Fig. 5: Nras^G12D/Pten^KO cancer cells activate GM-CSF signaling in the TME.

a–c Differential expression analyses from bulk tumor (Fig. 1e) and cancer cell RNA-seq datasets comparing Nras^G12D/Pten^KO (n = 3) and Nras^G12V/Pten^KO HCC (n = 3). a DEGs between bulk tumors (y-axis) and cancer cells (x-axis). Colored dots represent genes significantly deregulated in both datasets. b Enriched signaling pathways for genes in (a). c Overlap of up-regulated genes and proteins identified by RNA-sequencing and cytokine arrays, respectively. d GM-CSF levels in conditioned media of genetically-distinct HCC cells (Myc^OE/Trp53^KO n = 4, Myc^OE/Pten^KO n = 4, Nras^G12D/Pten^KO n = 6, Nras^G12V/Pten^KO n = 4). e GM-CSF signature⁸⁹ enrichment (Csf2rb^KO-WT) in DEG from transcriptional signatures of Nras^G12D/Pten^KO (n = 4) and Nras^G12V/Pten^KO (n = 3) bulk tumors (top), and Nras^G12D/Pten^KO (n = 3) and Nras^G12V/Pten^KO (n = 3) CD45⁺ cells (bottom). Colors represent DEG overlap from Nras-driven models and GM-CSF signature. f DEGs from CD45⁺ cell RNA-seq of Nras^G12D/Pten^KO (n = 7) relative to Nras^G12V/Pten^KO (n = 7) overlapping with GM-CSF signature⁸⁹. Colors represent direction of GM-CSF regulation, relevant genes from GM-CSF signature are depicted (Supplementary Data 9). g Csf2ra, Csf2rb and Ccl6 expression from scRNA-seq (Fig. 4a). h GM-CSF signature⁸⁹ enrichment for the indicated immune cell subsets identified by scRNA-seq. i. Survival of TCGA:LIHC patients¹⁰³ segregated into high correlation with Nras^G12D/Pten^KO or Nras^G12V/Pten^KO transcriptional signatures (Fig. 2c) (Nras^G12D/Pten^KO n = 65, Nras^G12V/Pten^KO n = 66). high- and low-correlating patients are unique to each signature. Tables show patient numbers at the indicated time points. j Enrichment of GM-CSF positively-regulated genes (n = 628) from the GM-CSF signature⁸⁹ in TCGA:LIHC patients¹⁰³ segregated into high correlation with Nras^G12D/Pten^KO or Nras^G12V/Pten^KO transcriptional signatures (Fig. 2c) (Nras^G12D/Pten^KO n = 65, Nras^G12V/Pten^KO n = 66). k Survival of TCGA:LIHC patients¹⁰³ segregated into high/low enrichment of GM-CSF positively-regulated genes (n = 628) from the GM-CSF signature⁸⁹ (High n = 103, Low n = 82). Tables show patient numbers at the indicated time points. Graph show mean ± SEM (d). Statistical significance: two-sided hypergeometric test with Benjamini-Hochberg multiple testing correction (b, e, h), one-way ANOVA with Tukey’s multiple comparison test (d), log-rank test (i, k), unpaired two-sided Student’s t-test (j). Vertical lines at −log₁₀(FDR) = 2.5 indicate significance threshold (b, e, h). The shading represents 95% confidence interval (i, k). Source data are provided as a Source Data file.