ESTRO 2023 - Abstract Book


Saturday 13 May

ESTRO 2023

L. Kerkhove 1 , F. Geirnaert 1 , A. Rifi 1 , H. Vandenplas 2 , K.L. Law 3 , T. Gevaert 3 , I. Dufait 3 , M. De Ridder 3 1 VUB, Radiotherapy, Brussels, Belgium; 2 UZ Brussel, Medical Oncology, Brussels, Belgium; 3 UZ Brussel, Radiotherapy, Brussels, Belgium Purpose or Objective 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. Materials and Methods 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. Results 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. Conclusion 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. OC-0098 Developing modulators of tumour hypoxia through inhibition of cellular oxygen consumption N. Machado 1 , J. Holt-Martyn 2 , G. Rodriguez-Berriguete 1 , E. Traynor 1 , R. Puliyadi 3 , C. Schofield 2 , G. Higgins 1 1 University of Oxford , Department of Oncology, Oxford, United Kingdom; 2 University of Oxford , Department of Chemistry, Oxford, United Kingdom; 3 University of Oxford , Department of Oncology , Oxford, United Kingdom Purpose or Objective Tumor hypoxia is strongly associated with poor clinical outcomes and radiation therapy resistance (1). Previous studies have demonstrated that reducing the oxygen consumption rate (OCR) of tumour cells can be an effective strategy in increasing local oxygen availability to overcome hypoxia (2-4). Recent screening work by the Higgins lab has identified a prodrug, referred to as compound A, to strongly reduce OCR in vitro (4). However, compound A is completely metabolized in vivo to compound B, which has no effect on OCR (4). In the present work, derivatives of compound A that evade metabolism but maintain OCR inhibition have been assessed for their efficacy as tumour hypoxia modulators. Materials and Methods OCR in Fadu and HCT116 cells is measured after 24 hour treatments with compound A derivatives 719 and 1059, compound A, compound B at 30 µ M, 10 µ M, and 5 µ M. Hypoxia is assessed in HCT116 spheroids using EF5 and quantified by immunofluorescent staining after 24 hour incubation with 719, 1059, compound A, and compound B at 10 µ M and 2 µ M. 719 and 1059 are administered via oral gavage in HCT116 nude mice xenografts to assess tumour hypoxia alleviation via EF5 staining. 719 is delivered once daily at 15 mg/kg for 8 days (n=4-5) and 1059 is delivered twice daily at 60 mg/kg for 7 days (n=7-8). OCR in HCT116 cells is measured after 24-hour treatments with a new, more soluble series of compound A derivatives. Results 719 and 1059 significantly reduce OCR in both cell lines and alleviate hypoxia in spheroids, outperforming compound A at 10 µ M or lower. Oral delivery of 719 and 1059, at dosing schedules determined by toxicity and drug solubility limits, do not reduce hypoxia in vivo . 17 out of the 24 new compound A derivatives, optimized for improved solubility, produce a more than 50% reduction in OCR in HCT116 cells. Conclusion Compound A derivatives 719 and 1059 are potent inhibitors of OCR and alleviate hypoxia in 3D spheroids in vitro. Oral delivery of 719 and 1059 is ineffective in reducing hypoxia in vivo . Further screening in 2D and 3D in vitro models of a pharmacokinetically favorable series of compound A derivatives will guide the selection of a suitable in vivo candidate molecule for tumour hypoxia modification. This work will identify potential hypoxia modifiers to help improve radiotherapy efficacy in clinic. References (1) Wilson WR, et al. Nat Rev Cancer. 2011 Jun;11(6):393-410. (2) Jordan BF, et al. Front Pharmacol. 2012 May;3:94. (3) Zannella VE, et al. Clin Cancer Res. 2013 Dec 15;19(24):6741-50. (4) Ashton TM, et al. Nat Commun. 2016 Jul 25;7:12308.

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