Analytical cell adhesion chromatography reveals impaired persistence of metastatic cell rolling adhesion to P-selectin

Abstract

Selectins facilitate the recruitment of circulating cells from the bloodstream by mediating rolling adhesion, which initiates the cellcell signaling that directs extravasation into surrounding tissues. To measure the relative efficiency of cell adhesion in shear flow for in vitro drug screening, we designed and implemented a microfluidicbased analytical cell adhesion chromatography system. The juxtaposition of instantaneous rolling velocities with elution times revealed that human metastatic cancer cells, but not human leukocytes, had a reduced capacity to sustain rolling adhesion with P-selectin. We define a new parameter, termed adhesion persistence, which is conceptually similar to migration persistence in the context of chemotaxis, but instead describes the capacity of cells to resist the influence of shear flow and sustain rolling interactions with an adhesive substrate that might modulate the probability of extravasation. Among cell types assayed, adhesion persistence to P-selectin was specifically reduced in metastatic but not leukocyte-like cells in response to a low dose of heparin.In conclusion, we demonstrate this as an effective methodology to identify selectin adhesion antagonist doses that modulate homing cell adhesion and engraftment in a cell-subtype-selective manner.

P-Selectin and ICAM-1 synergy in mediating THP-1 monocyte adhesion in hemodynamic flow is length dependent

“…2D–F) exhibited a length dependence at 1 μg/mL P-selectin + ICAM-1 and both length and time dependence at 2.5 and 10 μg/mL P-selectin + ICAM-1 at the lowest shear stress tested. Notably, increases in firm adhesion on P-selectin and ICAM-1 co-functionalized substrates from FOV 1 to 3 (78–411% at 0.5 dyn/cm 2 ) could not be accounted for by the less than 5% increase in the number of cells settled on the substrate surface 39 between these locations. This suggests that the length of a substrate available for facilitating cell contact in flow regulates the extent of monocyte firm adhesion, particularly at low wall shear stress.…”

“…Substrates were functionalized uniformly with adhesive molecules presented in situ on the inflamed endothelium and since leukocyte recruitment occurs pathophysiologically under low fluid forces typical of the venous circulation 36–38 , wall shear stresses ranging from 0.5 to 2.0 dyn/cm 2 were assayed. In conjunction with high speed video microscopy, post-processing of perfusion experiments conducted in such flow systems allowed for quantification of the extents of THP-1 cell rolling adhesion, which is characterized by slow, unsteady forward translation at speeds less than the free flow velocity (free flow velocity > translational velocity > 0) 39 , and firm adhesion (translational velocity = 0) (Fig. S5A).…”

Implications of Lymphatic Transport to Lymph Nodes in Immunity and Immunotherapy

“…In this context, length scales relate not only to the initiation of rolling adhesion but also to the formation of firm adhesions, further emphasizing the important role of HEV expansion to increased lymphocyte homing and accumulation in inflamed lymph nodes. We recently demonstrated that the persistence with which cells mediate rolling adhesion to P-selectin is cell subtype dependent (95). Whether the persistence of rolling adhesion to HEVs via other receptors diverges between lymph node–homing cell subtypes, and whether its combined effect with HEV expansion/contraction affects the recruitment of specific lymphocyte subtypes, remains to be elucidated.…”

Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis

“…Of the identified extravasation pathways, many are redundant with those of leukocyte homing (Strell and Entschladen, 2008), including interactions between metastatic cell-presented selectin ligands (e.g., sialofucosylated CD44 [Hanley et al, 2006;Subramaniam et al, 2007] and carcinoembryonic antigen [CEA] [Thomas et al, 2008], among others ) with corresponding endothelial-, platelet-, or leukocyte-presented E-, P-, or L-selectin ( Figures 1Aiii and 1Aiv). Given the similarities of these mechanisms with physiologically important homeostatic processes, elucidating cancer cell-specific mechanisms of dissemination is thus crucial to the development of effective therapeutic interventions with highly selective anti-metastatic activity (Li and King, 2012;Oh et al, 2015) and also to the identification of biomarkers for the differentiation of patients with aggressive disease (Baumann et al, 2005;Lech et al, 2016).…”

“…Current methods for analyzing single-cell velocities are limited to interrogation via videomicroscopy-based tracking techniques ( Figures 1C and 2). These techniques are not only time consuming but also represent a measured property that cannot be retained by cells for simultaneous off-chip analysis with the molecular mediators that might underlie the heterogeneity of adhesive phenotypes exhibited by metastatic cells that interact with selectins in flow fields ( Figure 1D) and are thought to underlie the process of colon cancer hematogenous metastasis (Edwards et al, 2017;Oh et al, 2015;Reyes-Reyes et al, 2006). Fluorescently tagged antibodies offer a mechanism for labeling cells based on expression of any chosen marker, for example, various adhesive ligands ( Figure 1E), but no such technique exists to ''label'' cells in proportion to their velocity ( Figure 1F).…”