Fig 2: Cotargeting CAIX/XII and xCT blunts signaling through AMPK to enhance ferroptosis in a pH-dependent manner.(A and B) pHi measurements in hypoxic SUM159PT cells following the indicated treatments for 72 hours. (A) SLC-0111 (0, 25, 50, and 100 µM,), compound 11 (2 µM), and compound 13 (50 µM); (B) NT, untreated; S, S0859 (25 µM); N, NH4Cl (2.5 mM). Cell viability of hypoxic (C) SUM159PT and (D) LM2-4 treated with the indicated concentrations of S0859 and erastin for 72 hours. Cell viability of SUM159PT cells treated with SLC-0111 (50 µM) and erastin (0.5 µM) (E) or S0859 (50 µM) and erastin (F) in the presence of NH4Cl (1, 2.5, 5, and 10 mM) for 72 hours in hypoxia. Indicated protein levels of SUM159PT cells treated with (G) SLC-0111 (12.5 µM) and erastin (0.25 µM) or (H) S0859 (12.5 µM) and erastin (0.25 µM) for 72 hours in hypoxia. Cell viability data of SUM159PT treated with erastin and SLC-0111 (I) or S0859 (J) together with the indicated compounds for 72 hours in hypoxia. AICAR (0.25, 0.5, and 1.0 µM), TOFA (12.5, 25, and 50 µM), and rosiglitazone (ROSI; 1.0, 5.0, and 10 µM). Cell viability data of SUM159PT treated with siRNA to ACC1 together with SLC-0111 (K) or S0859 (L) and erastin for 72 hours in hypoxia. (M) Model depicting role of CAIX and sodium-driven bicarbonate cotransporters in pH homeostasis and suppression of ferroptosis. Bars indicate means ± SEM. Shown are representative experiments from experiments performed at least twice. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical significance was assessed using t test with Welch’s correction (F) or one-way (A, B, E, I, and J) or two-way ANOVA (C, D, K, and L) followed by Tukey’s multiple comparisons test post hoc.

Fig 3: NFS1 suppression combined with CAIX/XII inhibitors triggers ferroptosis.(A to C) Cell viability data of the indicated cell lines following NFS1 depletion in combination with SLC-0111 in the presence or absence of Fer-1 (2 µM) for 72 hours in hypoxia. (A) Representative IncuCyte images of the indicated treatments. Green, cytotoxicity; red, nuclei. (B and C) Quantitation of IncuCyte data for the indicated cell lines treated and cultured in hypoxia for 72 hours. (D and E) Lipid peroxidation (BODIPY C11Ox fluorescence) of the indicated cell lines treated as in (B) and (C) by flow cytometry. (F and G) Cell viability of the indicated cell lines following NFS1 depletion and treatment with compound 13 for 72 hours in hypoxia. (H) Tumor growth curve of orthotopic SUM159PT control (shNS) or NFS1-depleted (shNFS1) tumors treated with vehicle or SLC-0111 following induction of shRNA with Dox. n = 4 to 6 per group. V, vehicle; S, SLC-0111. (I) Waterfall plot depicting percent change in individual tumor volume of the groups color coded as in (H) following 14 days of treatment. (J) Model depicting CAIX inhibition combined with NFS1 depletion triggering ferroptosis. Bars represent means ± SEM (B to H). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical significance was assessed using two-way ANOVA followed by Tukey’s multiple comparison test post hoc (B to G) or by ANOVA followed by Bonferroni correction (H). Shown are representative data from experiments performed at least twice. Scale bar, 50 µm.

Fig 4: SLC-0111 inhibits sunitinib-induced Carbonic Anhydrase IX (CAIX) expression in liver metastases. (a) Representative 3D maximum projections of whole mount liver tissue slices from animals administered agents as indicated, showing rhodamine-labeled dextran (red) and vimentin-positive metastases (cyan). Scale bar = 100 µm. (b) Analysis of vimentin expression in the whole mount liver tissue sections described in panel a. Data show the mean ± standard error of the mean (SEM). n = 9–12 images/group: ** p < 0.01, *** p < 0.001. (c) Representative 3D maximum projections of whole mount liver tissue slices from animals administered agents as indicated, showing rhodamine-labeled dextran (red) and levels of expression of CAIX (green). Scale bar = 100 µm. (d) Analysis of CAIX expression in whole mount liver tissue sections described in panel c. Data show the mean ± SEM. n = 9–12 mages/group. *** p < 0.001.

Fig 5: Sunitinib induces hypoxia and Carbonic Anhydrase IX (CAIX) expression in primary Triple Negative Breast Cancer (TNBC) tumors. (a) Images of MDA-MB-231 LM2-4Luc+ primary tumor tissue sections harvested at increasing tumor volumes from animals administered either vehicle or 60 mg/kg sunitinib and immunohistochemically stained for the indicated markers. Scale bars: upper panels, 1 mm; lower panels, 200 µm. (b to e) Image-based quantification of (b) CD31+ blood vessels, (c) CAIX expression, (d) pimonidazole and (e) proliferation. Data show the mean ± standard error of the mean (SEM). n = 3 animals/group with 10 images/animal. * p < 0.05, *** p < 0.001.