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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 2. — IMPA1-derived inositol is required for maintaining ALDH^high PCSCs and 3D prostate tumor organoids from CRPC TRAMP mouse model. (A) The flow chart shows an experimental outline to demonstrate the tumor-initiating ability of ALDH^high cells sorted from LNCaP or PC3 cells stably expressing shLuc or shIMPA1 (#1 and #2), followed by transplanting 10⁴, 10³, and 10² cells into male nude mice for each group. (B and C) The image of tumor formation from limiting dilution transplantation using LNCaP (B) and PC3 (C) cells. (D and E) Table outlining differences in tumor-initiating frequency of ALDH^low/− or ALDH^high LNCaP cells (D) and PC3 cells (E) upon limiting dilution transplantation. Tumors arose from transplantation of 10⁴, 10³, and 10² ALDH^low/− or ALDH^high LNCaP cells upon IMPA1 loss. Tumor-initiating frequency was calculated by the ELDA software application for limiting dilution analysis (Hu and Smyth, 2009). (F) The flow chart shows prostate tumor organoids are established from male TRAMP mice, followed by infection with lentivirus shRNAs of IMPA1. (G) Representative images show the growth of prostate tumor organoids from male TRAMP mice at the age of 6 mo with indicated days. Scale bar, 200 μm. (H) H&E staining and IHC staining with IMPA1 antibody of prostate organoids from WT and TRAMP mice at the age of 6 mo. Scale bar, 100 μm for H&E staining; 50 μm for IHC staining. (I) Immunoblotting of tumor organoids from male TRAMP mice with indicated antibodies. Immunoblotting data were verified in at least two independent experiments. (J) Representative images of tumor organoids expressing shLuc or shIMPA1 (#1, #2 and #3) from male TRAMP mice. Scale bar, 200 μm. (K) Quantification of number of tumor organoids from J was shown as the mean ± SEM of three independent experiments for each group. ***, P < 0.001 by two-tailed unpaired t test. (L) Tumor organoids from male TRAMP mice expressing shLuc or shIMPA1 with vehicle (Veh) or 25 μM of inositol (Ins) treatment. Scale bar, 200 μm. (M) Quantification of the number of tumor organoids from L for TRAMP mice was shown as the mean ± SEM of three independent experiments for each group. **, P < 0.01; ***, P < 0.001 by two-tailed unpaired t test. Source data are available for this figure: SourceData F2.