Nanoscale analysis reveals distinct mechanical properties in Chronic Lymphocytic Leukemia cells

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in the Western world, with an incidence of 5 new cases per 100.000 individuals annually. The disease affects mainly elderly people, however one third of the new cases are diagnosed before the age of 55 years. CLL is an incurable ailment characterised by an intense trafficking and accumulation of mature CD5+ B lymphocytes in peripheral blood, bone marrow (BM) and secondary lymphoid organs (spleen and lymph nodes). It is known that the ability of lymphocytes to recirculate strongly depends on their capability to rapidly rearrange their cytoskeleton and adapt to external cues; however, little is known about the differences occurring between CLL and healthy B (HB) cells during these processes. To investigate this point, we performed a comparison between primary healthy, leukemic B cells by applying nanomechanical approaches (Atomic Force Microscopy). By means of single cells optical super resolution microscopy we demonstrated that CLL cells have a specific actomyosin complex organization and altered mechanical properties in comparison to their healthy counterpart. To evaluate the clinical relevance of our findings, we treated the cells in vitro with the Bruton’s tyrosine kinase inhibitors and we found for the first time that the drug restores the CLL cells mechanical properties to a healthy phenotype and activates the actomyosin complex. We further validated these results in vivo on CLL cells isolated from patients undergoing ibrutinib treatment. Our results suggest that CLL cells’ mechanical properties are linked to their actin cytoskeleton organization and might be involved in novel mechanisms of drug resistance, thus becoming a new potential therapeutic target aiming at the normalization of the mechanical fingerprints of the leukemic cells. Furthermore, we employed representative cellular lines of CLL (MEC Wild-Type) cells, which are indicative of the disease characteristics. Additionally, we generated a knockout model using CRISPR-Cas9 for the Hematopoietic Lineage Cell-Specific protein, HS1, an interactor of cytoskeleton. This model allows us to investigate the role of HS1 in the cytoskeletal interactions within CLL cells. MEC WT cell lines were adopted to conduct creeping and stress relaxation experiments adopting pyramidal tips to further investigate and elucidate the cellular properties. The primary objective of these measurements was to develop a viscoelastic model that could comprehensively characterize not only the cellular elasticity (Young's Modulus) but also the viscous properties of the cells under basal and pathological conditions, as well as in response to pharmacological treatments.