Figure 3.

Single-cell transcriptomic analysis of CMM cell differentiation into immunogenic myeloid cells in vivo

CMM leukemia-bearing mice were treated using i.v. injections of CpG-STAT3d (5 mg/kg) or PBS three times every other day. (A–H) Single-cell RNA-seq analysis was performed on the sorted GFP⁺ leukemic cells from four individually treated mice or PBS control (88%–94% purity). (A) UMAP plots of AML cells in control (top) and CpG-STAT3d-treated (bottom) mice, clustered using the Leiden algorithm, revealing nine distinct populations. (B) Cell clusters were labeled based on the expression of lineage and immune marker genes. Among these, four populations represented CMM cells, while the remaining five exhibited signatures of differentiated immune cell types. (C) Stacked bar plots depict percentages of various cell clusters to highlight the enrichment of differentiated cell subsets in CpG-STAT3d-treated mice. (D) Module score plots indicating a correlation between reduced proliferative potential of macrophage and B cell-like clusters together with augmented antigen-presenting potential. (E) The upregulation of antigen-presentation and immune signaling gene sets coupled with a decrease in DNA synthesis and proliferation-related genes in CMM cells from treated vs. control mice as assessed using GSEA. (F) Heatmap of transcription factor expression levels revealing the downregulation of pro-tumor TFs (e.g., Runx1, Myb, Myc) and the upregulation of TFs driving cell differentiation, antigen-presentation and immune activity (e.g., Cebpa, Irf8, Stat1, Ciita). (G) UMAP plot of Kit expression levels in the LSC subset of AML cells. (H) UMAP plot of Irf8 expression levels overlapping with monocyte/macrophage clusters. (I–K) To achieve inducible Irf8 silencing in AML cells, we generated CMM-tetON-shIrf8 cells expressing doxycycline (Dox)-inducible Irf8shRNA (tetON-shIrf8). Mice engrafted with CMM-tetON-shIrf8 cells were treated three times daily with PBS, CpG-STAT3d, or Dox+CpG-STAT3d. Dox-inducible IRF8 knockdown decreased CpG-STAT3d-driven IRF8 upregulation in splenic CMM cells at mRNA (I) and protein (J) levels. (K) Inducible Irf8 knockdown abrogates differentiation of AML cells after CpG-STAT3d treatment. The data (I–K) are representative of the results obtained in three independent experiments. Statistically significant differences were indicated by asterisks: ∗∗, p < 0.01; ∗, p < 0.05; shown are means ± SEM (n = 3–5 mice/group).