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Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an intense trafficking of the leukemic cells between the peripheral blood and lymphoid tissues. 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 cells during these processes. To investigate this point, we applied a single-cell optical (super resolution microscopy) and nanomechanical approaches (atomic force microscopy, real-time deformability cytometry) to both CLL and healthy B lymphocytes and compared their behavior. 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.

Chronic lymphocytic leukaemia (CLL) is one of the most common and incurable B cell leukaemia. CLL cells traffic between peripheral blood, bone marrow and secondary lymphatic tissues where interact with a supportive microenvironment. These processes are affected by the mechanical forces present in the environment and by the capability of the cells to sense the forces. In order to migrate from a fluid environment like the blood to a significantly more viscous surrounding, B lymphocytes need to modify their cytoskeleton and consequently their rigidity. Those processes are known to be regulated by Hematopoietic-cell-specific Lyn-substrate-1 (HS1) protein, which promotes the homing processes and is involved in cell-cell communication and focal adhesions formation. From a nanomechanical point of view, cytoskeleton rearrangements can be observed as a difference in the force distribution along the cell and, ultimately, as a change of the overall rigidity of the cell. To measure this change, we used Atomic Force Microscopy (AFM) as an external pressure stimulus and we observed the cell deformation, which finally determines the value of its stiffness. By AFM we measured primary B lymphocytes from selected CLL patients and healthy donors, and we found a significant decrease of stiffness in leukaemia cells compared to healthy ones. We also performed Real-Time Deformability Cytometry (RT-DC) to test the rigidity of cells in flow condition. Finally, we tested the effect of a first line drug (Ibrutinib) on the mechanical properties of leukemic and healthy cells with both techniques in order to understand its effect on the mechanics of cells along their path to development of the disease.

Chronic lymphocytic leukaemia (CLL) is one of the most common and incurable B cell leukaemia. CLL cells traffic between peripheral blood, bone marrow and secondary lymphatic tissues where interact with the microenvironment. These processes are affected by the mechanical-forces present in the environment and by the capability of the cells to sense the forces. In this context we demonstrated that Hematopoietic-cell-specific Lyn-substrate-1 (HS1) protein is a cytoskeletal regulator and a prognostic factor in CLL. We proved that interfering with HS1 expression impacts on the progression and homing of CLL cells. To further study HS1 role in CLL we knocked-down HS1 expression by CRISPR/CAS9 technology in a CLL cells line (MEC1). By RNAseq and network analysis on MEC1HS1ko vs MEC1UT we found that HS1-KO significantly affects the expression of molecules involved in: cell motility, adhesion, cell-cell communication, focal adhesion formation. In particular, we found LEF1, FAK and Cortactin genes are up-regulated in MEC1HS1ko, suggesting their involvement in the mechano-signalling pathway. To study the nano-mechanical properties of those cells we are performing AFM measurements of single cell. The results provide an evaluation of the cell stiffness that is related to its deformation in response to an externally applied force. By AFM we found that MEC1HS1ko cells are less stiff if compared with MEC1UT cells. This results demonstrates a putative role for HS1 in regulating the mechanical properties of CLL cells. Due to the prognostic value of HS1 we are currently performing AFM measurement on selected patients and healthy B cells. We are planning to study in depth the role of HS1 in regulating the mechanical properties of the leukemic cells and how this contributes to leukemia development, progression and resistance to therapy.

Chronic lymphocytic leukaemia (CLL) is one of the most common and incurable B cell leukaemia. CLL cells traffic between peripheral blood, bone marrow and secondary lymphatic tissues where interact with the microenvironment. These processes are affected by the mechanical-forces present in the environment and by the capability of the cells to sense the forces. In this context we demonstrated that Hematopoietic-cell-specific Lyn-substrate-1 (HS1) protein is a cytoskeletal regulator and a prognostic factor in CLL. We proved that interfering with HS1 expression impacts on the progression and homing of CLL cells. To further study HS1 role in CLL we knocked-down HS1 expression by CRISPR/CAS9 technology in a CLL cells line (MEC1). By RNAseq and network analysis on MEC1HS1ko vs MEC1UT we found that HS1-KO significantly affects the expression of molecules involved in: cell motility, adhesion, cell-cell communication, focal adhesion formation. In particular, we found LEF1, FAK and Cortactin genes are up-regulated in MEC1HS1ko, suggesting their involvement in the mechano-signalling pathway. To study the nano-mechanical properties of those cells we are performing AFM measurements of single cell. The results provide an evaluation of the cell stiffness that is related to its deformation in response to an externally applied force. By AFM we found that MEC1HS1ko cells are less stiff if compared with MEC1UT cells. This results demonstrates a putative role for HS1 in regulating the mechanical properties of CLL cells. Due to the prognostic value of HS1 we are currently performing AFM measurement on selected patients and healthy B cells.