Figure 2 from A RAS(ON) Multi-Selective Inhibitor Combination Therapy Triggers Long-term Tumor Control through Senescence-Associated Tumor-Immune Equilibrium in Pancreatic Ductal Adenocarcinoma
Combined RAS and CDK4/6 inhibition drives tumor cells into a senescence-like state. A, Scheme of experimental design for B–D and Supplementary Fig S3A–S3I (KPC1-ZsGreen orthotopic transplant into wild-type C57Bl/6 mice). B, Representative images of pancreatic tumor tissues following detection of β-galactosidase activity using the chromogenic substrate X-Gal. C, Quantification of IF staining of pancreatic tumor tissues for β-galactosidase activity in mice 4 hours, 3, 7, or 14 days after treatment initiation. Each dot represents an individual mouse (average of 3–5 ∼120,000 µm2 regions per mouse). Where data from two separate experiments were available, mice from experiments were merged (n = 3–8 mice per treatment group). Statistical testing: Ordinary one-way ANOVA, comparing preselected pairs of columns (within treatment time point vs. vehicle only) and correcting for multiple comparisons with a Bonferroni test. Only statistically significant comparisons are shown. D, Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) showing tumor cells from each treatment group classified as “Sen-High” in blue and those classified as “Sen-Low” in yellow. “Sen-High” cells are defined as those that are positive for three senescence signatures in Supplementary Fig. S3F–S3H). Pie charts below show the fraction of total cells classified as Sen-High versus Sen-Low in each treatment group. The number of sequenced tumor cells is displayed for each treatment group. E, Scheme of experimental design (KPC1-ZsGreen-Watermelon orthotopic transplant into NSG mice) to trace the proliferative history of tumor cells following the indicated treatments. The strategy for gating DAPI−, ZsGreen+ tumor cells based on H2B-mCherry levels is shown. F, Fraction of tumor cells with highly retained (stably arrested since treatment initiation), mid, or low H2B-mCherry following the indicated treatments. Statistical testing: Two-way ANOVA, comparing the mean of each treatment group within each H2B-mCherry with every other treatment group. Multiple comparisons testing was corrected for with a Tukey test, and statistically significant results are reported here. H2B-mCherry high: vehicle versus RMC-7977 + palbociclib (P = 0.0458). H2B-mCherry mid: vehicle versus RMC-7977 + palbociclib (P < 0.0001); RMC-7977 versus RMC-7977 + palbociclib (P < 0.0001). H2B-mCherry low: vehicle versus RMC-7977 (P = 0.0128); vehicle versus RMC-7977 + palbociclib (P < 0.0001); RMC-7977 versus RMC-7977 + Palbociclib P < 0.0001). (A and E, Created with BioRender.com.)