Figure 3 from A RAS(ON) Multi-Selective Inhibitor Combination Therapy Triggers Long-term Tumor Control through Senescence-Associated Tumor-Immune Equilibrium in Pancreatic Ductal Adenocarcinoma

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Combined RASi + CDK4/6i triggers favorable immune remodeling and poises tumors to respond to immunotherapy. For the scheme of experimental design, see Supplementary Fig. S5A (KPC1-zsGreen orthotopic transplant into wild-type C57Bl/6 mice). A, Fraction of CD45 cells out of total live cells by flow cytometry. Each dot represents an individual mouse. Statistical testing: Ordinary one-way ANOVA with multiple comparisons, comparing the means of each treatment group against the vehicle of the relevant time point and correcting for multiple comparisons using a Sidak test. Only statistically significant comparisons are shown. B, Fraction of indicated immune cell populations (%) out of total CD45+ immune cells at day 14 after treatment initiation. Each dot represents an individual mouse. Statistical testing: Two-way ANOVA with multiple comparisons, comparing the means of each treatment group against the vehicle and correcting for multiple comparisons using a Dunnett test. Only statistically significant comparisons are shown. C, Quantification of representative regions of IF staining for CD4 T cells (representative images shown in E) in tumors 4 hours, 3, 7, or 14 days after treatment initiation. Each dot represents an individual mouse (average of 3–5 ∼40,000 µm2 regions). Statistical testing: Ordinary one-way ANOVA with multiple comparisons, comparing the means of each treatment group against the vehicle at the relevant time point and correcting for multiple comparisons using a Bonferroni test. Only statistically significant comparisons are shown. D, Quantification of representative regions of IF staining for CD8 T cells (representative images shown in E) in tumors 4 hours, 3, 7, or 14 days after treatment initiation. Each dot represents an individual mouse (average of 3–5 ∼40,000 µm2 regions). Statistical testing: Ordinary one-way ANOVA with multiple comparisons, comparing the means of each treatment group against the vehicle of the relevant time point and correcting for multiple comparisons using a Bonferroni test. Only statistically significant comparisons are shown. E, Representative images of IF staining for T-cell markers CD3 (white), CD8 (red), and CD4 (yellow) following indicated treatments at indicated time points (D7 = 7 days after treatment initiation, D14 = 14 days after treatment initiation). F, Quantification of the number and area (µm2) of lymphoid aggregates (clusters of B cells, DCs, and other MHC-II positive cells) found in whole-slide scans of tumors. Each tick on the x-axis represents a single mouse. Each dot represents an individual lymphoid aggregate, and the size of each aggregate is reflected on the y-axis. The color of each dot indicates the treatment group, as shown in the figure legend. Arrows and corresponding letters point to lymphoid aggregates for which examples are shown in G–I. G, Example of a lymphoid aggregate from an RMC-7977 + palbociclib–treated mouse at t = 7 days after treatment initiation. H and I, Example of a lymphoid aggregate from an RMC-7977 + palbociclib–treated mouse at t = 14 days after treatment initiation. J, Highly multiplexed IF images of TLSs in tumors from mice treated with RMC-7977 + palbociclib for 7 days. Images were acquired by the Cell Dive and stained for endothelial cell (CD31), fibroblast (Podoplanin: PDPN), and activated fibroblast (α-smooth muscle actin: αSMA) markers. K, Highly multiplexed IF images of TLSs in tumors from mice treated with RMC-7977 + palbociclib for 7 days. Images were acquired by the Cell Dive and stained for B-cell (B220) and proliferation (Ki67) markers. L, Highly multiplexed IF images of TLSs in tumors from mice treated with RMC-7977 + palbociclib for 7 days. Images were acquired by the Cell Dive and stained for the indicated markers. M, Low magnification image of lymphoid aggregates shown in H and I following 14 days of RMC-7977 + palbociclib treatment.

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