Dominique Higgins

Ferroptosis sensitivity in cancer cells is directly governed by cellular metabolism and nutritional inputs, including amino acid availability, lipid composition, and redox homeostasis. This suggests that dietary interventions might systemically shift the metabolic landscape of tumors toward ferroptotic susceptibility. Specifically, dietary restriction of methionine and cysteine, iron supplementation, selenium or lipid modulation, ketogenic diets, and fasting-mimicking regimens each target distinct aspects of the ferroptosis regulatory network and offer pharmacologic parallels that converge on complementary mechanisms. In glioblastoma (GBM), preclinical studies demonstrate that restriction of dietary methionine and cysteine sensitizes gliomas to ferroptosis and synergizes with glutathione peroxidase 4 (GPX4) inhibitors, while radiation itself triggers ferroptotic cell death. Emerging data extend these principles across multiple tumor types, including pancreatic, gastric, esophageal, and colorectal cancers, as well as leukemia, suggesting broad applicability. Here, we synthesize the rapidly growing literature that links these nutritional interventions to ferroptosis sensitization in cancer, with particular focus on GBM as a paradigmatic treatment-resistant malignancy.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumor in adults, with incidence peaking in later life. It is commonly treated with surgery followed by administration of ionizing radiation and the DNA alkylating agent temozolomide (TMZ). Even though this regimen confers some progression-free survival, there is essentially no cure with the median survival with the standard of care being about 12 months. Currently, several alternative approaches are being developed to improve upon this outcome. We have already shown EdU alone effectively treats GBM, and we now study efficacy of TMZ+EdU combination therapy. TMZ+EdU significantly improves antitumor efficacy compared to either single-agent therapy against GBM cell lines in vitro , against three different orthotopic GBM xenograft models, and against passage-zero patient GBM tumor tissues engrafted within an organotypic brain slice culture (OBSC)-based platform. Together, these data suggest that EdU could be effective alongside standard of care TMZ in patients with GBM. STATEMENT OF SIGNIFICANCE (50-WORD): By simultaneously engaging distinct DNA repair pathways, combination of TMZ and EdU produced unprecedented survival benefits and synergistic effects in preclinical GBM models, offering a promising new avenue in the treatment of GBM.

Figure S1: Illustration of the proposed futile cycle induced by the incorporation of EdU by DNA polymerases and removal of it by excision repair. EdU gets incorporated into the genome of highly proliferative GBM cells in the brain. Then, EdU-containing DNA is recognized and removed by nucleotide excision repair, leaving the DNA gap to be filled in. During the resynthesis, EdU reincorporates into this repair patch and the futile cycle repeats, leading to apoptosis and thus, suppression of the growth of GBM tumor.

Figure S2: Features of glioblastoma models used to assess therapeutic potential of EdU. The features in red are less than desirable, blue are moderately desirable, and green are ideal. As such, the compounds were tested on multiple cell culture platforms to supplement the weaknesses of individual platforms.

Figure S5: Semi-quantitative maximum intensity projection image of untreated GBM8 (Red), an mCherry-expressing patient-derived glioblastoma cell line, seeded on OBSC. Following a 4-day culture in brain slice media without EdU, GBM8-seeded OBSCs were fixed in 4% paraformaldehyde for an additional 72 hours, stained for EdU (Green) via click chemistry and imaged by confocal microscopy to observe cell proliferation (Red), nuclear staining (Blue), and EdU presence (Green).

Figure S4: Semi-quantitative Z-stack images of OBSC-seeded GBM8 (red), an mCherry-expressing patient-derived glioblastoma cell line, treated with 1000 μmol/L EdU (Green). Following a 3-day treatment with EdU, GBM8-seeded OBSCs were fixed in 4% paraformaldehyde for 72 hours, stained for EdU via click chemistry, and imaged by confocal microscopy to observe nuclear staining (Blue) and treatment efficacy (Red and Green). (see Materials & Methods for details).

Glioblastoma (GBM) is the most common type of malignant central nervous system tumor, and more than 300,000 people are diagnosed with GBM worldwide annually. Based on its recognition as damage by nucleotide excision repair, we now repurpose the DNA labeling agent 5-ethynyl-2′-deoxyuridine (EdU) as a treatment for GBM. We tested the efficacy of EdU in several different model systems, including not only GBM cell lines in in vitro cell culture and in vivo orthotopic mouse models of GBM, but also against living, uncultured tumor tissues of patients with GBM grown within our organotypic brain slice culture (OBSC) ex vivo platform. When compared with the standard-of-care drug temozolomide (TMZ) in in vitro GBM cell survival assays, EdU displayed ED50 values orders of magnitude lower than those of TMZ in all five GBM tumor lines tested. Against two in vivo orthotopic brain tumor models, EdU significantly extended survival relative to controls. EdU efficacy against a panel of patient GBMs largely correlated with the clinical Ki-67 status of each tumor, save for one tumor that remained unresponsive to treatment with both EdU and TMZ. Overall, these data suggest that (i) EdU has potential to be repurposed as an anticancer therapeutic and is especially adept at killing rapidly proliferating cells with low off-target toxicity; (ii) the OBSC platform can measure nuanced differences in efficacy of experimental therapeutics on heterogeneous patient tumor tissues; and (iii) OBSCs can continue to help identify potential responders and nonresponders to EdU treatment via functional precision testing of patient tumors ex vivo.

Figure S3: (A) Number of mice bearing intracranial allografts of CT2A and xenografts of U251 cells, nontreated or treated with different doses of EdU or thymidine, showing different mortality observations. Mice bearing intracranial allografts of CT2A and xenografts of U251 cells showed body weight loss (BW) as the main mortality observation, followed by crowning (CR),brain seizure (BS), moribund (MO, unknown cause, related symptoms: e.g. Bulging Eyes (BE)) and edema/distended abdomen (E/DA). (B-C) Histogram of mice bearing intracranial allografts of CT2A and xenografts of U251 cells, nontreated or treated with different doses of EdU or thymidine, showing onset of different mortality observations over the course of the study.

Glioblastoma (GBM) is the most common type of malignant central nervous system tumor, and more than 300,000 people are diagnosed with GBM worldwide annually. Based on its recognition as damage by nucleotide excision repair, we now repurpose the DNA labeling agent 5-ethynyl-2′-deoxyuridine (EdU) as a treatment for GBM. We tested the efficacy of EdU in several different model systems, including not only GBM cell lines in in vitro cell culture and in vivo orthotopic mouse models of GBM, but also against living, uncultured tumor tissues of patients with GBM grown within our organotypic brain slice culture (OBSC) ex vivo platform. When compared with the standard-of-care drug temozolomide (TMZ) in in vitro GBM cell survival assays, EdU displayed ED50 values orders of magnitude lower than those of TMZ in all five GBM tumor lines tested. Against two in vivo orthotopic brain tumor models, EdU significantly extended survival relative to controls. EdU efficacy against a panel of patient GBMs largely correlated with the clinical Ki-67 status of each tumor, save for one tumor that remained unresponsive to treatment with both EdU and TMZ. Overall, these data suggest that (i) EdU has potential to be repurposed as an anticancer therapeutic and is especially adept at killing rapidly proliferating cells with low off-target toxicity; (ii) the OBSC platform can measure nuanced differences in efficacy of experimental therapeutics on heterogeneous patient tumor tissues; and (iii) OBSCs can continue to help identify potential responders and nonresponders to EdU treatment via functional precision testing of patient tumors ex vivo.