Treatment with tmTNF-α antibody targets LSCs in vivo. (A) An overview of the experimental design. At the end of the first transplantation, leukemia cells were harvested. For the second transplantation, each NOD-SCID recipient mouse was transplanted with 1 × 10⁵ CD45⁺ cells from C1- or IgG1- treated mice (n = 5 each group). Engraftment of AML cells in NOD-SCID mice was determined 6 weeks posttransplantation. (B) Levels of AML cell engraftment into the BM of secondary recipient mice were determined by FCM. Vertical bars represent means ± SEM; ***P < .001 between control and C1- treated groups. (C) The percentage of tmTNF-α⁺ cells within the total engrafted AML cells of secondary recipient mice was determined by FCM; ***P < .001 between control and tmTNF-α-treated groups. (D) The percentage of CD34⁺ AML cells among total engrafted AML cells in the BM of secondary recipient mice was determined by FCM. Values represent means ± SEM; ***P < .001 between the control and C1-treated groups. (E) Heatmap of gene expression profiles associated with leukemia cells harvested from control or C1- treated groups at the end of the first transplantation. (F) The most highly enriched pathways exhibiting differential expression between the control- and C1- treated groups were identified using DAVID pathway analysis. Each column indicates the number of differentially expressed genes between the 2 groups. For the differentially expressed genes, the call value of expression had to be P < .05, with an expression ratio ≥1.3 or ≤0.7692. Both Q and P values are shown. (G) Typical genes that are differentially expressed between control- and C1- treated groups that were confirmed by qPCR. Values represent mean expression ratios comparing the C1- treated and control groups.