the dual role of cd44 as functional p-selectin

8/7/2019 THE DUAL ROLE OF CD44 AS FUNCTIONAL P-SELECTIN

1/36

THE DUAL ROLE OF CD44 ASFUNCTIONAL P-SELECTIN LIGAND

AND FIBRIN RECEPTOR IN COLON

CARCINOMA CELL ADHESIONChristina S. Alves, Monica M. Burdick,

SusanN. Thomas, Parag Pawar,

and Konstantinos Konstantopoulos

Department of Chemical and BiomolecularEngineering, The Johns Hopkins University,

Baltimore, Maryland Am J Physiol Cell Physiol

294: C907C916, 2008


2/36

INTRODUCTION

BLOOD-BORNE METASTASIS is a highly regulated and

dynamic process, in which cancerous cells separate from aprimary tumor and migrate across blood vessel walls into thebloodstream where they interact extensively with various hostcellsbefore they lodge in the target organ and form secondarymetastatic colonies.

The most compelling evidence is the inhibition of metastasis byeitherpharmacological or genetic depletion of platelets and therestoration of metastatic potential by platelet infusion.

Microscopic observations of tumor cells trapped in the lungvasculature of wild-type mice reveal the presence of a densecoat of platelets surrounding them as well as their intimateassociation with fibrin(ogen) . In contrast, the colon carcinomacells in P-selectin deficient mice had a looser and more limitedplatelet coat


3/36

Many cancer patients, including those with disseminated coloncancer, have detectable abnormalities of blood coagulationsuch as elevated levels of fibrinogen and fibrinopeptide A.

The most convincing evidence for the direct role of fibrin(ogen)in metastatic spread is the profound inhibition of experimentaland spontaneous metastasis in fibrinogen-deficient micecompared with wild-type controls.

It is believed that platelet/fibrin(ogen) clots surrounding tumorcells may protect them from immunological and physiologicalstresses in the bloodstream and facilitate their lodging to thepulmonary vasculature.

With selectins, fibrinogen does not play a role in the initialbinding/seeding of tumor cells within the pulmonaryvasculature . Instead, it appears to facilitate metastasis bymediating the sustained adhesion and survival of tumor cells inthe high shear environment of target organs


4/36

INTRODUCTION

Selectins and fibrin play key roles in the hematogenousdissemination of tumor cells and especially of colon

carcinomas.

Using human CD44-knockdown and control LS174T cells, we

demonstrate the pivotal involvement ofCD44 in the P-selectin-mediated binding to platelets in shear flow.

Quantitative comparisons of the binding kinetics ofLS174T

versus P-selectin glycoprotein ligand-1 (PSGL-1)-expressing

THP-1 cells to activated platelets reveal that the relative

avidity of P-selectin-CD44 binding is more than sevenfold

lower than that of P-selectin-PSGL-1 interaction.


5/36

Using CD44-knockdown LS174T cells and microspheres

coated with CD44 immunoprecipitated from control LS174T

cells, and purified fibrin(ogen) as substrate, we provide thefirst direct evidence that CD44 also acts as the major fibrin,

but not fibrinogen receptoron LS174T colon carcinoma cells.

Binding of plasma fibrin to CD44 on the colon carcinoma cell

surface interferes with the P-selectin-CD44 molecularinteraction and diminishes platelet-LS174T heteroaggregation

in the high shear regime.

Fibrinogen does not play a role in the initial binding/seeding of

tumor cells within the pulmonary vasculature. Instead, it

appears to facilitate metastasis by mediating the sustained

adhesion and survival of tumor cells in the high shear

environment of target organs


6/36

We investigate in a comprehensive and systematic manner

the effects of plasma proteins in the modulation of these

heterotypic adhesive interactions, and we demonstrate for the

first time that CD44 functions as a fibrin, but not fibrinogen,

receptor


7/36

METHODOLOGY

CD44-knockdown or control LS174T cells were harvested bymild trypsinization (0.25% trypsin/EDTA for 5 min at 37C) and

subsequently incubated at 37C for 2 h to allow regeneration

of surface glycoproteins. Carcinoma cell suspensions (107

cells/ml) were incubated with SNARF(dye) for 60 min at 37C.

LS174T cells were then washed once to remove excess dye,

resuspended in Dulbeccos phosphate-buffered saline (DPBS)

containing Ca2/Mg2, and stored at 4C for no longer than 4 h

before use in aggregation assays.

Similarly THP-1 cells were grown in above suspension and

stained with ssSNARF.


8/36

Human blood samples were drawn by venipuncture from

healthy volunteers into heparin (10 units/ml) anticoagulant.

Platelet-rich plasma (PRP) was prepared by centrifugation of

whole blood at 160 g for 15 min.

Platelet-poor plasma (PPP) was obtained by centrifugation

(1,900 g for 15 min) of the blood remaining after PRP

removal. The final platelet count was adjusted to the desired

levels by dilution with eitherHEPES-Tyrode buffer or PPP.

Platelet and SNARF-stained LS174T colon carcinoma or

THP-1 cell suspensions were allowed to equilibrate separately

to 37C for 2 min. Thereafter, 50 l of LS174T or THP-1 cells(2 x 107 cells/ml) along with 150 l of platelets (2 x 107

platelets/ml) were placed onto the stationary plate of a cone-

and plate rheometer to achieve the desired ratio of platelets

to LS174T or THP-1 cells (3:1). Shear rates were varied from

100 /s to 5,000 /s for 30 or 60 s.


9/36

To potentiate platelet activation, platelet specimens wereincubated for 10 min before shear exposure with thrombin inthe presence of the fibrin polymerization inhibitor glycine-

proline-arginine-proline (GPRP)-NH2.

The inclusion of GPRP-NH2 prevented not only fibrinpolymerization but also the formation ofhomotypic plateletaggregates, even after the exposure of specimens to thrombinand/or relatively high shear rates up to 5,000 /s.

Purified fibrinogen (1 mg/ml final concentration), vWf (7.5g/ml), human IgG (5 mg/ml), or human albumin (5 mg/ml) wasadded to the platelet suspension before thrombin/GPRP-NH2incubation. In select experiments, fibrinogen (1 mg/ml) wasadded to the cell suspension on the plate 1 s before the onsetof shear.

For inhibition studies, platelets were pretreated for 10 min withEP5C7 and/orXV454


10/36

The particle distribution and cellular composition of stableaggregates generated in the rheometric assay weredetermined by a dual-color flow cytometric methodology.

SNARF-stained cells and FITC-labeled platelets wereidentified on the basis of their characteristic forward-scatter,side-scatter, and fluorescence profiles in a FACSCalibur flowcytometer.

LS174T whole cell lysate was prepared by membranedisruption using 2% Nonidet P-40 followed by differentialcentrifugation. CD44 was immunoprecipitated from coloncarcinoma cell lysate with an anti-CD44 MAb, 2C5, using

recombinant protein G agarose beads.

Immunoprecipitated CD44 from LS174T whole cell wasdiluted to desired concentrations with binding buffer (0.2Mcarbonate/bicarbonate buffer, pH 9.2) and was incubated with10-m polystyrene microspheres.


11/36

RESULTS

Platelet-LS174T (or THP-1) cell adhesion efficiency is defined asthe fraction of heterotypic shear-induced collisions that result instable heteroaggregate formation, and it is determined by theintercellular collision frequency and the capture efficiency ofthese collisions.

This index was determined by fitting the aggregation data overthe first 30 s after application of shear with a mathematical modelbased on the Smoluchowski two-body collision theory, aspreviously described .

Data are expressed as means+/-SE. Statistical significance ofdifferences between means was determined by ANOVA orStudents t-test wherever appropriate. If means were shown to besignificantly different, multiple comparisons were performed bythe Tukey test.


12/36


13/36

For comparison purposes, we examined the adhesive behavior

of PSGL-1-expressing THP-1 cells with washed platelets in

shear flow. Our data reveal that the extent of platelet recruitment

by THP-1 cells increased with increasing shear rate, plateauedat 800/s, and remained essentially unaltered up to 5,000/s.

Maximal adhesion efficiencies were observed at the lowest

shear rate of100/s, at which 18 versus 9 of 100 collisions led to

stable platelet-THP-1 versus platelet-LS174T cell aggregate

formation.

In contrast with the monotonic decrease of the platelet-LS174T

adhesion efficiency with increasing shear, the efficiency ofplatelet recruitment by THP-1 cells was essentially constant

between 400 and 800/s before decreasing modestly at higher

shear rates.


14/36

A: effects of hydrodynamic shear and shear exposure time on the

extent of heterotypic aggregation of LS174T or THP-1 cells with

thrombin-activated washed platelets


15/36

B: adhesion efficiency of platelet binding to LS174T vs. THP-1

cells as a function of hydrodynamic shear calculated at the 30-s

time point in the presence of thrombin and GPRP-NH2


16/36

C and D: distribution of thrombin-GPRP-NH2-activated platelets (Plt)

bound to LS174T colon carcinoma cells (C) or THP-1 monocytic-like

cells (D) after exposure of free-cell suspensions to prescribed levels

of hydrodynamic shear for 60 s.


17/36

Our data also show that THP-1 relative to LS174T cells

displayed a higher efficacy to capture thrombin activated

platelets especially in the high shear regime (Fig. 1B), which

can be explained by the increased percentage of THP-1 cells

with adherent platelets (Fig. 1A) and the higher number ofplatelets bound per THP-1 cell (Fig. 1, C and D).


18/36

2.CD44 is the primary P-selectin ligand on

LS174T carcinoma cells.

Platelet P-selectin and IIb3 -integrins act primarily in a sequential

mannerto mediate maximal heterotypic binding under shear on

activation with thrombin/GPRP-NH2.

As shown in Fig. 2A, individual blockade of either platelet Pselectin orIIb3 -integrin function was equally effective in suppressing the extent

ofplatelet recruitment by LS174T colon carcinoma cells at all shear

rates, except for 100 and 5,000/s, at which heteroaggregation in the

absence of MAbs was near basal levels.

In contrast, the use of the platelet IIb3 specific antagonist XV454

alone failed to impairthe extent ofplatelet-THP-1 cell binding at all

shear rates (Fig. 2B).


19/36

Antibody interference assays also reveal that P-selectin is

necessary and sufficient to support adhesion of activatedwashed platelets to THP-1 cells at 5,000/s (Fig. 2B).

Simultaneous blockade of P-selectin and IIb3-integrin function

abolished platelet-THP-1 cell binding (Fig. 2B), suggesting an

auxiliary role forIIb3-integrins in this adhesion process.

IIb3-integrins stabilize the adhesion of free-flowing THP-1 cells

to immobilized platelets in shear flow and P-selectin binding to

PSGL-1 is solely responsible for maximal adhesion of activatedplatelets to monocytes in shear flow


20/36

A and B: effects of platelet P-selectin and IIb3 antagonists on

platelet-LS174T cell (A) or platelet-THP-1 cell (B) heterotypic

aggregate formation


21/36

Using purified P-selectin as a substrate, its discovered that

sialofucosylated CD44v represents the majorfunctional P-

selectin ligand on a variety of colon carcinoma cells, includingLS174T.

Thrombin-GPRP-NH2-activated washed platelets were

combined with stable CD44-knockdown LS174T cells in the

cone-and plate rheometer and were subjected to prescribed

levels of shear for 60/s.

As a control, activated platelets were mixed in the linear shear

field of a cone-and-plate rheometer with stable LS174Ttransfectants generated by using a mammalian scramble

control plasmid .


22/36

As shown in Fig. 2C, the CD44- knockdown LS174T cells

displayed a markedly reduced capacity relative to mammalian

scramble controls to recruit activated platelets in free-cellsuspensions, disclosing that CD44 is the predominant ligand for

platelet bound P-selectin.


23/36

3.Effect of plasma proteins on platelet

binding to LS174T colon carcinoma cells in

shear flow Washed platelets resuspended in eitherHEPES-Tyrode buffer or

PPP and stimulated with thrombin/GPRP-NH2 for 10 min, werecombined with LS174T cells and subjected to controlled levels ofhydrodynamic shear for 60/s.

The presence of plasma markedly affected the platelet-LS174Theteroaggregation process. More specifically, peakheteroaggregation in the presence and absence of blood plasmawas detected at 100/s and 800/s, respectively (Fig. 3A).

The extent ofheteroaggregation was significantly lowerinplasma-containing specimens at all shear rates except at the lowlevel of100/s, at which the extent of platelet recruitment byLS174T cells was modestly higherin the presence rather than theabsence ofplasma (Fig. 3A).


24/36

To identify inhibitory effect ofkey plasma proteins in this

heteroaggregation process, purified fibrinogen , vWf, IgG or

human serum albumin was added to washed platelet

suspensions in HEPES-Tyrode buffer during the 10-minincubation with thrombin/GPRP-NH2.

At the shear rate of800/s, exogenously added purified vWf, IgG,

and HSA did not interfere with the extent ofplatelet capture by

LS174T cells.

The presence offibrin alone markedly suppressed

heteroaggregation(Fig. 3, A and B).

The extent of heteroaggregation remained intact when purified

fibrinogen was added to the platelet-LS174T cell suspensions

just before the onset of shear (Fig. 3B).


25/36

The presence of fibrin did not alterthe heteroaggregation

levels at 100/s (Fig. 3B) IgG and HSA mildly increased the

extent of platelet recruitment by LS174T colon carcinoma cells

at low shear conditions via P-selectin independent/RGD-

dependent pathway, whereas the addition ofvWfand

fibrinogen did not have any effect on this process (Fig. 3B).

For comparison as shown in Fig. 3C, neither of theseexogenously added proteins significantly affected the extent of

platelet capture by THP-1 cells at 100 or 800/s.

Our data discloses that the presence offibrin, but not

fibrinogen or any other plasma proteins, selectively interfereswith platelet binding to LS174T colon carcinoma cells at

elevated levels of hydrodynamic shear.


26/36


27/36

B and C: effect of exogenously added purified proteins on platelet-

LS174T (B) or THP-1 (C) cell heterotypic aggregate formation.


28/36

4.CD44 is the primary fibrin receptor on

LS174T colon carcinoma cells

Fig. 4A shows that CD44-knockdown LS174T cells relative to

mammalian scramble or untreated controls displayed a

markedly reduced capacity (25% of control) to tether and

adhere to purified fibrin at a wall shear stress of 0.5 dyn/cm2.

LS174T colon carcinoma cells interacted minimally with

immobilized fibrinogen in shear flow (Fig. 4A).

As a control for proper fibrin(ogen) coating, free-flowing THP-1monocyticlike cells interacted effectively with both fibrinogen

and fibrin under dynamic flow conditions (Fig. 4A).


29/36

A: wild-type and CD44-knockdown or mammalian scramble control

LS174T cells, or THP-1 cells, resuspended in buffer were perfused over

immobilized fibrin or fibrinogen at 0.5 dyn/cm2


30/36

To verify that LS174T CD44 interacts directly with fibrin, but not

fibrinogen, we used polystyrene beads, coated with CD44

immunopurified from LS174T cells, perfused over immobilized

fibrin and fibrinogen

CD44-coated beads interacted avidly and extensively with

immobilized fibrin but not fibrinogen at 0.25 and 0.5 dyn/cm2

(Fig. 4B).

To document the specificity of CD44-fibrin interaction, beads

coated with human IgG (10 mg/ml) were perfused over fibrin

and fibrinogen coated surfaces.

As shown in Fig. 4B, IgG-coated beads interacted minimally with

immobilized fibrin and fibrinogen in shear flow.


31/36

B: microspheres coated with CD44, immunopurified from LS174Tcolon carcinoma whole cell lysate, or IgG-coated control beads wereperfused over immobilized fibrin or fibrinogen at prescribedwallshear stresses.


32/36

C: SDS-PAGEanalysis of fibrin (laneA) and

fibrinogen (laneB) under nonreducedconditions and immunoblot analysis of fibrin

(lane C) and fibrinogen (lane D) using the

human anti-fibrin Mab MH-1 after transfer to

a nitrocellulose membrane.

To confirm the purity of fibrinogen and fibrin as well as the

generation of fibrin, we performed SDS-PAGE followed by

immunoblot analysis using an anti-fibrin MAb, MH-1. Fig. 4Cdiscloses that the molecular masses of fibrinogen and fibrin detected

by SDS-PAGE match very well with previously published data (56).

Our immunoblot data also confirm the generation of soluble fibrin

upon fibrinogen incubation with thrombin/GPRP-NH2.


33/36

Our data provide the first direct evidence that CD44 is the

primary fibrin, but not fibrinogen, receptor onLS174T colon

carcinoma cells.

Moreover, platelet P-selectin and fibrin compete for CD44

binding, a process that dictates the extent ofplatelet-colon

carcinoma heterotypic cell aggregation.


34/36

SUMMARY

CD44 on LS174T colon carcinoma cells is the major functionalligand for platelet bound P-selectin.

Using stable CD44-knockdown LS174T cells as well aspolystyrene beads coated with CD44 immunopurified fromcontrol (untreated) LS174T cells, we provided the first direct

evidence that CD44 acts as the primary fibrin, but notfibrinogen, receptor on colon carcinoma cells.

We assessed the influence of plasma proteins in the modulationof platelet-LS174T cell heteroaggregation under shear anddiscovered that the presence of fibrin, but not fibrinogen,

diminishes the extent ofheteroaggregation.

We provided a mechanistic interpretation for the reducedplatelet- LS174T heteroaggregation under shear in thepresence offibrin by showing that fibrin interferes with theplatelet P-selectin-CD44 molecular recognition.


35/36

To further support the validity of our concept, we used THP-1

monocytic cells as a negative control, which are known to

interact with P-selectin via PSGL-1, in a CD44-independent

manner.

LS174T relative to THP-1 cells display a lower efficacy to

capture activated platelets in shear flow due to the lower

affinity/avidity of the P-selectin-CD44 versus P-selectin-PSGL-

1 binding interaction.


36/36

THANK YOU

the dual role of cd44 as functional p-selectin

Documents