Leukaemic stem cells (LSCs) possess the capacity for self-renewal, pluripotency and quiescence. Interactions between LSCs and bone marrow stromal cells promote resistance to chemotherapy and favour relapse through a process called cell adhesion-mediated drug resistance. Expression of junctional adhesion molecule C (JAM-C) correlates with increased adhesion to stromal cells and poor leukaemia prognosis. This suggests that JAM-C is involved in chemotherapy resistance, but the mechanism of LSC adhesion and its therapeutic potential are unknown. Here, Arnauld Sergé and colleagues (Gorshkova et al. 2021) employ single-molecule tracking to characterise junctional adhesion molecule (JAM) dynamics at leuko-stromal contacts in co-cultures of murine stromal MS5 cells and human acute myeloblastic KG1 cells. Analysis of JAM trajectories at single-molecule resolution using a multi-target tracing algorithm developed by the authors reveals the dynamic nature of the interactions between leukaemic JAM-C and stromal JAM-B, including modulation of their diffusion, and the establishment and stabilization of cell contacts. Moreover, increased expression of JAM-C strengthens these interactions, whereas the presence of a blocking anti-JAM-C antibody perturbs contacts between LSCs and stromal cells, hampering JAM dynamics and recruitment. Taken together, these findings reveal the highly dynamic process of LSC adhesion and have potential therapeutic implications for the use of anti-JAM-C antibodies to overcome chemotherapy resistance.