A comprehensive set of 15 data panels (A to O) illustrates the efficacy and safety of H I F-2 alpha inhibitors (S S 1.21, S S 3.2, 1.21 S 9 N, and 3.2.16) across multiple cancer models, including H C T 116, C T 26, F a D u, S C C 7, B T-474, E M T 6, and P y M T. Across several mouse models (A, B, D, L), body weights remain stable, indicating that the treatments are well-tolerated without significant systemic toxicity. Tumor growth and weight are consistently reduced in xenograft and syngeneic models (C, G, H, J, N), with the combination of S S 1.21 and Oxaliplatin showing the most pronounced tumor suppression in H C T 116 (C). Molecular and histological analyses further support these findings: immunoblotting (E, K) shows a decrease in H I F-2 alpha and H I F-1 alpha protein levels; C D 31 plus staining (F) reveals reduced tumor vascularization; and sections (I) provide visual confirmation of altered tumor margins. Notably, the E M T 6 model (M) demonstrates a complete lack of tumor growth in rechallenged mice, suggesting the induction of long-term immune memory. Finally, R T-q P C R data (O) confirms that these inhibitors significantly suppress the hypoxia-induced expression of the Adm gene in primary mammary tumor cells under 1 percent oxygen conditions. All data is approximate.
CRC, HNSCC, and BrCa tumor models. Related to Figs. 4 and 5. (A–C) Additional data from the HCT116 tumor model, including mouse BW (A and B) and final tumor weight (C), are shown. Data are presented as the mean ± SD (n = 5) and analyzed by one-way ANOVA with the Sidak multiple comparisons. **P < 0.01; ***P < 0.001. (D–F) Additional data from the CT26 model (presented in Fig. 4. D–F) are shown including: mouse BW (D); HIF-2α immunoblot assay of tumor lysates (E); and CD31+ vessel area (square pixels per field) in tumor sections from mice treated with vehicle or SS1.21 (10 mg/kg QD) (F). Data are presented as the mean ± SD. **P < 0.01 by Student’s t test. (G–I) HNSCC tumor models were studied: FaDu human HNSCC xenografts in nude mice (G) and syngeneic SCC-VII tumors in C3H mice (H). Mice were treated with vehicle or SS1.21 (10 or 20 mg/kg BID IP × 5 days). Tumor volumes are presented as the mean ± SD (n = 5). **P < 0.01, two-way ANOVA with Tukey’s post-test; ns, not significant. Representative hematoxylin-and-eosin–stained FaDu tumor sections from mice treated with vehicle or SS1.21 (20 mg/kg) are shown illustrating resection margins (I). (J) Mice bearing BT-474 human orthotopic breast tumors were treated with vehicle or SS3.2 (10 mg/kg BID IP × 7 days). Data are presented as the mean ± SD (n = 5). *P < 0.05 by two-way ANOVA. (K) BT-474 tumor lysates were subjected to immunoblot assays using antibodies for HIF-1α, HIF-2α, HIF-1β, and β-actin. (L–N) Additional data from the EMT6 model, including mouse BW (L); results of rechallenge experiment in which control naïve mice or previously treated, tumor-free mice received an injection of tumor cells (M); and tumor growth in mice treated with vehicle, 1.21S9N (40 mg/kg BID), or 3.2.16 (40 mg/kg BID) with data presented as the mean ± SD (n = 5); *P <0.05 by two-way ANOVA with Tukey’s post-test (N). RNA and protein extracted from these tumors were analyzed in Fig. 5, I and J, respectively. (O) Primary cancer cells from MMTV-PyMT mammary tumors were treated with vehicle or the indicated HIFi at 20% or 1% O2 for 24 h, and Adm mRNA expression was analyzed by RT-qPCR. Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 by two-way ANOVA with Dunnett’s post-test. Scale bar, 500 µm. Source data are available for this figure: SourceData FS3.