Selectins facilitate the recruitment of circulating cells from the bloodstream by mediating rolling adhesion, which initiates the cell–cell 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 microfluidic-based 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.

Circulating cell adhesion amidst the high shear force environment of the vasculature is initiated by selectins, the molecular ‘brakes’ that slow cells down relative to bulk flow through ligand recognition to facilitate paracrine signaling, firm adhesion and eventual transmigration into the adjacent tissue bed. Deletion of P-selectin, which is expressed by activated platelets and endothelial cells, dramatically attenuates metastatic cell seeding and tumor formation within the lung in experimental mouse models of human colon carcinoma metastasis (Borsig et al., 2001, 2002; Kim et al., 1998), suggesting that P-selectin is an important regulator of circulating tumor cell (CTC) adhesion to and extravasation from the vasculature. Therapeutic interventions disrupting the molecular recognition of P-selectin by CTCs therefore represent a rational means to prevent formation of disseminated metastatic tumor foci, which persists as the largest clinical hurdle in the management of advanced malignancies. However, P-selectin functions in numerous physiological homing processes including leukocyte recruitment during inflammation (Mayadas et al., 1993; Robinson et al., 1999) as well as platelet accumulation and aggregation at areas of vascular injury (Frenette et al., 1995). Broadly inhibiting P-selectin would therefore severely compromise patient immune function and wound healing responses. An ideal anti-metastasis intervention would instead selectively attenuate CTC but not normal circulating cell recognition of P-selectin.

To this end, in vitro screening has the potential to repurpose drugs developed in recent years for applications in the treatment of inflammatory conditions and ischemia-reperfusion injury (Lowe and Ward, 1997) to prevent CTC dissemination into systemic organs. A challenge posed in this application as opposed to other conventional drug targets, however, is that P-selectin-mediated recognition functionally contributes to metastasis under fluid flow rather than static conditions (McCarty et al., 2000). Therefore, as has been appropriately argued in the literature, data obtained using static (no flow) binding assays might not be relevant to the fluid dynamic environment of the vasculature. Another challenge is that selectin-mediated adhesion is highly heterogenous even within a clonal cell population (Aigner et al., 1998), necessitating large sample sizes. A system that uniformly subjects large numbers of whole cells to well-controlled shear flow conditions is thus required to evaluate the influence of therapeutic drug doses on the efficiency of sustained P-selectin adhesion. Such a platform would also reduce the number of animals used in laborious, expensive and time-prohibitive metastasis models to screen and dose-test drug candidates.

Previous efforts developed a parallel-plate flow chamber system for the separation of cells based on their rolling adhesion behavior (Greenberg and Hammer, 2001), a so-called ‘cell adhesion chromatography’ platform. This methodology exploits the differences in rolling adhesion, defined as the transient interaction between a cell in fluid flow and an immobilized adhesive substrate. In such a system where the velocity of the cell while mediating rolling adhesion is significantly lower than its velocity would be in the free flow stream immediately proximal to the surface, cell subpopulations can be enriched. The work which developed this methodology utilized a cell-free system to estimate how CD34+ cells can be enriched from a mixture of adult bone marrow cells on an L-selectin-functionalized substrate (Greenberg and Hammer, 2001) based on the differential rolling adhesion behavior of CD34+ versus CD34 cells over L-selectin (Greenberg et al., 2000).

Based on these conceptual advances, but repurposed as an analytical rather than preparative chromatographic method, we report here the use of a microfluidic-based parallel-plate flow chamber device designed for use in conjunction with video microscopy to chromatographically interrogate adhesion efficiency of cells to P-selectin under physiological shear flow conditions as a novel drug screening platform. In order to achieve uniform cell–substrate contact of a pulse cell suspension input into a selectin-functionalized parallel-plate flow chamber, we designed a feature that enables settling to the chamber bottom of infused cells based on Stokes flow predictions. This simple modification increased the fraction of cells in contact with the substrate upon entry into the main chromatography channel to >95%, enabling the precise quantification of adhesion efficiencies to P-selectin under physiological levels of venular shear stress (∼1 dyn cm−2) (Konstantopoulos et al., 1998), mimicking conditions under which hematogenous metastasis principally occurs. By simultaneously monitoring individual cell rolling velocities and elution times, we unexpectedly observed that longer time- and distance-averaged velocities (determined by the cell elution time from the chamber) of cells do not necessarily correspond to their instantaneous velocities. Using this cell adhesion chromatography methodology, we define a new parameter termed ‘adhesion persistence’, which is conceptually consistent with migration persistence in the context of chemotaxis (Tranquillo et al., 1988) but instead describes the capacity of cells to resist the influence of shear flow and sustain rolling interactions with an adhesive substrate. Importantly, this is distinct from rolling velocity per se because we demonstrate that the adhesion persistence of rolling cell subtypes do not necessarily correspond with their instantaneous velocity. We report that metastatic cells exhibited marked deficiencies in adhesion persistence to P-selectin in shear flow relative to leukocyte-like cells, despite no significant differences in P-selectin ligand expression nor size between analyzed cell subtypes. This suggests that selectins separately regulate the strength versus persistence of cell adhesion, which might result in distinct homing behaviors during the processes of inflammation and wound healing compared with those in metastasis. We also provide evidence that this analytical adhesion chromatography system facilitates comparative dose-testing for the identification of P-selectin antagonists to selectively attenuate CTC but not leukocyte adhesion to P-selectin in shear fluid flow.

Flow-based cell adhesion chromatography

Within the circulatory system at the inflamed endothelium, activated endothelial cells upregulate surface expression of P-selectin to facilitate circulating cell capture from blood flow (Fig. 1A). For the in vitro investigation of adhesion dynamics of P-selectin-binding cells (Fig. 1B), parallel-plate flow chambers that induce a uniform level of shear stress are used in conjunction with video microscopy to interrogate cell adhesion to recombinant P-selectin-functionalized substrates (Fig. 1A). In conventional parallel-plate flow chambers, infused cells have a non-uniform distribution of y-axis positions upon entry into the chamber (Fig. 1C,D). Cell contact with the planar functionalized substrate occurs only after cell settling, which transpires at a rate proportional to the density difference between the perfusion medium and cells, as well as the flow rate (see Eqn 5 in the Materials and Methods). This means that within conventional channel lengths (∼2–5 cm), only a minority of cells are within a distance sufficient to support contact with the planar bottom substrate (Fig. 1E). In addition, any horizontal travel distance within the functionalized channel prior to complete cell settling results in non-uniform cell contact with the bottom substrate, complicating chromatographic analysis of cell adhesion behavior of the infused cell population.

Fig. 1.

Parallel plate flow chamber modification with a settling feature results in uniform cell contact with the channel substrate. (A) Parallel plate flow chambers with P-selectin-functionalized substrates are used to simulate the shear flow environment and P-selectin presentation by the inflamed vascular endothelium. (B) Cell types that exhibit P-selectin-binding activity to facilitate homing in inflammation and metastasis. In conventional parallel plate flow chambers (C), cells enter at non-uniform y-axis distributions resulting in a low proportion of cell contact with the bottom substrate (D). (E) Cell trajectories based on y-axis position at entry into a parallel plate flow chamber predicted by Stokes flow at 1.0 dyn/cm2 indicate complete cell settling requires over 4 cm of channel length. A settling feature (SF) incorporated upstream to the main parallel plate channel (F) increases the residence time of cells in flow, enabling cell settling to the bottom substrate before entering the main channel (F–H). Red streamlines in F indicate x-z trajectories of cells infused into the settling feature at 1.0 dyn/cm2. (I) 5 mm from the main channel inlet, over 95% of cells are within 1.8 times the cell radius from the bottom functionalized substrate when the settling feature is included compared to ∼40% in the same distance for unmodified chambers. (J) The mean cell distance from the channel bottom is uniform throughout the channel length when the parallel plate flow chamber is modified with a settling feature. Each point represents the mean±s.e.m. of n≥3 independently run experiments. (K) Schematic diagram of cell adhesion chromatography set up that enables simultaneous quantification of instantaneous velocity within the imaging FOV as well as elution time as a function of channel length for >104 individual cells in pulse input over the course of 120-min residence time distribution experiments. The settling feature is left uncoated whereas the main channel is functionalized with 25 μg/ml P-selectin. In D,H,I and J, perfusion experiments were performed in blank (1% BSA-coated) chambers at a shear stress of 1.0 dyn/cm2 in a 100-μm deep channel. In B,C,F,G,K, arrows indicate the direction of flow.

Fig. 1.

Parallel plate flow chamber modification with a settling feature results in uniform cell contact with the channel substrate. (A) Parallel plate flow chambers with P-selectin-functionalized substrates are used to simulate the shear flow environment and P-selectin presentation by the inflamed vascular endothelium. (B) Cell types that exhibit P-selectin-binding activity to facilitate homing in inflammation and metastasis. In conventional parallel plate flow chambers (C), cells enter at non-uniform y-axis distributions resulting in a low proportion of cell contact with the bottom substrate (D). (E) Cell trajectories based on y-axis position at entry into a parallel plate flow chamber predicted by Stokes flow at 1.0 dyn/cm2 indicate complete cell settling requires over 4 cm of channel length. A settling feature (SF) incorporated upstream to the main parallel plate channel (F) increases the residence time of cells in flow, enabling cell settling to the bottom substrate before entering the main channel (F–H). Red streamlines in F indicate x-z trajectories of cells infused into the settling feature at 1.0 dyn/cm2. (I) 5 mm from the main channel inlet, over 95% of cells are within 1.8 times the cell radius from the bottom functionalized substrate when the settling feature is included compared to ∼40% in the same distance for unmodified chambers. (J) The mean cell distance from the channel bottom is uniform throughout the channel length when the parallel plate flow chamber is modified with a settling feature. Each point represents the mean±s.e.m. of n≥3 independently run experiments. (K) Schematic diagram of cell adhesion chromatography set up that enables simultaneous quantification of instantaneous velocity within the imaging FOV as well as elution time as a function of channel length for >104 individual cells in pulse input over the course of 120-min residence time distribution experiments. The settling feature is left uncoated whereas the main channel is functionalized with 25 μg/ml P-selectin. In D,H,I and J, perfusion experiments were performed in blank (1% BSA-coated) chambers at a shear stress of 1.0 dyn/cm2 in a 100-μm deep channel. In B,C,F,G,K, arrows indicate the direction of flow.

To overcome these technical limitations, a non-functionalized settling feature was added upstream to the main parallel-plate channel (Fig. 1F,G) to increase the residence time of cells in the chamber. This ensures that cells flowing into the chamber as a pulse suspension input are able to settle to the bottom substrate before entering the main functionalized channel (Fig. 1G,H). This results in >95% cell contact with the substrate (calculated height based on observed velocity within 1.8× the predicted cell radius) after immediate entry into the functionalized main channel (Fig. 1H,I), as compared to only 40% cell contact achieved by conventional parallel-plate flow chambers (Fig. 1D,I). As a result, uniform cell contact is maintained throughout the entire length of the chamber (Fig. 1J), allowing legitimate chromatographic analysis of perfused cells over P-selectin substrates. This parallel plate design modification results in recovery of ∼75% of infused cells, as estimated from the number of detected cells in the field of view (FOV), as opposed to 40% recovery in chambers without an incorporated settling feature.

Using this configuration, the frequency distributions of observed cell-instantaneous velocities and times of elution from the flow chamber main channel to arrive within the video microscopy FOV over a 120 min perfusion window when perfused as a pulse cell suspension input can be generated (Fig. 1K). This methodology enables the comparison of the levels of P-selectin binding in flow between cell subtypes, defined as the fraction of total cells that interact via rolling adhesion (cells that exhibit instantaneous velocities within the FOV >0 µm s−1, as well as <125, 250 and 375 µm s−1 at 0.5, 1.0 and 1.5 dyn cm−2, respectively) with the functionalized substrate. Observed elution times of individual cells were also converted into what we term average velocities by Eqn 1:
formula
(1)
where Lchannel is channel length (either 1.3 or 14 cm), telution is the time of cell elution from the channel length given by the measured cell arrival time within the video microscopy FOV, and toffset is the mean time cells spend in the settling feature (calculated based on chamber geometry as well as shear stress level, which was experimentally confirmed).

Analysis of cell subtype adhesion efficiencies to P-selectin

We used this adhesion chromatography platform to evaluate the adhesive behavior of various human cell lines with previously reported P-selectin-binding activity (Alves et al., 2008; Hanley et al., 2006; Napier et al., 2007) in a simulated shear flow environment. LS174T and Colo205 are human colon carcinoma cells with a high propensity to form lung and liver metastases when infused intravenously or after solid tumor outgrowth (Dallas et al., 2012). THP-1 and HL-60 are immortalized leukocytes that are commonly used as model monocytes and leukocytes (Alves et al., 2008; Hanley et al., 2006). We found that for all cell types used, a broad distribution of instantaneous velocities of cells perfused over P-selectin-functionalized substrates existed (Fig. 2A,B), including a substantial number of cells that travelled through the FOV at free flow velocity (i) and cells that were interacting in a characteristic rolling fashion (ii). Of the rolling cells (Fig. 2B), differences between the frequency of relatively fast (iii) versus slow (iv) rolling cells were found between each cell subtype. In particular, metastatic cells (LS174T and Colo205) interacted with P-selectin via slow rolling adhesion at lower frequencies relative to leukocyte-like (THP-1 and HL-60) cell subtypes.

Fig. 2.

Metastatic colon carcinoma cells interact with P-selectin on average with similar efficiencies and similar-to-higher instantaneous rolling velocities to leukocyte-like cells. (A,B) Histograms of total observed cell instantaneous velocities over P-selectin at 1.0 dyn/cm2. A, i, cells in free flow (>250 μm s−1); A, ii, rolling cells (<250 μm s−1). B, iii, fast rolling cells (>20 μm s−1); B, iv, slow rolling cells (<20 μm s−1). (C,D) As a percentage of the total cell population, metastatic human colon carcinomas LS174T and Colo205 interact with P-selectin through rolling adhesion (as determined by measured instantaneous velocities) at similar frequencies to human monocytic THP-1 and leukocytic HL-60 cells at 0.5, 1.0 and 1.5 dyn/cm2 and is inhibited by 5 mM EDTA (C). (E) Median rolling velocities of cells interacting with P-selectin increase with increasing shear stress. (F) Of the rolling cell fraction, fewer metastatic cells interact with P-selectin in shear flow through slow rolling adhesion (as determined by measured instantaneous velocities) than do peripheral blood cells. In A and B, merged data (n≥2×104) from n≥3 independently run experiments is shown. In C and D, each point represents the percentage of total cells mediating rolling adhesion in independently run perfusion experiments (in D the mean±s.e.m. is also shown). E and F show the mean±s.e.m. of the percentage of total cells mediating rolling adhesion in each of n≥3 independently run experiments. **P<0.01, ***P<0.001 compared with LS174T and Colo205 cells (two-way ANOVA with post-hoc Dunn tests).

Fig. 2.

Metastatic colon carcinoma cells interact with P-selectin on average with similar efficiencies and similar-to-higher instantaneous rolling velocities to leukocyte-like cells. (A,B) Histograms of total observed cell instantaneous velocities over P-selectin at 1.0 dyn/cm2. A, i, cells in free flow (>250 μm s−1); A, ii, rolling cells (<250 μm s−1). B, iii, fast rolling cells (>20 μm s−1); B, iv, slow rolling cells (<20 μm s−1). (C,D) As a percentage of the total cell population, metastatic human colon carcinomas LS174T and Colo205 interact with P-selectin through rolling adhesion (as determined by measured instantaneous velocities) at similar frequencies to human monocytic THP-1 and leukocytic HL-60 cells at 0.5, 1.0 and 1.5 dyn/cm2 and is inhibited by 5 mM EDTA (C). (E) Median rolling velocities of cells interacting with P-selectin increase with increasing shear stress. (F) Of the rolling cell fraction, fewer metastatic cells interact with P-selectin in shear flow through slow rolling adhesion (as determined by measured instantaneous velocities) than do peripheral blood cells. In A and B, merged data (n≥2×104) from n≥3 independently run experiments is shown. In C and D, each point represents the percentage of total cells mediating rolling adhesion in independently run perfusion experiments (in D the mean±s.e.m. is also shown). E and F show the mean±s.e.m. of the percentage of total cells mediating rolling adhesion in each of n≥3 independently run experiments. **P<0.01, ***P<0.001 compared with LS174T and Colo205 cells (two-way ANOVA with post-hoc Dunn tests).

Expressed as a proportion of total cells, metastatic and leukocyte-like cells mediated rolling interactions with P-selectin with similar frequencies at each shear stress evaluated (Fig. 2C,D). As expected, the median instantaneous velocity of rolling cells increased with shear stress for all cell subtypes, with metastatic cell subtypes demonstrating the highest median instantaneous velocities of rolling cells and THP-1 cells rolling on average with higher median instantaneous velocities relative to HL-60 cells (Fig. 2E). Of the rolling cell fraction, a markedly larger portion of leukocyte-like cells compared to metastatic cells exhibited instantaneous velocities characteristic of slow rolling (e.g. instantaneous velocity <20 µm s−1) at all shear stress levels evaluated (Fig. 2F). These interactions were specific to cation-dependent P-selectin-mediated recognition of cell-surface-expressed selectin ligands given that co-infusion with 5 mM of the cation chelator EDTA resulted in a significant diminution of the frequency of adhesion and an increase in instantaneous velocities of rolling cells (Fig. 2C).

Analysis of average velocities based on cell elution times from P-selectin-functionalized channels

We also analyzed the elution time behavior of cell suspensions comprised of the human cell lines in a simulated shear flow environment. Based on measured instantaneous velocities in flow, a channel length (measured as starting after the settling feature to the imaging FOV) of 1.3 cm was required to elute >95% of the infused population of leukocyte-like cell subtypes within 120 min, presumably because of the high frequency of these cells that exhibit slow rolling behavior (Fig. 2F). Accordingly, chambers comprised of a settling feature upstream of a 1.3-cm long channel resulted in elution of the entire population of cells within the experimental timeframe during residence time distribution experiments (Fig. 3A). However, whereas leukocyte elution persisted throughout the course of experimentation, metastatic cancer cells eluted very quickly and at approximately one quarter the mean elution time of leukocytes (Fig. 3A), presumably due to their on average higher instantaneous velocities over P-selectin (Fig. 2E). In order to improve the resolution of residence time distributions obtained for metastatic cells using this chromatographic method, the chamber channel length was extended to 14 cm in order to delay the elution times of metastatic cells such that they occurred over the entire course of experimentation (Fig. 3B). The extension of the chamber had no impact on the average number of cells recovered nor on the measured median instantaneous velocity. Additionally, in both chamber lengths, co-infusion with 5 mM EDTA resulted in cells eluting as quickly from P-selectin-coated channels as non-functionalized (blank) channels (Fig. 3A,B), indicating that the delay in elution was the result of cation-dependent P-selectin-mediated cell adhesion.

Fig. 3.

Longer time- and length-scale-averaged velocities of metastatic colon carcinoma cell interactions with P-selectin are significantly higher than those from peripheral blood cells. (A,B) Representative residence time distribution plots based on cell elution times from blank or P-selectin-functionalized substrates in either 1.3-cm (A) or 14-cm (B) length channels with and without 5 mM EDTA. (C–E) Of the total (C,D) and rolling (E) cell subpopulations, metastatic cells exhibit dramatically higher average velocities, as calculated by the channel length per elution time, relative to leukocyte-like cells. Average velocities of cells interacting with P-selectin increase with increasing shear stress and remain higher for metastatic cell types at all shear stresses tested (D). A–C and E show merged data (n≥2×104) from n≥3 independently run experiments performed at a wall shear stress of 1.0 dyn/cm2. D, mean±s.e.m. of the median of n≥3 independently run experiments. ***P<0.001 (two-way ANOVA with post-hoc Dunn tests).

Fig. 3.

Longer time- and length-scale-averaged velocities of metastatic colon carcinoma cell interactions with P-selectin are significantly higher than those from peripheral blood cells. (A,B) Representative residence time distribution plots based on cell elution times from blank or P-selectin-functionalized substrates in either 1.3-cm (A) or 14-cm (B) length channels with and without 5 mM EDTA. (C–E) Of the total (C,D) and rolling (E) cell subpopulations, metastatic cells exhibit dramatically higher average velocities, as calculated by the channel length per elution time, relative to leukocyte-like cells. Average velocities of cells interacting with P-selectin increase with increasing shear stress and remain higher for metastatic cell types at all shear stresses tested (D). A–C and E show merged data (n≥2×104) from n≥3 independently run experiments performed at a wall shear stress of 1.0 dyn/cm2. D, mean±s.e.m. of the median of n≥3 independently run experiments. ***P<0.001 (two-way ANOVA with post-hoc Dunn tests).

Based on these measured elution times, the average velocity of individual cells during their transit through the chamber can be calculated using Eqn 1 and frequency distributions and/or medians compared across cell subtypes. Strikingly, the average velocities of LS174T and Colo205 cells were elevated approximately three- to eight-fold relative to THP-1 and HL-60 cells (Fig. 3C) at all shear stresses tested (Fig. 3D). The considerable dissimilarity in average velocities of metastatic versus leukocyte-like cells was not the result of subtle differences in the frequency of adhesive cells of each individual subtype, given that when accounting for the behavior of rolling cells only, this dramatic separation between the average velocity of metastatic versus leukocyte-like cells remained (Fig. 3E). Nor could these striking differences in average velocities between cell subtypes be solely attributed to the increase in instantaneous cell velocities exhibited by metastatic relative to leukocyte-like cells (Fig. 2E), which were increased by only 20–200%.

Instantaneous and average adhesive interactions diverge

To investigate this discrepancy, we evaluated the instantaneous velocity distributions of eluting cells. We discovered that at any given elution time, cells from a clonal population exhibited different instantaneous velocities within the FOV (Fig. 4A), including instantaneous velocities indicative of both cells being in free flow (i) and mediating rolling adhesion (ii). This means that even for cells with identical average velocities (given their identical elution time), cells might not interact with P-selectin with the same instantaneous velocity (Fig. 4A). This is supported by the bimodal distribution of elution times predicted by observed instantaneous cell velocities compared to the monomodality of the observed elution profile (Fig. 4B).

Fig. 4.

Comparisons on a per cell basis of instantaneous velocity to elution times or average velocities reveals that instantaneous velocities measured within the imaging FOV are not necessarily indicative of cell adhesive state over longer time and length scales throughout the P-selectin-functionalized channel. (A) At any given elution time, cells exhibit a dichotomy between adhesion behavior, e.g. both rolling (i) and in free flow (no adhesion) (ii). (B) Predicted elution times based on cell velocities within the FOV do not correspond to observed elution times. (C) The slope of average velocity (based on elution time) versus instantaneous velocity of LS174T and THP-1 cells mediating rolling adhesion with P-selectin in shear flow. Ratios of average (vavg) to instantaneous (vinst) velocity for individual cells within the elution point FOV mediating rolling adhesion (D,F) or in free flow (E,F) perfused over P-selectin at 1.0 dyn/cm2. A–C show representative elution time and/or instantaneous rolling velocity data for perfusion experiments with (A–C) LS174T and (C) THP-1 cells. In C, *** indicates the slope of linear regression fit is statistically different from zero (P<0.001) and the differences between slopes are statistically significant (P<0.001). D–F show merged data (n≥2×104) from n≥3 independently run experiments. In F, data represent the median±interquartile range. ***P<0.001 (two-way ANOVA and post-hoc Dunn tests).

Fig. 4.

Comparisons on a per cell basis of instantaneous velocity to elution times or average velocities reveals that instantaneous velocities measured within the imaging FOV are not necessarily indicative of cell adhesive state over longer time and length scales throughout the P-selectin-functionalized channel. (A) At any given elution time, cells exhibit a dichotomy between adhesion behavior, e.g. both rolling (i) and in free flow (no adhesion) (ii). (B) Predicted elution times based on cell velocities within the FOV do not correspond to observed elution times. (C) The slope of average velocity (based on elution time) versus instantaneous velocity of LS174T and THP-1 cells mediating rolling adhesion with P-selectin in shear flow. Ratios of average (vavg) to instantaneous (vinst) velocity for individual cells within the elution point FOV mediating rolling adhesion (D,F) or in free flow (E,F) perfused over P-selectin at 1.0 dyn/cm2. A–C show representative elution time and/or instantaneous rolling velocity data for perfusion experiments with (A–C) LS174T and (C) THP-1 cells. In C, *** indicates the slope of linear regression fit is statistically different from zero (P<0.001) and the differences between slopes are statistically significant (P<0.001). D–F show merged data (n≥2×104) from n≥3 independently run experiments. In F, data represent the median±interquartile range. ***P<0.001 (two-way ANOVA and post-hoc Dunn tests).

When juxtaposing the measured average versus instantaneous velocities of rolling cells, we found that at any given instantaneous velocity, LS174T (Fig. 4C) and Colo205 (data not shown) cell subtypes exhibited a significantly higher average velocity relative to that of THP-1 (Fig. 4C) and HL-60 (data not shown) cell subtypes. This resulted in a slope of approximately seven for LS174T cells whereas THP-1 (Fig. 4C) and HL-60 (data not shown) were much closer to one. On a per cell basis, we next considered the ratio of average to instantaneous velocities, where a value of one implies that the instantaneous cell velocity persists throughout the channel and matches that of the average cell behavior. Cells exhibiting instantaneous velocities within the imaging FOV indicative of rolling adhesion (instantaneous velocities <250 μm s−1 at 1.0 dyn cm−2) exhibited a ratio of average to instantaneous velocities substantially greater than one for metastatic LS174T and Colo205 cells subtypes but approached an average closer to one for THP-1 and HL-60 cells (Fig. 4D,F). This implies that THP-1 and to a lesser extent HL-60, but not LS174T nor Colo205, cells on average sustained rolling adhesion throughout the channel length. By contrast, LS174T and Colo205 cells interacting with P-selectin through rolling adhesion at the time of elution eluted much earlier than would be predicted from observed instantaneous velocities, indicating that these cells were instead not interacting with the P-selectin substrate for significant portions of the channel length. These data suggest that although metastatic cancer cells might have a capacity to interact with P-selectin at a given rolling velocity, rolling adhesion might not be sustained over prolonged times or distances. The small increase in vavg/vinst over a value of one also suggests the same to some extent for HL-60 cells.

The trend in vavg/vinst ratio of cells in free flow at the time of observation within the imaging FOV (>250 μm s−1 at 1.0 dyn cm−2) was as follows: the ratio of average to instantaneous velocity approached a median of one for metastatic LS174T and Colo205 cells but was markedly less than one for THP-1 and HL-60 cells (Fig. 4E,F). This indicates that LS174T and Colo205 cells moving at free flow velocities at the time of elution travelled through the majority of the P-selectin-functionalized channel at that velocity, therefore mediating little to no adhesion. THP-1 and HL-60 cells moving at free flow velocities within the FOV, by contrast, eluted much later than would be predicted from observed instantaneous velocities, indicating that these cells were instead interacting with the P-selectin substrate for significant portions of the chamber length, but not within the imaging FOV.

Impaired persistence of rolling adhesion to P-selectin by metastatic cells

These data indicate that instantaneous velocities recorded within the imaging FOV represent only a snapshot of cell adhesion behavior and are not necessarily representative of cell adhesion throughout the length of the channel (Fig. 5A). Using a mass balance evaluation of the predicted elution time based on the measured instantaneous velocity of rolling cells (Fig. 4Aii), the percentage time a cell spent bound to the substrate could be calculated (Eqn 2):
formula
(2)
where vfree flow is the predicted cell velocity when not adherent (in free flow) and vinst is the measured instantaneous velocity. With increasing elution time, we found the percentage binding time of cells mediating rolling adhesion on P-selectin under shear fluid flow in the imaging FOV to increase (Fig. 5B), given that cells that can sustain interactions for longer are more likely to elute later. We next compared the relative capacity of different cell subtypes to sustain interactions with P-selectin (as indicated by high percentage binding times) and found a dichotomy between human metastatic cancer cells versus leukocyte-like cells (Fig. 5C) that was conserved over all shear stress levels tested. Strikingly, this deficiency in adhesion persistence by rolling metastatic cells relative to leukocyte-like cells did not correlate with any difference in mean instantaneous velocity (Fig. 5C), suggesting that adhesion persistence is independently regulated from instantaneous rolling velocity at the cellular level.
Fig. 5.

Percentage binding time analysis reveals that adhesion persistence is dramatically compromised in metastatic but not leukocyte-like cell adhesion to P-selectin in shear flow. (A) Ratios of average (vavg) to instantaneous (vinst) velocity for individual LS174T and THP-1 cells indicate that cells must not be continuously translating through the chamber with the instantaneous velocity observed within the imaging FOV. (B) As expected, rolling cells with higher adhesion persistence (high percentage binding time) elute at later times. (C) The mean percentage binding time of metastatic rolling cells to P-selectin is significantly reduced relative to leukocyte-like cells and does not significantly change between shear stress levels 0.5 (circles), 1.0 (squares) and 1.5 (triangles) dyn/cm2. B shows representative elution time and calculated percentage binding time data for perfusion experiments with LS174T cells over P-selectin at 1.0 dyn/cm2. In C, each point represents the mean of an independently run experiment. ***P<0.001 (two-way ANOVA and post-hoc Dunn tests).

Fig. 5.

Percentage binding time analysis reveals that adhesion persistence is dramatically compromised in metastatic but not leukocyte-like cell adhesion to P-selectin in shear flow. (A) Ratios of average (vavg) to instantaneous (vinst) velocity for individual LS174T and THP-1 cells indicate that cells must not be continuously translating through the chamber with the instantaneous velocity observed within the imaging FOV. (B) As expected, rolling cells with higher adhesion persistence (high percentage binding time) elute at later times. (C) The mean percentage binding time of metastatic rolling cells to P-selectin is significantly reduced relative to leukocyte-like cells and does not significantly change between shear stress levels 0.5 (circles), 1.0 (squares) and 1.5 (triangles) dyn/cm2. B shows representative elution time and calculated percentage binding time data for perfusion experiments with LS174T cells over P-selectin at 1.0 dyn/cm2. In C, each point represents the mean of an independently run experiment. ***P<0.001 (two-way ANOVA and post-hoc Dunn tests).

No correlation of P-selectin ligand density and size with cell subtype adhesivity metrics

To determine whether the differences in metrics of cell adhesivity to P-selectin could be directly attributed to differences in cell subtype densities of P-selectin ligand or sialyl-Lewis x/a (sLex/a) expression, flow cytometry was performed. We found that both metastatic and leukocyte-like cells stained prodigiously and to a relatively similar extent with recombinant P-selectin (Fig. 6A) and exhibited no notable differences in sLex/a expression (Fig. 6B). We also found only modest to subtle differences between diameters of each cell type in suspension, which ranged on average between 11 and 13 μm (Fig. 6C,D) and no appreciable differences in cell aspect ratios (Fig. 6D). Pearson correlation analysis also revealed no statistically significant relationships between cellular P-selectin ligand and sLex/a densities, diameter, as well as aspect ratio with metrics of P-selectin adhesivity that were measured for individual cell subtypes in flow including adhesion efficiency (the fraction of total cells that mediate rolling adhesion in flow), median instantaneous velocity, median average velocity and percentage binding time. These results indicate that neither adhesive ligand expression nor cell size or geometry could predict the results of functional P-selectin adhesion assays a priori.

Fig. 6.

Cellular P-selectin ligand densities and size features do not predict the measured metrics of P-selectin adhesivity in flow. Flow cytometry fluorescence intensity distributions for (A) P-selectin ligand and (B) sLex/a expression levels. Measured cell size distributions (C,D) and aspect ratios (D) of cell subtypes. Pearson correlation analysis reveals no statistically significant relationships (P>0.05 with P=0.05 graphically indicated with a dotted line in E) between (A,B,E) P-selectin ligand and sLex/a densities (fluorescence signal per μm2 of cell surface area), (C,E) diameter or (D,E) aspect ratio of cell subtypes with their respective measured levels of adhesion efficiency, median instantaneous velocity of cells rolling within the imaging FOV, median average velocity of total cells or percentage binding time.

Fig. 6.

Cellular P-selectin ligand densities and size features do not predict the measured metrics of P-selectin adhesivity in flow. Flow cytometry fluorescence intensity distributions for (A) P-selectin ligand and (B) sLex/a expression levels. Measured cell size distributions (C,D) and aspect ratios (D) of cell subtypes. Pearson correlation analysis reveals no statistically significant relationships (P>0.05 with P=0.05 graphically indicated with a dotted line in E) between (A,B,E) P-selectin ligand and sLex/a densities (fluorescence signal per μm2 of cell surface area), (C,E) diameter or (D,E) aspect ratio of cell subtypes with their respective measured levels of adhesion efficiency, median instantaneous velocity of cells rolling within the imaging FOV, median average velocity of total cells or percentage binding time.

Dose effects of heparin on cell subtype interactions with P-selectin

We next evaluated the influence of P-selectin antagonist heparin on the capacity of metastatic cancer cells versus leukocytes to mediate and sustain adhesion to P-selectin in shear flow (Fig. 7A,B). We found that escalating concentrations of heparin resulted in a larger reduction in the frequency of malignant versus leukocyte-like cell rolling over P-selectin in shear flow at 1.0 dyn cm−2 (Fig. 7C) and a very significant versus slight reduction in the frequency of slow rolling cells for LS174T and THP-1 cells, respectively (Fig. 7D).

Fig. 7.

Analysis of cell instantaneous velocity and percentage binding time reveals that a low dose of heparin attenuates rolling adhesion of LS174T cells to P-selectin at 1.0 dyn/cm2 through multiple related mechanisms without impairing adhesion of THP-1 cells. (A,B,G,H) Cumulative distribution frequencies of LS174T (A,G) and THP-1 (B,H) cell instantaneous velocities (A,B) and percentage binding times (G,H) with escalating doses of heparin. (A–D) Increasing heparin concentration decreases the proportion of total (C) and slow rolling (D) LS174T cells to a significantly greater extent relative to THP-1 cells. As assessed by flow cytometry, (E) pre-incubation of P-selectin with increasing doses of heparin prior to co-incubation with cells interferes with cell labeling but (F) substantially overestimates the attenuation of cell adhesion to P-selectin-functionalized planar substrates in flow. (G,H) Increasing heparin concentration dramatically reduces the percentage binding time of rolling LS174T but not THP-1 cells to P-selectin. In A and B, the dotted line indicates the rolling cell instantaneous velocity threshold. A,B,G and H show merged data (n≥1×104–2×104) from independently run experiments. C and D show the mean±s.e.m. of the mean of each of independently run experiments. E shows the mean±s.e.m. of two independently run experiments. In F, a value of one is graphically indicated with a dotted line and **P<0.01, ***P<0.001 (two-way ANOVA and post-hoc Dunn tests).

Fig. 7.

Analysis of cell instantaneous velocity and percentage binding time reveals that a low dose of heparin attenuates rolling adhesion of LS174T cells to P-selectin at 1.0 dyn/cm2 through multiple related mechanisms without impairing adhesion of THP-1 cells. (A,B,G,H) Cumulative distribution frequencies of LS174T (A,G) and THP-1 (B,H) cell instantaneous velocities (A,B) and percentage binding times (G,H) with escalating doses of heparin. (A–D) Increasing heparin concentration decreases the proportion of total (C) and slow rolling (D) LS174T cells to a significantly greater extent relative to THP-1 cells. As assessed by flow cytometry, (E) pre-incubation of P-selectin with increasing doses of heparin prior to co-incubation with cells interferes with cell labeling but (F) substantially overestimates the attenuation of cell adhesion to P-selectin-functionalized planar substrates in flow. (G,H) Increasing heparin concentration dramatically reduces the percentage binding time of rolling LS174T but not THP-1 cells to P-selectin. In A and B, the dotted line indicates the rolling cell instantaneous velocity threshold. A,B,G and H show merged data (n≥1×104–2×104) from independently run experiments. C and D show the mean±s.e.m. of the mean of each of independently run experiments. E shows the mean±s.e.m. of two independently run experiments. In F, a value of one is graphically indicated with a dotted line and **P<0.01, ***P<0.001 (two-way ANOVA and post-hoc Dunn tests).

In order to probe the molecular underpinnings of these differences, we incubated LS174T and THP-1 cells with P-selectin in the presence of increasing doses of heparin. Flow cytometry revealed that heparin interferes with P-selectin labeling in a dose-dependent manner for both cell types, with LS174T cells exhibiting a more dramatic attenuation in P-selectin staining with low doses of heparin than THP-1 cells (Fig. 7E), mirroring very closely the effect mediated by heparin on the frequency of cells rolling slowly over P-selectin (Fig. 7D). However, the percentage of P-selectin labeled cells (of the total population) significantly overestimated the attenuation in the total adhesion of (not just slow) rolling LS174T cells to P-selectin-functionalized planar substrates in flow (Fig. 7C), as exhibited by high ratio of LS174T cells exhibiting rolling adhesion to the number of cells staining positive for P-selectin at increasing doses of heparin (Fig. 7F).

Finally, using the percentage binding time as a newly defined metric of adhesion persistence, we determined that although low heparin doses significantly decrease the capacity of LS174T cells to sustain adhesion to P-selectin (Fig. 7G), THP-1 cell adhesion is only affected (and only modestly so) at the highest dose tested (Fig. 7H). These data indicate that this newly described cell adhesion chromatography platform and analytic methodology to quantify adhesion efficiency (based on frequency of rolling cells of total) and persistence (based on juxtaposing measured velocity versus elution time) can be used to measure the relative efficacy of agent dose in impeding P-selectin adhesion under physiological shear conditions.

P-selectin contributes to the hematogenous dissemination of CTCs by mediating interactions with host endothelial cells and platelets. For example, P-selectin expressed by the activated endothelium can facilitate CTC adhesion to the vessel wall (Konstantopoulos and Thomas, 2009). After entry into the bloodstream, CTCs also form complexes with platelets (Borsig et al., 2001, 2002), which masks CTCs from cytotoxic natural killer cells (Nieswandt et al., 1999) or bridges CTC adhesion to the vascular endothelium (Burdick and Konstantopoulos, 2004), thus assisting carcinoma escape from the circulation and seeding of metastatic foci. Given that genetic knockdown of P-selectin dramatically reduces metastasis (Borsig et al., 2001, 2002; Kim et al., 1998) interference with P-selectin recognition by CTCs is a rational therapeutic approach to attenuate cancer dissemination in patients with advanced malignancies.

The biophysical and biomechanical regulation of instantaneous selectin ligand recognition at either a whole-cell or purified single-molecule level has been widely interrogated (Alon et al., 1995; Fritz et al., 1998; Hanley et al., 2003, 2004; Marshall et al., 2003; McCarty et al., 2000) given that selectins facilitate high kon and koff rate interactions under hemodynamic force (Chen and Springer, 2001). However, the capacity of cells to sustain interactions with the vasculature during their transit through the circulatory system, which is mediated through selectins, influences their capacity to respond to chemokine signaling, firmly adhere to the endothelium (Diacovo et al., 1996; Veerman et al., 2007) and extravasate to take up residence in the surrounding tissue (Hanna and Etzioni, 2012). Thus, the lack of quantitative insight into the force regulation of selectin-mediated adhesion over longer time and distance scales limits the design of effective therapeutic interventions to selectively modulate cell homing in a cell-subtype-selective manner, such as in anti-metastasis applications.

Herein, we report the design (Fig. 1) and implementation of an analytical cell adhesion chromatography platform that enables the independent analysis of instantaneous versus longer distance and time scale adhesion behavior (Figs 24). This microfluidic platform allows for the quantification of cell adhesion to planar substrates functionalized with adhesive ligands in shear flow, thus recapitulating several of the salient features relevant to selectin-mediated cell adhesion in the vasculature. We have modified the conventional parallel-plate flow chamber configuration with a settling feature (Fig. 1F,G,K), which facilitates accurate quantification of the frequency of cells within an infused cell pulse that mediates rolling interactions, as determined by their measured instantaneous velocity within the video microscopy FOV. This approach offers the substantial advantage over other conventional parallel-plate flow chamber systems for the evaluation of cell adhesion efficiencies because it allows for the entire population of perfused cells to have contact with the substrate, in contrast to only ∼40% cell contact with typical chamber geometries. Indeed, although the number of interacting cells within a short experimental timeframe (typically ∼2–5 min) is a widely reported metric used for quantifying the extent of selectin-mediated adhesion, these levels will vary from system to system based on chamber geometry and imaging location. Furthermore, these experiments are typically performed under a constant perfusion of cell solution, complicating mass balance calculations (Munn et al., 1994).

Using this system with a pulse cell suspension input, we can recover the substantial majority of perfused cells, and thus quantify adhesion efficiencies in a manner that is reproducible and can be used to compare cell subtypes or treatment groups. Thus, rather then separating cell subpopulations based on rolling adhesion (Greenberg and Hammer, 2001), we utilize the chromatographic nature of this system to analyze the adhesive behavior of cells in flow. We find that metastatic human colon carcinoma LS174T and Colo205 cells interact with P-selectin under venous levels of shear stress at efficiencies on par with human leukocyte-like THP-1 and HL-60 cells (Fig. 2C,D). However, leukocytes interact with P-selectin through slow rolling interactions at higher frequencies than metastatic cancer cells (Fig. 2F), despite nearly identical levels of selectin ligand expression (Fig. 6A,B) and size features (Fig. 6C,D). These differences might result from P-selectin glycoprotein ligand-1 versus variant isoforms of CD44 (among others, see Napier et al., 2007) functioning as the predominant P-selectin ligands on THP-1 (van Genderen et al., 2006) and HL-60 (Norman et al., 1995) versus LS174T and Colo205 cells (Napier et al., 2007; Thomas et al., 2008), respectively, and their corresponding differences in bond tensile strength and unstressed off rate (Hanley et al., 2003; Raman et al., 2011). Additionally, although average per cell densities of selectin ligands are similar between cell types (Fig. 6B), whether P-selectin ligands expressed by tumor cells in suspension are clustered into microdomains on the cell surface is unknown. Thus, differences in the cell surface distribution of adhesion receptors, which influences the initiation of adhesive interactions but not rolling velocity nor the strength of established rolling adhesion (Stein et al., 1999), might contribute to differential persistence of rolling adhesion between metastatic and leukocyte-like cells. Other as-of-yet undefined biochemical (glycocalyx) and biophysical (cell deformability) differences might also play a role.

In addition to enabling uniform cell contact with the planar substrate to facilitate the quantification of cell adhesion efficiency in flow, our simple chamber design innovation homogenizes the channel length traveled by perfused cells within a pulse input. Given that all cells are in contact with the adhesive substrate for identical lengths in a given experiment using this configuration, the average velocity of a cell can be calculated based on its elution time (Fig. 1K). We found that although metastatic cells interact with P-selectin at similar frequencies to leukocyte-like cells (Fig. 2C,D), the longer distance- and time-averaged velocities of metastatic cells were dramatically higher relative to those of leukocytes (Fig. 3C–E). Given that metastatic LS174T and Colo205 colon carcinoma cells interact on average with P-selectin in shear flow at higher instantaneous velocities relative to THP-1 and HL-60 cells (Fig. 2E), these results would not be unexpected at first glance. However, the incongruent fold difference in instantanous versus average velocities between cell subtypes suggests that another more-dominant mechanism is present. Our finding of co-eluting cells exhibiting instantaneous velocities indicative of cells being in both rolling adhesion and free flow (Fig. 4A) suggests that an observed instantaneous velocity of a cell is not necessarily indicative of its capacity to mediate adhesion. Rather, instantaneous velocity describes the adhesive state of a cell (rolling versus non-interacting) and not the type (adhesive versus non-adhesive). This is best exemplified by our observation of cells with identical measured instantaneous velocities within the imaging FOV exhibiting significantly different average velocities depending on their subtype (Fig. 4C). Hence, despite individual cells exhibiting the same rolling adhesion characteristics over short time and length scales, their capacities to sustain that adhesion diverges between cell subtypes (Fig. 4C). This concept is furthermore supported by our findings that HL-60 cells on average exhibit slower instantaneous velocities (Fig. 2E) but higher average velocities (Fig. 3D) relative to THP-1 cells, corresponding to slightly lower mean percentage binding times (Fig. 5C).

Strikingly, for both the populations of cells rolling and in free flow within the imaging FOV, the median of the per cell ratio of average to instantaneous velocity is roughly tenfold higher for metastatic LS174T and Colo205 colon carcinoma cells than for THP-1 leukocytes. Furthermore, instantaneous velocity within the FOV appears to misconstrue the capacity of cells to sustain adhesive interactions, as this measure indicates too little adhesive interaction by THP-1 and HL-60 cells and too much adhesive interaction by LS174T and Colo205 (and to a much less extent HL-60) cells based on their respective elution time distributions. The percentage binding time metric, which quantifies rolling cell adhesion persistence in shear flow, might therefore represent a previously overlooked aspect of selectin-mediated adhesion that regulates homing activity throughout the circulation. It remains to be determined whether diminished adhesion persistence is a characteristic of P-selectin adhesion in other pathologic states.

The subtype-dependent differences in adhesivity metrics of cells to P-selectin in flow showed no statistically significant correlation with measured selectin ligand densities and size features (Fig. 6E). Although insignificant, cell size did demonstrate a stronger correlation with cell instantaneous and average velocities (Fig. 6E), which is expected given that larger cells are subjected to higher levels of hemodynamic force when interacting with a planar substrate in flow. Interestingly, a somewhat stronger correlation, which was still statistically insignificant, between cell size and percentage binding time was found (Fig. 6E). This suggests that a biophysical mechanism underpins this metric of adhesivity, a concept supported by the lower percentage binding time (as well as a higher vavg/vinst ratio) to P-selectin exhibited by HL-60 relative to THP-1 cells despite P-selectin glycoprotein ligand-1 functioning as the predominant P-selectin ligand on both cell subtypes (Norman et al., 1995; van Genderen et al., 2006). However, the underlying mechanism of this adhesive behavior remains to be elucidated.

Numerous agents have been or are in development for modifying selectin–ligand interactions in inflammatory and reperfusion injury states (Lowe and Ward, 1997). Given that P-selectin-mediated recognition is hypothesized to primarily contribute to metastasis and disease progression in the blood stream, these agents have the potential to be applied to use in treatment or management of disseminated cancers. A drug that has been proposed for this use is the antithrombotic agent heparin (Bendas and Borsig, 2012). It is a potent inhibitor of P-selectin binding at concentrations substantially lower than required for its anticoagulant activity (Koenig et al., 1998). Furthermore, in vivo studies have demonstrated considerable efficacy in the reduction of tumor metastasis by heparin and other sulfated compounds (Bendas and Borsig, 2012; Borsig et al., 2001). We found that a low dose of heparin significantly diminished the efficiency of adhesion, the proportion of slow rolling cells, and percent binding time of LS174T but not THP-1 cells to P-selectin (Fig. 7A–D,G,H). This differential response might arise due to the heparin-binding domain of P-selectin being more crucial to cell adhesion when mediated by the P-selectin ligands expressed by metastatic cells, as opposed to P-selectin glycoprotein ligand-1 expressed by leukocyte-like cells (Konstantopoulos and Thomas, 2009; Wang and Geng, 2003). Alternatively, LS174T cells might be more significantly affected by heparin-induced reductions in P-selectin levels available for adhesion due to the lower tensile strengths and higher unstressed off rates exhibited by whole LS174T cells (Hanley et al., 2003) or purified CD44v (Raman et al., 2011) relative to that of P-selectin glycoprotein ligand-1 binding to P-selectin. The long length of microvilli presenting P-selectin glycoprotein ligand-1 (Yago et al., 2002) might also confer a geometrical advantage over carcinoma-expressed selectin ligands for contact with P-selectin in the presence of heparin. These results suggest that heparin might provide robust anti-metastasis efficacy with only marginal effects on monocyte homing activity given the modest diminution in THP-1 cell adhesion efficiency and negligible effects on THP-1 instantaneous velocities and adhesion persistence behavior. Although flow cytometry also indicates a cell-subtype-dependent attenuation of P-selectin binding in the presence of heparin (Fig. 7E), it substantially underestimates the fraction of total cells that mediate rolling adhesion with P-selectin in flow (Fig. 7F). Taken together, these results indicate the potential of our cell adhesion chromatography platform for in vitro dose testing of pharmacologic agents with selective anti-metastasis activity and suggest that this platform has an advantage over single-variable molecular methods such as flow cytometry.

In conclusion, this cell adhesion chromatography-based analytical methodology facilitates the quantification of cell adhesion efficiencies under physiological fluid flow conditions. We demonstrate that this technique represents an effective platform to dose-test P-selectin antagonists and quantify their relative influence on cell-subtype-specific adhesion to P-selectin. Through the juxtaposition of the predicted rolling cell elution time based on instantaneous velocity with observed elution time, we find that metastatic colon carcinoma cells, but not leukocyte-like cells, exhibit a diminished capacity to sustain adhesion to P-selectin in shear flow. Strikingly, this reduced adhesion persistence is not solely reflected by cell rolling at higher instantaneous velocities. Taken together, these data support the notion that the aforementioned biophysical parameters are independently regulated at the cellular level. The analytical capabilities of cell adhesion chromatography will facilitate future work aimed at studying how this differential regulation might contribute to overall cell response to paracrine signaling and other factors regulating the multi-step adhesive cascade of cell homing, as well as identifying specific and targeted anti-metastatic therapies.

Reagents and materials

Cell culture reagents were purchased from Life Technologies. Heparin (1000 units ml−1) was from SAGENT Pharmaceuticals. Anti-human-IgG (Fc specific) antibody and bovine serum albumin (BSA) were from Sigma-Aldrich. P-selectin-IgG Fc (P-selectin) was purchased from R&D Systems. HECA-452, Rat IgM isotype control and FITC-conjugated mouse anti-rat-IgM antibodies were purchased from BD Pharmingen (San Jose, CA, USA) and Cy3-conjugated anti-human IgG Fc specific antibody was purchased from Sigma-Aldrich. Polydimethylsiloxane (PDMS) base and curing agent were from Ellsworth Adhesives (Germantown, WI, USA).

Chamber fabrication

The chamber was composed of a PDMS block with an open 2-mm wide by 100-μm deep channel either 1.3 or 14 cm long attached to a non-tissue culture treated polystyrene plate (Corning), forming a closed channel with a planar substrate. A settling feature, designed to enable complete cell settling to the chamber bottom before entry into the main channel was designed in a circular configuration with an inner and outer radius of 10.5 and 11.75 mm, respectively, and a 10.9-mm linear inlet region before a bifurcation at the 2.5-mm wide circular channel. The entire settling feature depth was 100 μm. The molds with a negative feature for the PDMS block were produced by micro-machining an aluminium block (Alloy 6013) with a micro-milling machine (OM 1-A, HAAS, Oxnard, CA, USA). Silicone elastomer base and curing agent at a ratio of 9:1 was poured into the molds and cured at 90°C for 3 to 4 h. After the curing process, a biopsy punch was used to make holes for an inlet and an outlet. Then, glass slides (VWR, Randor, PA, USA) were spin-coated (WS-400BZ-6NPP-LITE, Laurell, North Wales, PA, USA) with a mixture of PDMS elastomer base and curing agent at a ratio of 10:1. The bottom of the PDMS block, at the location of the channel, was stamped on the glass slides to apply uncured PDMS mixture. The PDMS block with its bottom covered with the uncured PDMS mixture (functioning as glue) was placed on a polystyrene plate. Care was taken to ensure that there were no air bubbles formed at the interface between the PDMS block and the polystyrene plate. Then, the whole assembly was treated in a 50°C oven overnight to completely cure the PDMS glue mixture, thus finalizing chamber bonding. After the curing process, the chamber was cooled to room temperature and either used immediately or functionalized with proteins for adhesion experiments.

Cell culture

All cell lines were obtained from the American Type Culture Collection. Human colorectal adenocarcinoma cell lines LS174T and Colo205 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic or RPMI 1640 supplemented with 10% heat-inactivated FBS and 1% antibiotic-antimycotic, respectively. Each line was subcultured when they reached 90% confluency. For perfusion and flow cytometry experiments, LS174T and Colo205 cells were harvested by mild trypsinization (0.25% Trypsin-EDTA at 37°C) and subsequently incubated in complete medium (at 107 cells ml−1) at 37°C for 2 h to allow regeneration of surface glycoproteins. In the case of Colo205 cells, the suspension cell fraction was first collected and subsequently combined with trypsin-harvested adherent cells. After regeneration, cells were centrifuged at 300 g for 5 min, washed with Dulbecco's phosphate-buffered saline (D-PBS) containing 0.9 mM Ca2+ and 0.5 mM Mg2+, and resuspended after centrifugation in 0.1% BSA in D-PBS containing Ca2+ and Mg2+ at a final concentration of 5×105 cells ml−1. Human monocytic THP-1 cells and human promyeloblast HL-60 cells were suspension cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or RPMI 1640 supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin, respectively. THP-1 and HL-60 cells were subcultured every 3 days at a dilution of 1:4 or 1:5. The density of THP-1 and HL-60 cells was maintained at between 105 and 106 cells ml−1. For perfusion and flow cytometry experiments with these cell types, cell surface glycoprotein regeneration was not required. Collected cell suspensions were washed with D-PBS containing Ca2+ and Mg2+ and resuspended in 0.1% BSA in D-PBS containing Ca2+ and Mg2+ at 5×105 cells ml−1.

Perfusion experiments

The D-PBS-filled chamber with a non-functionalized (1% BSA blocked) substrate, installed with the inlet reservoir and tubing connected to the outlet, was placed on an optical microscope (Eclipse Ti, Nikon). The tubing from the outlet was then connected to the syringe on the syringe pump through a luer-lock connection. To determine the mean cell distance from the bottom planar substrate during transit through the FOV, a suspension of 5×105 LS174T cells per ml was added to the inlet reservoir and perfused at the desired flow rate. The focal plane was determined in advance by focusing on the bottom of the chamber and raising it ∼10 µm [as indicated by the NIS-Elements software (Nikon)]. After reaching steady state, 5-min movies imaged at nine evenly spaced positions along the chamber length were taken and subsequently analyzed using the equation:
formula
(3)
to calculate the predicted distance of each cell based on its velocity within the FOV. To perform residence time distribution experiments, the desired imaging site was located and the chamber was fixed on the stage of the microscope. The focal plane was determined in advance by focusing on the bottom of the chamber and raising it 5 µm. Prepared cell suspensions were then added to the inlet reservoir (50 μl of cell suspension concentrated at 5×105 cells ml−1 in 0.1% BSA solution for a total of 2.5×104 cells) as a pulse before initiating the syringe pump at the desired flow rate to initiate imaging and the experiment. When the cell solution was almost completely removed from the reservoir, 0.1% BSA solution was added, effectively creating a cell pulse in the perfusion chamber. As all residence time distribution experiments were run for 2 h, 0.1% BSA solution was periodically added to the reservoir. In select experiments, cell suspensions and perfusion medium contained 5 mM EDTA or heparin at 0.5, 5 and 25 U ml−1. Video acquisition was performed using NIS-Elements (Nikon). For all perfusion experiments, identical camera and software settings were employed. The frames per second used was 25, the exposure time was 0.281 μs, the objective magnification was 10×, the video image size was 960 pixels by 500 pixels, and the image was binned 2×2. Videos generated by NIS-element were saved in an audio video interleaved format for subsequent viewing and analysis.

Video analysis

5-min videos of cell perfusion experiments performed at prescribed chamber locations were analyzed by using a custom MATLAB-based cell tracking software. Contrast was first applied to detect cell edges and the x and y positions of each detected cell were stored in the first frame. Cell position, shape and size were subsequently recorded until the cell could no longer be detected in the FOV. Tracking was constrained by considering less than half of the pixels of the previously detected cell diameter forward and a divergence angle 15° to the flow direction. Data was only used for cells tracked for more than five frames and debris smaller than 10 μm was excluded. Cell velocity, based on the total tracked distance and video frames per second, as well as the mean aspect ratio, x and y cell radius and projected area of each cell were calculated using these data. The metric yvel/0.5(rx+ry) (the vertical location of the cell from the bottom of the chamber over an effective cell radius) was calculated from Eqn 1 and the mean x and y radius measured during the course of tracking each cell.

Videos from residence time distribution experiments were analyzed by modifying the OpenCV-based Traffic Flow Analyzer (https://github.com/telescope7/TrafficFlowAnalysis). Briefly, the background was subtracted using a mask weight of 0.0005, resulting in an approximate 80 s moving average. A blurring factor of 13 and a thresholding factor of 5 were used. The contour detection algorithm defines cells as >5 μm in diameter that are stored in the Moving Object database and compared to other previously tracked objects and mapped to the appropriate moving instance based on forward movement (using either a look-ahead window or an overlapping object boundary analysis). Once the object is no longer tracked or exits the FOV, the object data is read to file. Data were included in the cell velocity analysis if the tracking distance was >400 pixels, the cell diameter between 14 and 60 μm and the cell within 50 μm of the chamber bottom (as calculated based on its tracked velocity). Free-flow cell velocities were approximated as 529, 1059 and 1588 at 0.5, 1.0 and 1.5 dyn cm−2, respectively. Cells were considered to be rolling if their velocities were approximately one quarter of the free flow cell velocity. The average velocity was calculated from the chamber length (1.3 or 14 cm) divided by elution time less the offset time (which accounts for the mean time cells spend in the settling feature before entering the main channel). For the percentage binding time calculations, only rolling cells are considered. Graphs labeled as ‘Total Cells’ or ‘Rolling Cells’ represent individual, pooled, or averaged cell velocity data from all observed cells or cells with instantaneous velocities less than the maximum rolling velocity threshold, respectively.

Cell settling trajectory prediction

To predict the required distances for cells to settle within a flow field relative to a planar bottom substrate by Stoke's flow, a MATLAB (ver. 12.7.0) script was written to simulate particle trajectories. The parameters required to predict settling distance using Stokes' law were mass densities of the cells and of the medium, dynamic viscosity of the medium, cell size, gravity and wall shear stress, which were 1.032×103 kg m−3 (Bizzari et al., 1983), 1.0233×103 kg m−3 (Zhang and Neelamegham, 2003), 9×10−4 Pa s (Coumans et al., 2013), 14 μm, 9.81 m s−2, and either 0.5, 1.0 or 1.5 dyn cm−2, respectively. To predict how cell entry height into the chamber influences the distance it travels before coming in contact with the substrate, the trajectories of cells at prescribed chamber entry distances from the bottom substrate evenly distributed over the chamber height (100 μm) were predicted. A time increment of 0.1 s was used to calculate the x and y trajectories of cells using Eqns 4–7 until the particles reached the bottom of the chamber. Hydrodynamic wall effects were assumed to be negligible. Alternatively, the cell settling trajectories of cells located at the top of chambers of different heights (20–300 μm) were calculated:
formula
(4)
formula
(5)
formula
(6)
formula
(7)
where vx is the x-direction velocity of the cell (depending on the y position of the cell), Q is the volumetric flow rate, w is the channel width, h is the chamber height, vset is the settling velocity of the cell, ρcell is the mass density of the cells, ρmedium is the mass density of the perfusion medium, µmedium is the dynamic viscosity of the perfusion medium, rcell is the radius of the cell, g is the gravity, x2 is the next x position of the cells after traveling for time t, y2 is the next y position of the cells after settling for time t. Please note Eqn 5 is derived from Zhang and Neelamegham, 2002.

Velocity flow profile modeling

The flow velocity profile within the parallel-plate flow chamber was simulated using COMSOL (version 4.2.1.110, COMSOL Multiphysics, Stockholm, Sweden). The chamber was modeled in SolidWorks (version 2013 SP4.0, Dassault Systèmes SolidWorks Corporation, Waltham, MA, USA) and exported as a STL file to be imported into COMSOL. After importing the chamber model into COMSOL, the fluid material was chosen to be water (liquid), and the chamber flow profile solution model set to laminar flow. The boundary walls were chosen to be the outer surface of the liquid domain, the initial velocity of the flow was set to 0 m s−1, and the location of the inlet was selected as a surface normal to the inlet circumference. The normal inflow average velocity to the inlet was set to 5.2×10−5 m s−1, which was obtained by dividing a volumetric flow rate by an inlet area, and the outlet of the flow was selected to be a surface normal to the outlet circumference. Geometry meshing was performed (maximum and minimum element sizes were 343 μm and 22.4 μm and the maximum element growth rate was set to 1.08). The streamline profile for the settling feature was plotted by adding a streamline module. 100 streamline positions were defined at the inlet.

Channel functionalization

The main channel of the chamber was first incubated with anti-IgG (Fc specific) antibody solution made at desired concentration in D-PBS overnight in 4°C by infusing 50 µl of the solution from the outlet to just cover the main channel. The main channel was subsequently washed with D-PBS, blocked with 1% BSA in D-PBS for 1 h at room temperature, washed with D-PBS, and incubated with the desired concentration of P-selectin in D-PBS for 2 h at room temperature (all infused from the outlet). The main channel was subsequently washed with D-PBS, the whole chamber (settling feature included) blocked with 1% BSA for 1 h at room temperature (infused from the inlet), washed again with D-PBS (infused from the inlet), and stored at room temperature for use the same day. In select experiments, chambers were instead functionalized with BSA alone by overnight incubation with 1% BSA in D-PBS at 4°C. D-PBS used in all steps of the functionalization contained Ca2+ and Mg2+ except for the anti-IgG incubation step when cation-free D-PBS was used instead.

Flow cytometry

Cellular P-selectin ligand and sLex/a expression levels were measured via flow cytometry. P-selectin-IgG Fc or HECA-452 were premixed with Cy3-conjugated anti-human-IgG or FITC conjugated mouse anti-rat-IgM antibodies, respectively, for 1 h at room temperature. In select experiments, premixes also included 0.5, 5, or 25 units ml−1 of heparin. Cells were incubated with the premixed antibody solutions at 5×106 cells ml−1 for 1 h on ice. Following incubation with antibody premixes, cells were washed, and resuspended in PBS for analysis of 104 events on the Accuri C6 (BD Biosciences, San Jose, CA, USA) for P-selectin ligand expression or the LSRII for sLex/a expression (BD Biosciences). Specificity of P-selectin binding was confirmed by incubation of cells in P-selectin premixed with secondary antibody in 30 mM EDTA or incubation with the secondary antibody alone. Specificity of HECA-452 binding was confirmed by incubation of cells in the isotype control premix as well as incubation with the secondary antibody alone. Data was analyzed using FlowJo (Ashland, OR, USA).

Cell size

Cell diameter measurements were acquired using a coulter counter (Multisizer III, Beckman Coulter).

Statistical analysis

Data were analyzed using RStudio (0.97.551, Boston, MA, USA) and plotted in Prism (version 5.0b, GraphPad Software). Statistical tests were performed in Prism by ANOVA followed by post-hoc Dunn tests (***P<0.001, **P<0.01 and *P<0.05).

We thank M. Thomas for technical assistance.

Author contributions

J.O. and E.E.E. conducted all flow cytometry, adhesion and microfluidic studies as well as collection of data. J.O., P.M.M. and S.N.T. developed the microfluidic methods and performed data analysis. S.N.T. and P.M.M. developed the concept, and together with J.O. and E.E.E. contributed to the planning and design of the project. S.N.T. wrote the manuscript and all authors discussed the results and commented on the manuscript.

Funding

Funding was provided by a National Science Foundation Award [grant number 1342194]; an award from the Regenerative Engineering and Medicine Research Center; a National Institutes of Health Biotechnology Cell and Tissue Engineering Training Grant [grant number T32 GM-008433]; Air Products; and the Georgia Institute of Technology Undergraduate Research Opportunity Program. Deposited in PMC for release after 12 months.

Aigner
,
S.
,
Ramos
,
C. L.
,
Hafezi-Moghadam
,
A.
,
Lawrence
,
M. B.
,
Friederichs
,
J.
,
Altevogt
,
P.
and
Ley
,
K.
(
1998
).
CD24 mediates rolling of breast carcinoma cells on P-selectin
.
FASEB J.
12
,
1241
-
1251
.
Alon
,
R.
,
Hammer
,
D. A.
and
Springer
,
T. A.
(
1995
).
Lifetime of the P-selectin-carbohydrate bond and its response to tensile force in hydrodynamic flow
.
Nature
374
,
539
-
542
.
Alves
,
C. S.
,
Burdick
,
M. M.
,
Thomas
,
S. N.
,
Pawar
,
P.
and
Konstantopoulos
,
K.
(
2008
).
The dual role of CD44 as a functional P-selectin ligand and fibrin receptor in colon carcinoma cell adhesion
.
Am. J. Physiol. Cell Physiol.
294
,
C907
-
C916
.
Bendas
,
G.
and
Borsig
,
L.
(
2012
).
Cancer cell adhesion and metastasis: selectins, integrins, and the inhibitory potential of heparins
.
Int. J. Cell Biol.
2012
,
676731
.
Bizzari
,
J. P.
,
Mackillop
,
W. J.
and
Buick
,
R. N.
(
1983
).
Cellular specificity of NB70K, a putative human ovarian tumor antigen
.
Cancer Res.
43
,
864
-
867
.
Borsig
,
L.
,
Wong
,
R.
,
Feramisco
,
J.
,
Nadeau
,
D. R.
,
Varki
,
N. M.
and
Varki
,
A.
(
2001
).
Heparin and cancer revisited: mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis
.
Proc. Natl. Acad. Sci. USA
98
,
3352
-
3357
.
Borsig
,
L.
,
Wong
,
R.
,
Hynes
,
R. O.
,
Varki
,
N. M.
and
Varki
,
A.
(
2002
).
Synergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis
.
Proc. Natl. Acad. Sci. USA
99
,
2193
-
2198
.
Burdick
,
M. M.
and
Konstantopoulos
,
K.
(
2004
).
Platelet-induced enhancement of LS174T colon carcinoma and THP-1 monocytoid cell adhesion to vascular endothelium under flow
.
Am. J. Physiol. Cell Physiol.
287
,
C539
-
C547
.
Chen
,
S.
and
Springer
,
T. A.
(
2001
).
Selectin receptor-ligand bonds: formation limited by shear rate and dissociation governed by the Bell model
.
Proc. Natl. Acad. Sci. USA
98
,
950
-
955
.
Coumans
,
F. A. W.
,
van Dalum
,
G.
,
Beck
,
M.
and
Terstappen
,
L. W. M. M.
(
2013
).
Filtration parameters influencing circulating tumor cell enrichment from whole blood
.
PLoS ONE
8
,
e61774
.
Dallas
,
M. R.
,
Liu
,
G.
,
Chen
,
W.-C.
,
Thomas
,
S. N.
,
Wirtz
,
D.
,
Huso
,
D. L.
and
Konstantopoulos
,
K.
(
2012
).
Divergent roles of CD44 and carcinoembryonic antigen in colon cancer metastasis
.
FASEB J.
26
,
2648
-
2656
.
Diacovo
,
T. G.
,
Roth
,
S. J.
,
Buccola
,
J. M.
,
Bainton
,
D. F.
and
Springer
,
T. A.
(
1996
).
Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18
.
Blood
88
,
146
-
157
.
Frenette
,
P. S.
,
Johnson
,
R. C.
,
Hynes
,
R. O.
and
Wagner
,
D. D.
(
1995
).
Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin
.
Proc. Natl. Acad. Sci. USA
92
,
7450
-
7454
.
Fritz
,
J.
,
Katopodis
,
A. G.
,
Kolbinger
,
F.
and
Anselmetti
,
D.
(
1998
).
Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy
.
Proc. Natl. Acad. Sci. USA
95
,
12283
-
12288
.
Greenberg
,
A. W.
and
Hammer
,
D. A.
(
2001
).
Cell separation mediated by differential rolling adhesion
.
Biotechnol. Bioeng.
73
,
111
-
124
.
Greenberg
,
A. W.
,
Kerr
,
W. G.
and
Hammer
,
D. A.
(
2000
).
Relationship between selectin-mediated rolling of hematopoietic stem and progenitor cells and progression in hematopoietic development
.
Blood
95
,
478
-
486
.
Hanley
,
W.
,
McCarty
,
O.
,
Jadhav
,
S.
,
Tseng
,
Y.
,
Wirtz
,
D.
and
Konstantopoulos
,
K.
(
2003
).
Single molecule characterization of P-selectin/ligand binding
.
J. Biol. Chem.
278
,
10556
-
10561
.
Hanley
,
W. D.
,
Wirtz
,
D.
and
Konstantopoulos
,
K.
(
2004
).
Distinct kinetic and mechanical properties govern selectin-leukocyte interactions
.
J. Cell Sci.
117
,
2503
-
2511
.
Hanley
,
W. D.
,
Napier
,
S. L.
,
Burdick
,
M. M.
,
Schnaar
,
R. L.
,
Sackstein
,
R.
and
Konstantopoulos
,
K.
(
2006
).
Variant isoforms of CD44 are P- and L-selectin ligands on colon carcinoma cells
.
FASEB J.
20
,
337
-
339
.
Hanna
,
S.
and
Etzioni
,
A.
(
2012
).
Leukocyte adhesion deficiencies
.
Ann. N. Y. Acad. Sci.
1250
,
50
-
55
.
Kim
,
Y. J.
,
Borsig
,
L.
,
Varki
,
N. M.
and
Varki
,
A.
(
1998
).
P-selectin deficiency attenuates tumor growth and metastasis
.
Proc. Natl. Acad. Sci. USA
95
,
9325
-
9330
.
Koenig
,
A.
,
Norgard-Sumnicht
,
K.
,
Linhardt
,
R.
and
Varki
,
A.
(
1998
).
Differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. Implications for the use of unfractionated and low molecular weight heparins as therapeutic agents
.
J. Clin. Invest.
101
,
877
-
889
.
Konstantopoulos
,
K.
and
Thomas
,
S. N.
(
2009
).
Cancer cells in transit: the vascular interactions of tumor cells
.
Annu. Rev. Biomed. Eng.
11
,
177
-
202
.
Konstantopoulos
,
K.
,
Kukreti
,
S.
and
McIntire
,
L. V.
(
1998
).
Biomechanics of cell interactions in shear fields
.
Adv. Drug Deliv. Rev.
33
,
141
-
164
.
Lowe
,
J. B.
and
Ward
,
P. A.
(
1997
).
Therapeutic inhibition of carbohydrate-protein interactions in vivo
.
J. Clin. Invest.
100
,
S47
-
S51
.
Marshall
,
B. T.
,
Long
,
M.
,
Piper
,
J. W.
,
Yago
,
T.
,
McEver
,
R. P.
and
Zhu
,
C.
(
2003
).
Direct observation of catch bonds involving cell-adhesion molecules
.
Nature
423
,
190
-
193
.
Mayadas
,
T. N.
,
Johnson
,
R. C.
,
Rayburn
,
H.
,
Hynes
,
R. O.
and
Wagner
,
D. D.
(
1993
).
Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice
.
Cell
74
,
541
-
554
.
McCarty
,
O. J.
,
Mousa
,
S. A.
,
Bray
,
P. F.
and
Konstantopoulos
,
K.
(
2000
).
Immobilized platelets support human colon carcinoma cell tethering, rolling, and firm adhesion under dynamic flow conditions
.
Blood
96
,
1789
-
1797
.
Munn
,
L. L.
,
Melder
,
R. J.
and
Jain
,
R. K.
(
1994
).
Analysis of cell flux in the parallel plate flow chamber: implications for cell capture studies
.
Biophys. J.
67
,
889
-
895
.
Napier
,
S. L.
,
Healy
,
Z. R.
,
Schnaar
,
R. L.
and
Konstantopoulos
,
K.
(
2007
).
Selectin ligand expression regulates the initial vascular interactions of colon carcinoma cells: the roles of CD44v and alternative sialofucosylated selectin ligands
.
J. Biol. Chem.
282
,
3433
-
3441
.
Nieswandt
,
B.
,
Hafner
,
M.
,
Echtenacher
,
B.
and
Mannel
,
D. N.
(
1999
).
Lysis of tumor cells by natural killer cells in mice is impeded by platelets
.
Cancer Res.
59
,
1295
-
1300
.
Norman
,
K. E.
,
Moore
,
K. L.
,
McEver
,
R. P.
and
Ley
,
K.
(
1995
).
Leukocyte rolling in vivo is mediated by P-selectin glycoprotein ligand-1
.
Blood
86
,
4417
-
4421
.
Raman
,
P. S.
,
Alves
,
C. S.
,
Wirtz
,
D.
and
Konstantopoulos
,
K.
(
2011
).
Single-molecule binding of CD44 to fibrin versus P-selectin predicts their distinct shear-dependent interactions in cancer
.
J. Cell Sci.
124
,
1903
-
1910
.
Robinson
,
S. D.
,
Frenette
,
P. S.
,
Rayburn
,
H.
,
Cummiskey
,
M.
,
Ullman-Cullere
,
M.
,
Wagner
,
D. D.
and
Hynes
,
R. O.
(
1999
).
Multiple, targeted deficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment
.
Proc. Natl. Acad. Sci. USA
96
,
11452
-
11457
.
Stein
,
J. V.
,
Cheng
,
G.
,
Stockton
,
B. M.
,
Fors
,
B. P.
,
Butcher
,
E. C.
and
von Andrian
,
U. H.
(
1999
).
L-selectin-mediated leukocyte adhesion in vivo: microvillous distribution determines tethering efficiency, but not rolling velocity
.
J. Exp. Med.
189
,
37
-
50
.
Thomas
,
S. N.
,
Zhu
,
F.
,
Schnaar
,
R. L.
,
Alves
,
C. S.
and
Konstantopoulos
,
K.
(
2008
).
Carcinoembryonic antigen and CD44 variant isoforms cooperate to mediate colon carcinoma cell adhesion to E- and L-selectin in shear flow
.
J. Biol. Chem.
283
,
15647
-
15655
.
Tranquillo
,
R. T.
,
Lauffenburger
,
D. A.
and
Zigmond
,
S. H.
(
1988
).
A stochastic model for leukocyte random motility and chemotaxis based on receptor binding fluctuations
.
J. Cell Biol.
106
,
303
-
309
.
van Genderen
,
H.
,
Wielders
,
S. J. H.
,
Lindhout
,
T.
and
Reutelingsperger
,
C. P.
(
2006
).
Rolling and adhesion of apoptotic monocytes is impaired by loss of functional cell surface-expressed P-selectin glycoprotein ligand-1
.
J. Thromb. Haemost.
4
,
1611
-
1617
.
Veerman
,
K. M.
,
Williams
,
M. J.
,
Uchimura
,
K.
,
Singer
,
M. S.
,
Merzaban
,
J. S.
,
Naus
,
S.
,
Carlow
,
D. A.
,
Owen
,
P.
,
Rivera-Nieves
,
J.
,
Rosen
,
S. D.
, et al. 
(
2007
).
Interaction of the selectin ligand PSGL-1 with chemokines CCL21 and CCL19 facilitates efficient homing of T cells to secondary lymphoid organs
.
Nat. Immunol.
8
,
532
-
539
.
Wang
,
J.-G.
and
Geng
,
J.-G.
(
2003
).
Affinity and kinetics of P-selectin binding to heparin
.
Thromb. Haemost.
90
,
309
-
316
.
Yago
,
T.
,
Leppänen
,
A.
,
Qiu
,
H.
,
Marcus
,
W. D.
,
Nollert
,
M. U.
,
Zhu
,
C.
,
Cummings
,
R. D.
and
McEver
,
R. P.
(
2002
).
Distinct molecular and cellular contributions to stabilizing selectin-mediated rolling under flow
.
J. Cell Biol.
158
,
787
-
799
.
Zhang
,
Y.
and
Neelamegham
,
S.
(
2002
).
Estimating the efficiency of cell capture and arrest in flow chambers: study of neutrophil binding via E-selectin and ICAM-1
.
Biophys. J.
83
,
1934
-
1952
.
Zhang
,
Y.
and
Neelamegham
,
S.
(
2003
).
An analysis tool to quantify the efficiency of cell tethering and firm-adhesion in the parallel-plate flow chamber
.
J. Immunol. Methods
278
,
305
-
317
.

Competing interests

The authors declare no competing or financial interests.