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. 2024 Oct 29;221(11):e20231832. doi: 10.1084/jem.20231832

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© 2024 Hsu et al.

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Figure 10. — Pharmacological inhibition of IMPA1 or IMPDH2 in combination with enzalutamide efficiently eradicates CRPC progression. (A) Colony formation assay of 22RV1 cells upon vehicle (Veh), 2 or 5 μM of enzalutamide (Enza), 0.5 or 1 μM of MPA, 0.5 or 1 μM of MZB, and 20 or 40 μM of LiCl treatment for 14 days. (B) Quantification of the number of colonies from 22RV1 by ImageJ is shown in A. (C) MTT assay of 22RV1 cells upon vehicle (Veh), 10 μM of enzalutamide (Enza); 250 nM, 500 nM, or 1 μM of MPA treatment for 24 h. (D) MTT assay of TRAMP-C2 cells upon vehicle (Veh), 10 μM of enzalutamide (Enza); 250 nM, 500 nM, or 1 μM of MPA treatment for 24 h. (E) MTT assay of 22RV1 cells upon vehicle (Veh), 10 μM of enzalutamide (Enza); 2, 5, or 10 μM of MZB treatment for 24 h. (F) MTT assay of TRAMP-C2 cells upon vehicle (Veh), 10 μM of enzalutamide (Enza); 2, 5, or 10 μM of MZB treatment for 24 h. All statistical data are shown as the mean ± SEM of three independent experiments for each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, non-significant by two-tailed unpaired t test. (G) Male nude mice subcutaneously injected with 22RV1 cells were intraperitoneally injected with vehicle (Veh), 5 mg/kg of enzalutamide (Enza) daily for 21 days, 200 mg/kg of MZB daily for 7 days; no treatment for 4 days; 100 mg/kg of MZB daily for 10 days or 200 mg/kg of LiCl daily for 21 days, and tumor volume was measured with indicated days. Five mice were included in each experimental group. (H) Male NSG mice subcutaneously injected with PDX prostate tumor were intraperitoneally injected with vehicle (Veh), 5 mg/kg of enzalutamide (Enza) daily for 21 days, 200 mg/kg of MZB daily for 7 days; no treatment for 4 days; 100 mg/kg of MZB daily for 10 days or 200 mg/kg of LiCl daily for 21 days, and tumor volume was measured with indicated days. Five mice were included in each experimental group. (I) Surgically castrated NOD/SCID mice subcutaneously injected with 22RV1 cells were intraperitoneally injected with vehicle or 5 mg/kg of enzalutamide (Enza), 200 mg/kg of LiCl, 100 mg/kg of MZB, 5 mg/kg of enzalutamide (Enza) in combination with 200 mg/kg of LiCl and 5 mg/kg of enzalutamide (Enza) in combination with 100 mg/kg of MZB three times a week for 6 wk and tumor volume was measured with indicated days. Five mice were included in each experimental group. (J) Tumor weight from I was measured on day 57. At least three xenograft tumors in each group were quantified. **, P < 0.01; ***, P < 0.001 by two-tailed unpaired t test. (K) Body weight from I was measured as indicated days. Five mice were included in each experimental group. (L) H&E staining of prostate tissues from surgically castrated TRAMP mice at the age of 6 mo administrated with 5 mg/kg of enzalutamide (Enza), 100 mg/kg of MZB, 200 mg/kg of LiCl, 5 mg/kg of Enza combination with 100 mg/kg of MZB and 5 mg/kg of Enza combination with 200 mg/kg of LiCl three times a week for 18 wk. Five mice were included in each experimental group. Scale bar, 100 μm. (M) Histological analysis for the phenotype of carcinoma in surgical castrated TRAMP mice upon the administration of indicated inhibitors. Each group includes 5 mice. (N) Tumor organoids from TRAMP mice upon Veh, 5 μM of Enza, 1 μM of MPA, 1 μM of MZB, or 500 μM of LiCl treatment for 4 days. Scale bar, 100 μm. (O) Quantification of organoid number from N by Image J was shown as the mean ± SEM of three independent experiments for each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed unpaired t test. (P) Tumor organoids from PDX tumors with Veh, 5 μM of Enza, 1 μM of MZB, or 500 μM of LiCl treatment for 4 days. Scale bar, 50 μm. (Q) Quantification of organoid number from P by ImageJ was shown as the mean ± SEM of three independent experiments for each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed unpaired t test.