Fig. 6. MM-102 impairs self-renewal, induces apoptosis, and attenuates in vivo tumorigenicity of PCSCs.

a Chemical structure of MM-102. b Schematic illustration depicting the treatment timeline of individual cells of CSC-enriched pancreatic tumorspheres with DMSO (control) and MM-102 for the assessment of H3K4me2 and H3K4me3 protein levels and in vitro functional analysis. The schematic illustration was created with Biorender scientific illustration software. c Western blot analysis of H3K4me2 and H3K4me3 protein levels in SSEA4⁺ A13A cells following treatment with different doses (0 – 75 µM) of MM-102 for 5 days. Total Histone H3 was used as an internal loading control for extracted histones. Data are representative of 2 biologically independent experiments with similar results. d Sphere limiting dilution analysis to examine the sphere-forming capacity of ABCG2⁺ L3.6sl cells treated with 75 µM MM-102 versus DMSO. Data are presented as the mean value ± SD (n = 3 biologically independent experiments). Statistical analysis was performed using two-way ANOVA with multiple comparisons with Tukey correction. e–h Brightfield images demonstrating the effect of MM-102 treatment at a concentration of 75 µM on the sphere-forming ability of ABCG2⁺ L3.6sl (f) and SSEA4⁺ A13A (h) cells versus ABCG2⁺ L3.6sl (e) and SSEA4⁺ A13A (g) cells treated with DMSO (control). Scale bar, 500 µm. i, upper panel: Representative contour plots of flow cytometry analysis of chromatin condensation in ABCG2⁺ L3.6sl cells treated with DMSO (left panel) and 75 µM MM-102 (right panel) for 5 days followed by Vybrant DyeCycle Violet and SYTOX AADvanced staining. i, lower panel: Scatter graphs demonstrating the percentages (%) of viable (left panel) and necrotic/late apoptotic (right panel) cells, as determined by flow cytometry analysis of SYTOX AADvanced and Vybrant DyeCycle Violet-stained ABCG2⁺ L3.6sl cells following DMSO and MM-102 treatments. Data are presented as the mean value ± SEM (n = 6 biologically independent experiments). Statistical analysis was performed using a two-tailed t test with Welch’s correction for unequal variances. j, upper panel: Representative contour plots of flow cytometry analysis of apoptosis following PE-Annexin V and 7-AAD staining of ABCG2⁺ L3.6sl cells treated with DMSO (left panel) and 75 µM MM-102 (right panel) for 5 days. j, lower panel: Graphical presentation of the percentages (%) of viable, early apoptotic, late apoptotic, and necrotic ABCG2⁺ L3.6sl cells treated with DMSO and 75 µM MM-102 for 5 days, as determined by flow cytometry analysis of PE-Annexin V and 7-AAD-stained ABCG2⁺ L3.6sl cells. Data are presented as the mean value ± SEM (n = 6 biologically independent experiments). Statistical analysis was performed using a two-tailed t test with Welch’s correction for unequal variances. k Experimental timeline for testing the in vivo effects of MM-102 in subcutaneous xenografts of ABCG2⁺ L3.6pl cells in athymic mice homozygous for Foxn1<nu>. The schematic illustration was created with Biorender scientific illustration software. l, Body weights (grams) of athymic mice xenografted subcutaneously with ABCG2⁺ L3.6pl cells and intraperitoneally injected with either vehicle or MM-102 (30 or 50 mg/kg/day) every 3 days. Data are presented as the mean value ± SEM (n = 6 mice). Statistical analysis was performed using a two-tailed t test with Welch’s correction for unequal variances. m Volume (mm³) of subcutaneous pancreatic tumors, as assessed by a digital caliper and calculated using the formula: V = 1/2 (Length × Width²). Data are presented as the mean value ± SEM (n = 6 mice). Statistical analysis was performed using a two-tailed t test with Welch’s correction for unequal variances. n Weight (grams) of subcutaneous pancreatic tumors resected from euthanized mice. Data are presented as the mean value ± SEM (n = 6 mice). Statistical analysis was performed using a two-tailed t test with Welch’s correction for unequal variances. Box plots show the median (center line), upper and lower quartiles (box), and range of the data excluding outliers (whiskers). The upper and lower whiskers represent the maximum (non-outlier) and minimum (non-outlier) values, respectively. o Image of resected subcutaneous pancreatic tumors from euthanized mice (n = 6) following treatment with either vehicle or MM-102 (30 or 50 mg/kg/day) every 3 days for 14 days. Corresponding tumor weight (grams) is annotated above each resected tumor.