 cell line but not in the HCT116 cell line. Consequently, the radiomodulatory effect of SSZ was more striking in DLD-1 compared to HCT116. Furthermore, SSZ synergizes with RT in vivo. However, the exact contribution of ferroptosis herein is still being studied. Accordingly, FINS directly targeting defense mechanisms, such as GPX4, will be investigated.