Figure S17. Cell deconvolution and cell specificity of spatial transcriptomic expression.

Figure S11. Identified ligand-receptor interactions involving CT-26 KRASG12C cells following no treatment.

Figure S14. Analysis of T-cell function, expansion and TCR clonotype analysis after KRAS(G12C)i and MEKi.

Figure S5. Classification of immune cell states in treated CT-26 KRASG12C tumors.

Table S1. Mouse T cell phenotyping antibody panel; Table S2. Mouse macrophage phenotyping antibody panel; Table S3. Mouse dendritic cell phenotyping antibody panel; Table S4. Mouse T cell function antibody panel; Table S5. FACS panel for RNA-seq; Table S6. Antibodies used to BMDC co-culture.

Abstract Although mutant-specific KRAS inhibitors are approved to treat cancer, a deeper understanding of intratumoral changes driven specifically by KRAS inhibition is needed to maximize therapeutic responses. In this study, we used single-cell RNA sequencing, flow cytometry, and spatial transcriptomics to distinguish mechanisms of tumor control after KRASG12C inhibition [KRAS(G12C)i] or MEK inhibition (MEKi). Despite both inhibiting the MAPK pathway, KRAS(G12C)i and MEKi drive the adaptation of distinct neoplastic cell fates affecting metabolism and cell-cycle regulation, and additive tumor suppression is observed after co-administration. KRAS(G12C)i results in the emergence of a specific, cDC1-driven mature conventional dendritic cell (cDC) state. Coculture of treated neoplastic cells with cDC1s is sufficient to upregulate maturation markers such as CCR7, and intercellular communication analyses suggest that activation is augmented through nonimmune mediators. Both KRAS(G12C)i and MEKi increase infiltration of cytotoxic T cells, but MEKi, which also targets nonmalignant cells, is associated with a reduced capacity for T-cell proliferation and degranulation, consistent with distinct adaptive immune activation mechanisms. We observe that combination treatment of KRAS(G12C)i with anti–PD-1 immunotherapy further expands effector T-cell states, increases clonal persistence, and induces proinflammatory macrophages associated with higher overall survival that were largely absent after KRAS(G12C)i alone. Furthermore, combination treatment enhances intercellular communication networks among non–PD-1+–expressing cells that can perpetuate cDC activation. Our findings delineate distinct tumor and immune responses to KRAS and MEK inhibition and identify molecular features of the responding tumor microenvironment that may be leveraged to improve therapeutic efficacy.

Figure S1. Quality filtering of scRNA-seq of all cells from CT-26 KRASG12C syngeneic tumors and classification of cell types.

Figure S18. Spatial transcriptomic analysis of immune signatures and enhanced intercellular communication following KRAS(G12C)i with anti-PD-1 blockade.

Figure S4. Classification of immune cell types in treated CT-26 KRASG12C syngeneic tumors.

Figure S6. Characterization of classical dendritic cell states (cDC) in treated tumors.

Figure S7. Flow cytometry analyses of dendritic cell infiltration and maturation.

Figure S8. In-vitro bone marrow-derived dendritic cell (BMDC) co-culture with treated KRASG12C cell lines.

Figure S2. Characterization of CT-26 KRASG12C neoplastic cell states in vivo.

Figure S10. Identified ligand-receptor interactions involving neoplastic cells following KRAS(G12C)i and MEKi treatment.

Figure S3. Quality assessment of single-cell RNA-seq in CD45+ tumor-infiltrating immune cells.

Figure S15. Cell state dynamics of intratumoral monocytes and macrophages after KRAS(G12C)i and MEKi.

Figure S9. Summary of intratumoral inter-cellular signaling interactions in CT-26 KRASG12C.

Figure S16. CD8 T cell intratumoral dynamics following KRAS(G12C)i and anti-PD1 combination therapy.

Figure S12. Intercellular communication between neoplastic cells and non-immune cells in CT-26 KRASG12C tumors.

Figure S13. Tumor infiltrating T cell states and expression dynamics after KRAS(G12C)i and MEKi.