RESEARCH ARTICLE Analyzing normal and disrupted leukemic stem cell adhesion to bone marrow stromal cells by single-molecule tracking nanoscopy Oksana Gorshkova 1 , Jessica Cappaï 1 , Loriane Maillot 2 and Arnauld Serge ́ 1,2, * ABSTRACT Leukemic stem cells (LSCs) adhere to bone niches through adhesion molecules. These interactions, which are deeply reorganized in tumors, contribute to LSC resistance to chemotherapy and leukemia relapse. However, LSC adhesion mechanisms and potential therapeutic disruption using blocking antibodies remain largely unknown. Junctional adhesion molecule C (JAM-C, also known as JAM3) overexpression by LSCs correlates with increased leukemia severity, and thus constitutes a putative therapeutic target. Here, we took advantage of the ability of nanoscopy to detect single molecules with nanometric accuracy to characterize junctional adhesion molecule (JAM) dynamics at leuko- stromal contacts. Videonanoscopy trajectories were reconstructed using our dedicated multi-target tracing algorithm, pipelined with dual-color analyses (MTT2col). JAM-C expressed by LSCs engaged in transient interactions with JAM-B (also known as JAM2) expressed by stromal cells. JAM recruitment and colocalization at cell contacts were proportional to JAM-C level and reduced by a blocking anti-JAM-C antibody. MTT2col revealed, at single-molecule resolution, the ability of blocking antibodies to destabilize LSC binding to their niches, opening opportunities for disrupting LSC resistance mechanisms. KEY WORDS: Adhesion, Leukemic stem cell, Nanoscopy, Single- molecule tracking, Super-resolution microscopy INTRODUCTION Advances in fluorescence microscopy over the past decades have allowed for detecting a single molecule (SM) with nearly nanometer accuracy. These innovations, initially conceptualized by Werner Heisenberg (Heisenberg, 1949), and which led to the award of the Nobel Prize to Eric Betzig (Betzig, 1995; Betzig et al., 2006), Stephan Hell (Hell, 2007; Hell and Wichmann, 1994) and William Moerner (Dickson et al., 1997; Moerner and Kador, 1989) in 2014, are termed super-resolution microscopy or nanoscopy. These approaches, first optimized on fixed cells, then extended to living cells, recently revealed an intense and unexpected dynamics of macromolecular structures, such as synapses and focal adhesions (Sergé, 2016). This completely revised the classic view of these as static structures, with a molecular composition essentially constant over time. Thus, it is now thought that major players, like integrins and their adapters, are finely regulated by mechanisms such as association/dissociation, local diffusion within the membrane and/ or direct targeting by local exocytosis (Bakker et al., 2012; Ishibashi et al., 2015; Rossier et al., 2012). This dynamic conception of cell adhesion has started to reveal an unsuspected plasticity that is modulated by physiopathological conditions and signals received by the cell. Hematopoiesis is the process by which hematopoietic stem cells (HSCs) replenish platelets, red blood cells and immune cells over lifetime. It occurs in the bone marrow (BM) of adult mammals and requires the retention of HSCs in stromal niches, constituting specialized microenvironments controlling HSC quiescence, proliferation and differentiation into transient amplifying progenitors. Acute myeloid leukemia (AML) cells are hierarchically organized in a similar manner to normal hematopoietic cells. The bulk of leukemic cells originates from leukemic stem cells (LSCs), which retain their capacity for self-renewal, pluripotency and quiescence. LSCs, which are responsible for cancer relapse, are functionally defined as leukemic-initiating cells, and are a subset of leukemic cells able to initiate AML in xenografted mice. Crosstalk interactions between HSCs and their surrounding stroma leads to reciprocal exchange, to the benefit of the metabolism of both cells (De Grandis et al., 2015). This process is particularly important for LSCs, promoting a higher resistance to chemotherapy and favoring relapse after treatment (Schepers et al., 2015), a process called cell-adhesion- mediated drug resistance (Meads et al., 2008). Of note, cell adhesion to the extracellular matrix has already been studied by single-molecule tracking (SMT) and super-resolution approaches, notably addressing the dynamics of integrins and associated molecules (Sergé, 2016). However, cell –cell adhesion is experimentally more challenging to investigate since it inherently occurs between the two membranes of contacting cells, or by replacing one cell by a lipid bilayer, while integrin-mediated cell adhesion can be studied on extracellular matrix fibers deposited directly on the coverslip. The junctional adhesion molecule (JAM) family contains three main members, JAM-A (Martin-Padura et al., 1998), JAM-B (Aurrand-Lions et al., 2001) and JAM-C (Arrate et al., 2001), also known as JAM1, JAM2 and JAM3, respectively. JAM-C expressed by normal HSCs interacts with JAM-B expressed by BM stromal cells, contributing to the maintenance of HSC quiescence (Arcangeli et al., 2011, 2014). In a follow-up study, we found that LSCs express JAM-C, a new biomarker for disease outcome in AML (De Grandis et al., 2017). LSC expression of JAM-C correlates with increased adhesion to BM stromal cells and is correlated with poor disease outcome. This suggests that JAM-C is involved in LSC resistance to chemotherapy by supporting adhesion of LSCs to the stroma. Thus, better understanding of the dynamics of LSC adhesion to BM stromal cells is critical for developing new therapeutic strategies targeting LSCs. Here, we characterized JAM dynamics during LSC interactions with stromal cells by advanced nanoscopy and analytic approaches. Handling Editor: Kathleen Green Received 1 April 2021; Accepted 2 August 2021 1 Centre de recherche en cancé rologie de Marseille (CRCM), Centre national de la recherche scientifique (CNRS), Institut national de la santé et de la recherche mé dicale (Inserm), Institut Paoli-Calmettes (IPC), Aix-Marseille Université , F-13273 Marseille, France. 2 Laboratoire adhé sion inflammation (LAI), Centre national de la recherche scientifique (CNRS), Institut national de la santé et de la recherche mé dicale (Inserm), Aix-Marseille Université , F-13288 Marseille, France. *Author for correspondence (arnauld.serge@univ-amu.fr) O.G., 0000-0001-5735-2574; L.M., 0000-0001-9936-2957; A.S., 0000-0003- 4271-3706 1 © 2021. Published by The Company of Biologists Ltd | Journal of Cell Science (2021) 134, jcs258736. doi:10.1242/jcs.258736 Journal of Cell Science