The disruption of endothelial heparan sulfate (HS) is an early event in tumor cell metastasis across vascular barriers, and the reinforcement of endothelial HS reduces tumor cell adhesion to endothelium. Our recent study showed that while vascular endothelial growth factor (VEGF) greatly reduces HS at an in vitro blood–brain barrier (BBB) formed by human cerebral microvascular endothelial cells (hCMECs), it significantly enhances HS on a breast cancer cell, MDA-MB-231 (MB231). Here, we tested that this differential effect of VEGF on the HS favors MB231 adhesion and transmigration. We also tested if agents that enhance endothelial HS may affect the HS of MB231 and reduce its adhesion and transmigration. To test these hypotheses, we generated an in vitro BBB by culturing hCMECs on either a glass-bottom dish or a Transwell filter. We first quantified the HS of the BBB and MB231 after treatment with VEGF and endothelial HS-enhancing agents and then quantified the adhesion and transmigration of MB231 across the BBB after pretreatment with these agents. Our results demonstrated that the reduced/enhanced BBB HS and enhanced/reduced MB231 HS increase/decrease MB231 adhesion to and transmigration across the BBB. Our findings suggest a therapeutic intervention by targeting the HS-mediated breast cancer brain metastasis.

Cell chirality is crucial for the chiral morphogenesis of biological tissues, yet its underlying mechanism remains unclear. Cell organelle polarization along multiple axes in a cell body, namely, apical–basal, front–rear, and left–right, is known to direct cell behavior such as orientation, rotation, and migration. Among these axes, the left–right bias holds significant sway in determining the chiral directionality of these behaviors. Normally, mouse myoblast (C2C12) cells exhibit a strong counterclockwise chirality on a ring-shaped micropattern, whereas they display a clockwise dominant chirality under Latrunculin A treatment. To investigate the relationship between multicellular chirality and organelle positioning in single cells, we studied the left–right positioning of cell organelles under distinct cell chirality in single cells via micropatterning technique, fluorescent microscopy, and imaging analysis. We found that on a “T”-shaped micropattern, a C2C12 cell adopts a triangular shape, with its nucleus–centrosome axis pointing toward the top-right direction of the “T.” Several other organelles, including the Golgi apparatus, lysosomes, actin filaments, and microtubules, showed a preference to polarize on one side of the axis, indicating the universality of the left–right asymmetrical organelle positioning. Interestingly, upon reversing cell chirality with Latrunculin A, the organelles correspondingly reversed their left–right positioning bias, as suggested by the consistently biased metabolism and contractile properties at the leading edge. This left–right asymmetry in organelle positioning may help predict cell migration direction and serve as a potential marker for identifying cell chirality in biological models.