B-chronic lymphocytic leukemia chemoresistance involves innate and acquired leukemic side population cells

B-cell chronic lymphocytic leukemia (B-CLL) therapy remains unsatisfactory due to repeated resurgences of the chemoresistant disease. In this study, we investigated the basis of this chemoresistance by applying the 'side population' (SP) analysis to blood samples from B-CLL patients. We report the existence of few natural SP cells, which harbors phenotypic and cytogenetic hallmarks of B-CLL in most patients with this disease (n=22). SP cells appeared resistant to conventional B-CLL treatments, such as Fludarabine, Bendamustin or Rituximab. Indeed, treatment with Fludarabine (16/18 cases) or Bendamustin (5/7 cases) resulted in complete elimination of non-SP, whereas cells displaying the SP phenotype were the only surviving. Although some B-CLL SP cells were innately chemoresistant, chemotherapy by Fludarabine selected not only innate SP cells but also induced some acquired SP cells, which arose from non-SP by drug-driven evolution. This SP selection by chemotherapeutic treatments is further supported by the overall increase of the SP percentage in patients who experienced chemotherapy in the preceding year. Functionally, proliferative stimulation of SP cells was able to partially replenish in vitro the non-SP cell compartment of the B-CLL disease. The chemoresistance of B-CLL relies, in our model, on the cellular heterogeneity of B-CLL SP cells and on their regenerating dynamics.