In vitro EC-cocultured T-ALL cells mirror in vivo T-ALL cells. (A) Schematic representation of the single-cell RNA-Seq analysis of 3119 T-ALL cells. Cells that were either cultured alone or cocultured with E4-ECs in vitro for 5 days were sequenced and combined with T-ALL cells freshly harvested from the spleen of a 3119 PDX mouse. (B) UMAP plot of the single-cell RNA-Seq data derived from panel A, color-coded by cluster identity. (C) UMAP plot of the single-cell RNA-Seq data derived from panel A, color-coded by sample origin. (D) Bar plot showing the proportions of in vitro and in vivo T-ALL cells (panel A) based on cluster identity. (E) Violin plots showing the enrichment score of the education signature derived from the in vitro experiment with the 3119 PDX model (Figure 3D). In vitro EC-cocultured and splenic T-ALL cells displayed similar enrichment across all clusters. (F) Dot plot displaying expression levels for a set of genes in common between RO2 and 3119 T-ALL “educated” elements (Figure 4G, T-ALL genes) in ex vivo splenic T-ALL cells compared with in vitro T-ALL elements (E4-ECs cocultured and cultured alone). The expression of these genes was very similar in splenic and E4-EC-cocultured T-ALL cells compared with T-ALL cultured alone, demonstrating that in vitro EC-educated elements are more similar to those freshly harvested ex vivo. The dot size encodes the percentage of cells expressing each gene, and the color encodes the average expression level across all cells. Expression levels were measured using the log-normalized counts. (G) Heat map of the genes from the 3119 “education signature” (Figure 3D) applied to cluster 0 from panel B containing enough cells from all 3 compartments (in vitro T-ALL cultured alone vs with E4-ECs and ex vivo splenic T-ALL). Cell proportions in cluster 0 are found in panel D. T-ALL cells from the spleen are clustered together with EC-cocultured T-ALL cells, based on the expression of the education signature. DEG, differentially expressed genes.