Key Points Diffuse large B-cell lymphomas are composed of clonogenic side population (SP) cells and non-SP cells organized in a dynamic equilibrium. Exosome-mediated Wnt signaling modulates transitions of cell states and tumor progression amenable to drug targeting.

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Introduction Patients with aggressive B-cell lymphoma are treated in curative intention. However, some patients experience fatal relapse, originating from refractory lymphoma cells with the capacity for clonogenic regrowth. We here addressed repopulation capacity of lymphoma cell subpopulations and the mechanisms regulating the populational composition in the growing tumor. Material & Methods We identified side population (SP) cells in diffuse large B-cell lymphoma cell lines and patient samples with the DNA-binding dye Hoechst33342, analyzed clonogenicity in vitro and in vivo and screened for differentially expressed genes and DNA-methylation patterns. A GFP-containing lentiviral vector construct was used to keep track of side population cells cultured among mixed cultures of SP and nonSP cells. Manipulation of canonical wnt-signaling was performed by lentiviral sh-RNA constructs as well as pharmacological tankyrase-inhibition by XAV-939. In vitro data were supported by in vivo experiments using a chorioallantoic membrane-assay. Results Colony assays and suspension cultures of sorted SP and nonSP cells revealed restriction of clonogenic potential to the SP cell population as well as resurgence of nonSP cells from purified SP cell progenitors, while mixed culture assays using a GFP-vector construct tracing the SP vs. nonSP-population revealed homeostasis between the two populations, showing both SP and nonSP cells contributing to either cell compartment. SP cells show enhanced canonical wnt-signaling and increased exosomal secretion of wnt3a. Suppression of canonical wnt-signaling resulted in reduced clonogenicity. Exosome stimulation of DLBCL cell lines resulted in increased clonogenicity, stabilization of beta catenin and enhanced TOP/FOP activity. Conclusion Here we show that tumor cells reversibly switch between states of autonomous and non-autonomous clonogenicity, and that such transitions are regulated by exosome-mediated wnt signaling. Disclosures: No relevant conflicts of interest to declare.