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

Abstract

The tightly orchestrated recruitment of monocytes, whose progeny are critical to the progression and resolution of various physiological and pathophysiological processes, is implicated in the time course, severity, and resolution of pathology. Using a microfluidic-based cell adhesion assay integrating spatiotemporal analyses and micropatterning of adhesive proteins, we interrogated the effects of adhesive molecule presentation length, which varies in vivo with disease and stage, on THP-1 monocyte cell rolling versus firm adhesion mediated by P-selectin and/or ICAM-1 in hemodynamic flow. Our results indicate that co-presentation of P-selectin and ICAM-1 substantially decreases the length of adhesive substrate required to sustain adhesion in flow and that P-selectin functions synergistically with ICAM-1 to substantially enhance THP-1 firm adhesion. This synergy was found to furthermore correlate with diminished cell rolling velocities and length-enhanced secondary cell capture. Our results suggest pathophysiological ramifications for local remodeling of the inflamed microvascular microenvironment in directing the efficiency of monocyte trafficking.

P-, but not E- or L-, selectin-mediated rolling adhesion persistence in hemodynamic flow diverges between metastatic and leukocytic cells

“…Our results revealed a striking similarity in the relationships of mean percent binding time of LS174T and Colo205 metastatic cells on P-selectin with instantaneous velocity, rolling percentages, and selectin concentration sensitivity with these same relationships for L-selectin-mediated adhesion (Figures 6A–6H and 7G–7HG ). Since L-selectin is primarily responsible for tethering and secondary cell capture of leukocytes at later time points in the cell recruitment processes [ 15 , 65 , 66 ] and can mediate adhesion with leukocytes in free flow [ 67 ], the similarity of metastatic cell adhesion persistence facilitated by P-selectin to that of L-selectin may suggest that P-selectin has a higher potential to functionally facilitate heterotypic aggregation of metastatic cells with activated platelets via platelet expressed P-selectin rather than mediating direct, sustained rolling adhesion on the inflamed endothelium. Indeed, McCarty et al reported that metastatic LS174T and Colo205 cells initially tether to P-selectin expressed by surface-immobilized platelets in a manner that is stabilized by engagement of von Willebrand factor [ 31 ].…”

“…In the context of vascular injury, monocyte and platelet accumulation and aggregation at denuded vascular regions also rely on adhesive interactions with P-selectin [ 12 ]. While L-selectin expressed on leukocytes can interact with corresponding ligands on the endothelium of high-endothelial venules or the inflamed endothelium of non-lymphoid tissues [ 13 , 14 ], when expressed by leukocytes adherent to the endothelium, it can additionally facilitate secondary capture of circulating cells [ 15 , 16 ].…”

Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis

“…Approximately 15% of cells were categorized as photoconverted positive (PC+), with the remaining in the photoconverted negative (PCÀ) population (gates defined based on the non-photoconverted cell population) when perfused over P-selectin functionalized surfaces at 0.5 dyn/cm 2 under exposure to 405 nm light, restricted to a length of 10 mm in the direction of flow (Figure 4F). These frequencies were altered by both adjustments to the laser power, flow rate (e.g., wall shear stress), as well as selectin type (P-, E-, versus L-), the latter mediating rolling adhesion at velocities that vary by orders of magnitude (Edwards et al, 2017;Napier et al, 2007;Oh et al, 2015;Thomas et al, 2008) (Table S3), indicating the sensitivity of this methodology to the kinetic process studied. A PC+ cell population isolated by perfusion through a P-selectin functionalized microchannel at 0.5 dyn/cm 2 under exposure to 405 nm light and sorting via FACS exhibited enhanced extents of rolling adhesion upon reperfusion under identical conditions (P-selectin functionalized substrate, 0.5 dyn/cm 2 wall shear stress) compared to both PCÀ and unsorted cell populations (though not to a statistically significant extent as this comparison lacked power at n = 6) ( Figure 4G).…”

“…Metastasis, which is the leading cause of mortality among patients with colon cancer (Massagué and Obenauf, 2016;Welch and Donaldson, 1979), comprises a highly complex sequence of time-and length-scale-regulated steps (Edwards and Thomas, 2017;Lote et al, 2017) that ultimately enable cancer cells to disseminate through the circulation and eventually form secondary tumors (Figure 1Ai). Hemodynamic force hence facilitates the transport of blood-borne metastatic cancer cells to distant organs (Figure 1Aii) but, in order to slow cells down for eventual arrest and extravasation, also stipulates that adhesion underpin the process of extravasation ).…”

“…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).…”