CLL cells roll, stick, and crawl within LN HEVs in vivo. (A) Visualization of the mouse inguinal LN microcirculation by intravital microscopy. HEV blood vessels were revealed by IV injection of Alexa 488–conjugated MECA-79 Ab. The different blood vessel orders are indicated (I-V). (B) Rolling and sticking of human CLL cells within mouse LN HEVs. The rolling fraction (left panel) and sticking fraction (right panel) of calcein-labeled CLL cells in the indicated venules are shown. Data represent the mean ± SD of independent experiments conducted with CLL cells from 7 CLL patients with no bulky disease (black) and 6 CLL patients with bulky disease and high circulating lymphocyte counts (≥40 000 cells/mm³; red). Unpaired t test; *P < .05, **P < .01, ***P < .001. (C) CLL cells from patients with bulky disease roll more slowly within LN HEVs. Data represent the percentages of rolling cells at or below a given velocity (V_roll) in the indicated venules. The median velocity of cells from CLL patients with no bulky disease was 220.7 and 124 µm.s⁻¹ in order IV and V, respectively. The median velocity of cells from CLL patients with bulky disease was 79.6 and 48.3 µm.s⁻¹ in order IV and V, respectively (50 cells/venular order from 5 patients were analyzed). Mann-Whitney test; ***P < .001. (D) Two-photon microscopy time-lapse images showing CFSE-labeled CLL cells from patients with bulky disease (arrowheads) crawling on HEV endothelium. Alexa 647–conjugated MECA-79 staining is shown on the first image. Yellow arrows indicate blood flow direction. Scale bar, 40 µm. (E) The median crawling speed (V_crawl) was determined from 2 independent experiments (n = 21 cells).