C3AR is biologically functional in NPM1-mutated AML. (A) Expression of pERK1/2 after 1-minute stimulation with 100 ng/mL of human C3a in 5 primary AML samples. Unstimulated shown in filled blue and stimulated in red. P = .06 using a Wilcoxon nonparametric paired test. (B) Number of primary NPM1-mutated AML cells and OCI-AML3 cells (C) after a 3-day culture with increasing concentrations of human C3a. Primary AML cells (100 000) and OCI-AML3 cells (20 000) were seeded per well in serum-free medium. The experiments were performed in triplicate. Bars in panels B-C show mean cell numbers with standard deviation. (D) Heat map showing the median FPKM value of C3-related complement genes in each AML subgroup, relative to the highest median value of that gene, in the Lund AML Cohort. AML subgroups with <3 patients were excluded from the analysis (n = 109). (E) Relative gene expression of bulk NPM1-mutated AML cells (n = 12 cases) compared with NBM MNCs (n = 5 cases), as assessed by scRNA-seq. (F) Amount of CFD in the cell culture supernatant 24 hour after seeding. One million cells per mL were seeded from 4 NPM1-mutated AML samples and 4 NBM MNC samples in S7 media. Bars show median with interquartile range. ∗∗P ≤ .01 using a Student t test in panel C. A nonparametric Mann-Whitney U test was used in panel F. chrom-splice, chromatin-spliceosome; FPKM, fragments per kilobase of exon model per million reads mapped.