Exosome-mediated Wnt3a signal transmission and β-catenin in clinical samples of DLBCL. (A) Enhanced CFU activity of OCI Ly3 SP cells by addition of autologous exosomes (2 mg/mL) in semisolid media (n = 3, ***P < .0001; *P = .046; 2-sided Student t test). (B) Enrichment of Wnt3a in the exosome fraction of lymphoma cell line supernatants (OCI Ly3, upper panel; further cell lines, lower panel; flottilin2 as an exosome marker). (C) Sedimentation of Wnt3a at a density of 1.16 g/mL together with flottillin2 in a sucrose gradient of OCI Ly3 supernatant. (D) Electron microscopy with immunogold staining showing Wnt3a at the surface of the exosomes (filled arrowhead, upper panel), with heterogeneous amounts between exosomes (lower panel). (E) Increased TOPflash (TOP) reporter activity by exosomes from OCI Ly3 lymphoma cells (2.3-fold, *P = .048, 2-sided t test), comparable to supernatant of L cells with ectopic Wnt3a expression or recombinant Wnt3a (representative example of 3 independent experiments). (F) Detection of β-catenin in aggressive lymphoma samples with cytoplasmic staining (right panel, empty arrowhead), perinuclear staining in most cases (middle panel, gray arrowhead), and rarely nuclear staining (right panel, black arrowhead; original magnification ×400; compare with supplemental Figure 10). (G) In the samples of the prospective DSHNHL B1/B2 protocols (n = 258), the proportions of positive cells per sample showed a unimodal distribution of β-catenin expression levels (compare with supplemental Figure 10B).