The Nanomechanical Properties of CLL Cells Are Linked to the Actin Cytoskeleton and Are a Potential Target of BTK Inhibitors.

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.

Capacity for LDL (Low-Density Lipoprotein) Retention Predicts the Course of Atherogenesis in the Murine Aortic Arch.

BACKGROUND: To cause atherosclerosis, LDLs (low-density lipoproteins) must first pass through the endothelium and then become retained in the arterial matrix. Which of these two processes is rate-limiting and predicts the topography of plaque formation remains controversial. To investigate this issue, we performed high-resolution mapping of LDL entry and retention in murine aortic arches before and during atherosclerosis development. METHODS: Maps of LDL entry and retention were created by injecting fluorescently labeled LDL followed by near-infrared scanning and whole-mount confocal microscopy after 1 hour (entry) and 18 hours (retention). By comparing arches between normal mice and mice with short-term hypercholesterolemia, we analyzed changes in LDL entry and retention during the LDL accumulation phase that precedes plaque formation. Experiments were designed to secure equal plasma clearance of labeled LDL in both conditions. RESULTS: We found that LDL retention is the overall limiting factor for LDL accumulation but that the capacity for LDL retention varied substantially over surprisingly short distances. The inner curvature region, previously considered a homogenous atherosclerosis-prone region, consisted of dorsal and ventral zones with high capacity and a central zone with low capacity for continued LDL retention. These features predicted the temporal pattern of atherosclerosis, which first appeared in the border zones and later in the central zone. The limit to LDL retention in the central zone was intrinsic to the arterial wall, possibly caused by saturation of the binding mechanism, and was lost upon conversion to atherosclerotic lesions. CONCLUSIONS: Capacity for continued LDL retention varies over short distances and predicts where and when atherosclerosis develops in the mouse aortic arch.

3D-STED Super-Resolution Microscopy Reveals Distinct Nanoscale Organization of the Hematopoietic Cell-Specific Lyn Substrate-1 (HS1) in Normal and Leukemic B Cells.

HS1, the hematopoietic homolog of cortactin, acts as a versatile actin-binding protein in leucocytes. After phosphorylation, it is involved in GTPase and integrin activation, and in BCR, TCR, and CXCR4 downstream signaling. In normal and leukemic B cells, HS1 is a central cytoskeletal interactor and its phosphorylation and expression are prognostic factors in chronic lymphocytic leukemia (CLL) patients. We here introduce for the first time a super-resolution imaging study based on single-cell 3D-STED microscopy optimized for revealing and comparing the nanoscale distribution of endogenous HS1 in healthy B and CLL primary cells. Our study reveals that the endogenous HS1 forms heterogeneous nanoclusters, similar to those of YFP-HS1 overexpressed in the leukemic MEC1 cell line. HS1 nanoclusters in healthy and leukemic B cells form bulky assemblies at the basal sides, suggesting the recruitment of HS1 for cell adhesion. This observation agrees with a phasor-FLIM-FRET and STED colocalization analyses of the endogenous MEC1-HS1, indicating an increased interaction with Vimentin at the cell adhesion sites. In CLL cells isolated from patients with poor prognosis, we observed a larger accumulation of HS1 at the basal region and a higher density of HS1 nanoclusters in the central regions of the cells if compared to good-prognosis CLL and healthy B cells, suggesting a different role for the protein in the cell types analyzed. Our 3D-STED approach lays the ground for revealing tiny differences of HS1 distribution, its functionally active forms, and colocalization with protein partners.