314 Research Article

IntroductionCD24, also known as heat-stable antigen (HSA) in mice, is asmall heavily glycosylated cell-surface protein that is linked tothe membrane by a glycosyl-phosphatidylinositol (GPI-)anchor (Pierres et al., 1987; Kay et al., 1990; Alterman et al.,1990). Mouse CD24 has a protein core of 27 amino acids withseven potential glycosylation sites, whereas human CD24consists of 31 amino acids with 16 potential O- and N-glycosylation sites. Owing to this extensive glycosylation,CD24 has mucin-like characteristics (reviewed by Kristiansenet al., 2004b).

CD24 is expressed in mouse hematopoietic cellsubpopulations including B lymphocytes, the majority ofthymocytes, erythrocytes and neutrophils. Because of itslineage-specific and developmentally regulated expression,CD24 was traditionally used as a differentiation marker for B-and T-cell ontogeny (Poncet et al., 1996; Egerton et al., 1990;Lu et al., 1998). Later studies revealed that CD24 is notexclusively expressed by hematopoietic cells but is also presentin the developing brain as well as in a broad range of epithelialcells (Shirasawa et al., 1993; Poncet et al., 1996; Magnoldoand Barrandon, 1996; Maric et al., 1996). In humans, CD24 isnot expressed on erythrocytes or thymocytes but is present ona wide variety of malignancies including B-cell lymphoma,renal cell carcinoma, small-cell and non-small-cell lungcarcinoma, nasopharyngeal carcinoma, hepatocellularcarcinoma, bladder carcinoma and glioma, epithelial ovarianand breast cancer (reviewed by Kristiansen et al., 2004b).

CD24-knockout mice are viable and display no obviousdefects. However, B lymphocytes from these mice displayed a

slight defect in bone marrow maturation with reduced numbersof pre-B lymphocytes, but normal numbers of mature Blymphocytes, in the periphery (Nielsen et al., 1997). Thissuggested a role for CD24 in stimulating proliferation andmaturation of pre-B lymphocytes within the bone marrow. Ithas also been described that CD24 expression on T cells isrequired for optimal proliferation in a lymphopenic host (Li etal., 2004). On the other hand, cross-linking of CD24 is able toinduce apoptosis in a human B-cell subset during the earlyactivation stage (Suzuki et al., 2001). Thus, probably bothoverexpression and lack of CD24 have an influence on cellularfunction.

Functionally, CD24 has been demonstrated to serve as aligand for P-selectin in mouse myeloid and in human tumourcells (Aigner et al., 1997). The role of human CD24 as a P-selectin ligand depends on the appropriate modification ofglycans as only sialylLex-modified CD24 can promote rollingand tumour cell colonization of the lungs (Friedrichs et al.,2000). The P-selectin ligand function has been preserved inmice and humans. For cancers of the breast, ovary, colon andprostate it has been demonstrated that CD24 expression isassociated with poor prognosis and shortened survival time(Kristiansen et al., 2002; Kristiansen et al., 2003; Kristiansenet al., 2004a). It has also been proposed that CD24-mediatedbinding to P-selectin on endothelial cells and platelets couldfacilitate the exit of tumour cells from the bloodstream andhence favour metastasis (Friedrichs et al., 2000; Kristiansen etal., 2004a).

A recent study of primary breast cancer cells isolated frompleural infusions suggested that CD24 might be a potential

CD24 is a small, heavily glycosylated cell-surface proteinwhich is linked to the membrane via a glycosyl-phosphatidylinositol (GPI-) anchor and therefore localizesin lipid rafts. CD24 is widely used as a cell-lineage markerfor hematopoietic cells. CD24 is also expressed on a varietyof human carcinomas, including epithelial ovarian, breast,prostate, colon and lung cancer and has been linked to poorprognosis. Except for its role as a ligand for P-selectin oncarcinoma and myeloid cells, a specific function for CD24has not been determined. Here we show that CD24 affectsthe function of the chemokine receptor CXCR4. Usingisolated CD19-positive bone marrow B cells from CD24-knockout mice and CD24–/– pre-B lymphocytic cell lines,we demonstrate that CD24 expression reduces SDF-1-

mediated cell migration and signalling via CXCR4. Weobserved that the loss of CD24 augmented cellularcholesterol levels and enhanced CXCR4 lipid raftassociation. Altered chemotactic migration and raftresidence was also observed in MDA-MB-231 breast cancercells expressing high and low levels of CD24 and CXCR4receptor. MDA-MB-231 cells expressing low levels of CD24also showed enhanced tumour formation in NOD/SCIDmice compared with cells overexpressing CD24. Theseresults demonstrate a novel role for CD24 as a regulator ofCXCR4 function that could be relevant for breast cancergrowth and metastasis.

Key words: CD24, Cholesterol, CXCR4, SDF-1

Summary

CD24 affects CXCR4 function in pre-B lymphocytesand breast carcinoma cellsHeidi Schabath, Steffen Runz, Safwan Joumaa and Peter Altevogt* Tumor Immunology Programme, D010, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany*Author for correspondence (e-mail: P.Altevogt@dkfz.de)

Accepted 17 October 2005Journal of Cell Science 119, 314-325 Published by The Company of Biologists 2006doi:10.1242/jcs.02741

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315CD24 and CXCR4 function

marker for breast tumour stem cells (Al-Hajj et al., 2003). Itwas shown that the ability to form tumours in NOD/SCID micewas greater in the CD44+CD24low/–-expressing cell fractioncompared with the CD44+CD24high fraction. Owing to theenhanced tumour-forming ability, the CD44+CD24low/– fractionwas proposed to represent ‘breast tumour stem cells’ (Al-Hajjet al., 2003). The mechanism of growth regulation by CD24 isas yet undefined.

Chemokines are a superfamily of cytokine-like smallpeptides that are subdivided into four distinct classesdepending on the arrangement of cysteine residues (Rossi andZlotnik, 2000). Chemokines and their respective receptors, afamily of seven transmembrane-spanning heterotrimeric G-protein-coupled molecules, are known to play an importantrole in immune and inflammatory responses as well ashematopoiesis and HIV infection (Taub et al., 1995; Gerardand Rollins, 2001; Moser et al., 1998; Broxmeyer, 2001;Berger et al., 1999). The stromal-cell-derived factor 1� (SDF-1�, also known as CXCL12) is the only known ligand forCXCR4 and was first characterized as a pre-B-cell growth-stimulating factor that is essential for B-cell lymphopoiesis,myelopoiesis, cardiogenesis and embryogenesis (Nagasawa,1996). The expression of CXCR4 in a wide variety of tumoursincluding breast cancer, coupled with the expression of SDF-1� in sites of breast cancer metastasis such as the lungs, liverand bone, suggests a crucial role for CXCR4/SDF-1 in organ-specific breast cancer metastasis (Muller et al., 2001; Smith etal., 2004).

For optimal signalling, CXCR4 must be embedded inmembrane lipid rafts (Manes et al., 2001; Shamri et al., 2002;Nguyen and Taub, 2002; Wysoczynski et al., 2005). Here wedemonstrate that the presence of CD24 in lipid rafts alters theraft residence and response of CXCR4. Our results in mousepre-B cell lines suggest that this occurs because of changesin membrane cholesterol, affecting CXCR4-triggered cellmigration and ERK1/2 phosphorylation. Furthermore, alteredlipid raft residence and chemotactic migration in response toSDF-1 was demonstrated in MDA-MB-231 breast carcinomacells expressing high and low levels of CD24. In addition weshow that low expression of CD24 augmented the growth oftumours from MDA-MB-231 breast cancer cells in NOD/SCIDmice. As CXCR4 is implicated in metastasis and growth ofbreast and other cancers, the regulatory role of CD24 onCXCR4 signalling may be of great interest for future research.

ResultsAltered SDF-1 responsiveness in bone-marrow-derivedCD19+ cells of CD24–/– miceThe production and characterization of CD24-knockout micewere described in detail before (Nielsen et al., 1996). Weisolated CD19+ B lymphocytes via depletion of non-Blymphocytes from the bone marrow and spleen of CD24–/– orCD24+/+ mice. The enrichment was monitored by FACSanalysis with appropriate markers (Fig. 1A). We compared theSDF-1-mediated migration of CD19+ cells derived from thebone marrow and spleen of CD24+/+ and CD24–/– mice. Bone-marrow-derived cells from CD24–/– mice showed an ~2.8-foldenhancement in migration compared with cells derived fromCD24+/+ mice (Fig. 1B). The difference in SDF-1-inducedmigration was not observed in CD19+ cells isolated from thespleen (Fig. 1C).

CD24 affects SDF-1-induced chemotaxis in pre-B cellsHaving established that CD24 can affect SDF-1-mediated cellmigration of bone-marrow-derived B cells, we investigatedwhether this was also detectable in pre-B-cell lines. The pre-B lymphocytic cell line N232.18 (CD24–/–) was establishedfrom CD24-knockout mice and retransfected with a CD24expression plasmid resulting in the cell line 18H18+ (CD24+/+)(Hahne et al., 1994). Additionally, a mock-transfected line18H18– (CD24–/–) was established. As depicted in Fig. 2A, thecell lines differed in CD24 expression but were similar inexpression of CXCR4. In chemotactic migration assaystowards SDF-1, we showed that N232.18 cells and 18H18–

cells migrated in response to SDF-1 in a dose-dependentmanner (Fig. 2B). By contrast, CD24+/+ cells were only weaklyresponsive to SDF-1 and showed a ~50% reduction inchemotaxis compared with CD24–/– cells (Fig. 2B).

In addition to SDF-1 we used the chemokines IP-10(CXCL10), MIP-3� (CCL20) and BLC (CXCL13) toinvestigate whether CD24 was able to influence migration. Allthree chemokines failed to induce migration of CD24+/+ andCD24–/– pre-B-cell lines (Fig. 2C). Similar results wereobtained with isolated CD19+ B cells from bone marrow (datanot shown).

We also examined the expression of CXCR4 by biochemicalmeans. Fig. 2D (upper panel) shows the analysis of cell lysatesof the three cell lines indicating that the CXCR4 expressionwas comparable. We next investigated whether there was achange in the binding affinity of SDF-1 to its receptor bycarrying out ligand binding with biotinylated SDF-1, thendetecting the bound ligand by FACS analysis. SDF-1 bindingwas indeed slightly elevated in both CD24–/– cell linescompared with CD24+/+ cells (Fig. 2E).

CD24 alters cholesterol levels in pre-B cellsIt has been reported that in lymphoid cells, the lack of GPI-anchored proteins can cause an increase in cellular cholesterol(Abrami et al., 2001). As CD24 is a prominent GPI-anchoredmolecule in pre-B cells, we hypothesized that the lack of CD24might have a similar effect. To test this, we measured thecellular cholesterol content in CD24–/– and CD24+/+ cell lines.The amount of cellular cholesterol was significantly higher inthe two CD24–/– lines compared with CD24+/+ cells (Fig. 3A).Cellular cholesterol can be visualized by staining with thepolyene antibiotic Filipin that specifically binds to unesterifiedcholesterol (Muller et al., 1984). We used FACS analysis ofFilipin-stained cells to assess membrane cholesterol levels inCD24+/+ versus CD24–/– cells. The mean fluorescence intensityof stained cells was significantly higher in CD24–/– cellscompared with CD24+/+ cells (Fig. 3B).

CD24 affects SDF-1 responsiveness by lowering thecholesterol levelThe function of the chemokine receptor CXCR4 is sensitive tomembrane cholesterol extraction (Nguyen and Taub, 2002;Nguyen and Taub, 2003). As CD24 appeared to affect SDF-1responsiveness, by possibly altering cholesterol levels, weexamined whether the rate of cell migration was sensitive tocholesterol manipulation. Therefore we incubated 18H18+

cells with soluble cholesterol to restore the diminished SDF-1signalling. This treatment resulted in an elevation ofchemotactic migration by approximately 40% compared with

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untreated cells (Fig. 4A, left panel). The high-cholesterol-containing cell lines N232.18 and18H18– were treated with the HMG-CoA-reductaseinhibitor fluvastatin in the presence of lipoprotein-deficient serum. This treatment inhibits cholesterolbiosynthesis as well as cholesterol uptake. BothCD24–/– cell lines became less responsive to SDF-1and displayed a reduction in chemotactic migrationby 80% and 60%, respectively, compared withuntreated cells (Fig. 4A, middle and right panels).Reduced SDF-1-triggered migration was also seenafter treatment of N232.18 and 18H18– cells with thecholesterol extracting agent methyl-�-cyclodextrin(MCD; data not shown).

CD24 impairs SDF-1-triggered ERK activationCXCR4 signalling via SDF-1 activates the MAP-kinase pathway by phosphorylation of ERK (Ganjuet al., 1998). SDF-1 triggered ERK phosphorylationin CD24–/– cells but not in CD24+/+ 18H18+ cells(Fig. 4B). To examine whether the ERKphosphorylation depended on cholesterol, werepeated the treatment with the HMG-CoA-reductase inhibitor fluvastatin for 72 hours withCD24-negative (18H18–) and CD24-positive(18H18+) cell lines. Fluvastatin treatment abolishedSDF-1-induced ERK1/2 phosphorylation comparedwith untreated cells (Fig. 4C). We also examinedthe potential of cholesterol loading to restore theSDF-1-induced phosphorylation of the CXCR4downstream target ERK1/2 in CD24-positive18H18+ cells. Incubation with soluble cholesterolleads to a partial restoration of ERK1/2phosphorylation in response to SDF-1 (data notshown).

MDA-MB-231 breast cancer cellsoverexpressing CD24 have reduced SDF-1-mediated migrationAs it has been published that CXCR4 is an importantfactor in breast cancer metastasis (Muller et al.,2001) we wanted to know whether the resultsobtained from pre-B cells could be transferred to abreast cancer cell line model. Therefore, weoverexpressed CD24 in MDA-MB-231 breast cancercells and established cells with CD24high andCD24low expression levels at the cell surface. Fig. 5A(left panel) depicts the different CD24 levels in thetwo MDA-MB-231 sub-lines, as examined by FACSanalysis. Overexpression of CD24 did not affect thelevel of other antigens such as the integrin �5�1or L1 adhesion molecule (data not shown).Interestingly, as measured by Filipin staining, nosignificant difference in membrane cholesterol wasdetected (data not shown).

Both cell lines expressed low levels of CXCR4 atthe cell surface (Fig. 5A, lower left panel). Wetherefore overexpressed a CXCR4-GFP fusionprotein by retroviral transduction in both sub-lines.Overexpression followed by FACS selection led to aclear enrichment of CXCR4 at the cell surface of the

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Fig. 1. Cell migration of CD19+ bone marrow and spleen-derived cells fromCD24–/– and CD24+/+ mice. (A) CD19+ B-lymphocytic cells from the bonemarrow and spleen of CD24–/– and control C57BL/6 mice were isolated usingmagnetic beads. An aliquot of cells was used for FACS analysis of CD24 andCD45 expression. A CD24 isotype control antibody was also used. (B,C)Chemotactic cell migration of CD24–/– versus CD24+/+ B lymphocytes fromthe bone marrow (B) and spleen (C) in response to SDF-1. *P�0.035;**P�0.02.

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Fig. 2. CD24 alters SDF-1-inducedchemotaxis in pre-B cell lines.(A) Cytofluorographic analysis ofN232.18, 18H18– (CD24–/–) and18H18+ (CD24+/+) cells withmonoclonal antibodies to CD24 (mAb79) followed by PE-conjugated goatanti-rat IgG and chemokine receptorCXCR4 with biotinylated mAb 2B11followed by streptavidin-PE. (B)Chemotactic cell migration of N232.18and 18H18– (CD24–/–) versus 18H18+

(CD24+/+) cells in response to SDF-1.*P�0.05; **P�0.03. (C) Chemotacticcell migration of N232.18 (CD24–/–)versus 18H18+ (CD24+/+) towardschemokines IP-10, MIP-3�, BLC andSDF-1. *P�0.033. (D) Detection ofCXCR4 receptor by western blotanalysis. Cell lysates from the indicatedcell lines were tested. Calnexin wasdetected as a loading control.(E) Binding of biotinylated SDF-1 toCD24–/– (N232.18 and 18H18–) andCD24+/+ (18H18+) cells. Cells werestained with biotinylated SDF-1followed by streptavidin-FITC andanalysed by FACS.

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CD24high and CD24low sub-lines (Fig. 5A, lower right panel).Next we analysed the chemotactic migration of all cell lines inresponse to SDF-1. Despite their low level of endogenousCXCR4 at the cell surface, the non-transduced MDA-MB-231cells showed a chemotactic response to SDF-1 (Fig. 5B, leftpanel). The CD24low-expressing MDA-MB-231 cells showed a~1.3- to 2-fold increase in migration compared with the MDA-MB-231 CD24high sub-line. This difference in migrationbehaviour owing to the expression of CD24 was furtherconfirmed in the CXCR4-GFP-overexpressing cell lines. Fig.5B (right panel) depicts an overall increase in motility owingto overexpression of CXCR4-GFP and approximately twofoldelevated migration in response to SDF-1 in MDA-MB-231CD24low cells compared with the corresponding CD24high-expressing cells.

To further corroborate these results, we used siRNA-mediated depletion of CD24 in CD24high-expressing cells.Transfection of the CD24-specific siRNA led to a significantreduction of CD24 expression on the cell surface as revealedby FACS staining (Fig. 5C, right panel). The CD24-depletedcells showed approximately threefold elevated migrationcompared with control-siRNA-transfected cells (Fig. 5C, leftpanel). Thus, CD24 expression levels can interfere withCXCR4-mediated cell migration.

CD24 expression attenuates growth of MDA-MB-231 celltumours in NOD/SCID miceThe SDF-1/CXCR4 axis can regulate the proliferation ofprogenitor cells (Smith et al., 2004; Zeelenberg et al., 2003;Orimo et al., 2005) but also promotes the growth of primaryand metastatic breast cancer cells (Smith et al., 2004.) We

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examined the tumour growth of our breast cancer cell lines inNOD/SCID mice. We injected subcutaneously MDA-MB-231CD24high and MDA-MB-231 CD24low as well as MDA-MB-231 CD24high CXCR4-GFP and MDA-MB-231 CD24low

CXCR4-GFP cells. The tumour volume was monitored over 40days after which the mice were sacrificed and tumours wereprepared. MDA-MB-231 CD24low cells formed tumours ofapproximately fivefold larger volume compared with theCD24high variant (Fig. 6A). MDA-MB-231 CD24low CXCR4-GFP tumours developed a significantly (approximatelythreefold) increased volume compared with that of theCD24high variant (Fig. 6B). Therefore it seems that low CD24expression favours tumour growth in NOD/SCID micepossibly by its influence on CXCR4 signalling.

We also determined the influence of SDF-1 on the growthof MDA-MB-231 cells in vitro using proliferation assays.MDA-MB-231 CD24high and MDA-MB-231 CD24low breastcancer cells were incubated with different concentrations ofSDF-1 and cell numbers were determined after 24 hours. In thepresence of SDF-1 MDA-MB-231 CD24low cells showedsignificantly higher proliferation compared with MDA-MB-231 CD24high cells (Fig. 6C).

CD24 alters CXCR4 lipid raft residenceWe finally assessed the distribution of the CXCR4 receptor atthe cell surface biochemically. CXCR4 is localized within lipidrafts that are rich in cholesterol (Nguyen and Taub, 2002;Wysoczynski et al., 2005). Therefore, we examined thedistribution of CXCR4 and CD24 in pre-B cell lines by sucrosedensity centrifugation after lysis in cold Triton X-100 buffer.As expected for a GPI-anchored protein, CD24 was detectedin the lipid raft fractions of CD24+/+ pre-B cells (Fig. 7A).CXCR4 was observed in the intermediate non-raft fractions butnot within the lipid raft fractions of the gradient (Fig. 7B). Bycontrast, in both CD24–/– cells the CXCR4 receptor waspartially found within lipid rafts (Fig. 7B). To control for equalloading, the western blot was reprobed with an antibodyagainst the raft-associated kinase Fyn. In all pre-B-cell linesFyn was equally distributed within lipid raft and inintermediate non-raft fractions (Fig. 7B).

To determine whether there was also a different CXCR4lipid raft pattern in breast cancer cells, we isolated lipid raftfractions of CD24high and CD24low-expressing breast cancercell lines. CXCR4 was present within the lipid raft fractionsof MDA-MB-231 CD24low cells (Fig. 7C, lower panel). Bycontrast, CXCR4 was absent in rafts of CD24high-expressingMDA-MB-231 cells (upper panel). The distribution of the raft-

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Fig. 3. CD24 affects cellularcholesterol levels in pre-B cells. (A)Cellular cholesterol levels in theindicated cell lines were determinedusing the Amplex Red colorimetricmethod. Equal numbers of cellswere used. (B) Non-esterifiedcholesterol levels were measured bystaining with the cholesterol-bindingdye Filipin and FACS analysis.

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associated kinase Fyn was equal in both sub-lines. Weconclude that CD24 affects the raft residence of the CXCR4receptor in both pre-B cells and in breast carcinoma cells.

DiscussionIn the present report we demonstrate that (1) CD24 expressionin pre-B cells affects the responsiveness of the CXCR4receptor; (2) a similar observation was made in MDA-MB-231breast carcinoma cells; (3) siRNA-mediated depletion of CD24in MDA-MB-231 CD24high-expressing cells enhancesCXCR4-receptor-mediated cell migration; (4) in pre-B cells,but not in carcinoma cells, the absence of CD24 affects thelevel of cellular cholesterol; (5) in carcinoma cells CD24expression reduced tumour growth in NOD/SCID mice; (6)CD24-expressing cells differed from non-expressing cells inthe residence of CXCR4 in membrane rafts. These findingssuggest that CD24 regulates CXCR4 receptor responsivenessby excluding it from membrane rafts. Our findings establish anew mechanism by which the function of CXCR4 can bemodulated without affecting protein expression levels.

Our study was inspired by the work of Abrami and co-workers who show that in mouse BW5147 cells deficient inGPI-anchor synthesis, the level of cellular cholesterol wasenhanced (Abrami et al., 2001). By contrast, in CHO cellclones rendered resistant to proaerolysin and displaying also a

lack in the synthesis of GPI-anchored proteins, a similarinfluence on cholesterol levels was not observed. Instead, theseclones revealed an enhanced content of caveolin-1 (but not ofother raft-associated proteins such as flotillin-1). It was arguedthat caveolin-1 was capable of counterbalancing the loss ofGPI-anchored proteins. In our study we hypothesized that theloss of a major B-cell GPI-anchored protein such as CD24might cause a similar effect in pre-B cells. Indeed, we found asignificantly enhanced level of cholesterol in CD24-deficientcells. The observed effect was surprising as murine pre-B cellsexpress other GPI-anchored proteins such as CD48, CD59,CD157 or Ly-6A/E. We speculate that CD24 represents a majorGPI-anchored protein and that the lack of expression maycause similar effects to those observed in cells in which GPI-anchor synthesis is blocked.

Cholesterol homeostasis in cells is subject of complexregulations consisting of uptake via the LDL receptor, exportvia HDL and lipoproteins or membrane vesicles and de novobiosynthesis (reviewed by Simons and Ikonen, 2000). It ispresently unclear, by which mechanisms CD24–/– cells acquireelevated cholesterol. Our preliminary results, using fluorescentLDL-uptake assays, suggest that both CD24–/– and CD24+/+

cells bind and take up LDL and that CD24+/+ cells display ahigher rate (H.S., unpublished results). This is expected giventhe lower level of cholesterol in these cells. As lymphoid cells

Fig. 4. Cellular cholesterol levels affectchemotactic cell migration. (A) CD24+/+

18H18+ cells were loaded with cholesterolby incubation with 30 �g/ml solublecholesterol and tested for chemotactic cellmigration in response to SDF-1. CD24–/–

cells N232.18 and 18H18– were treated withfluvastatin for 72 hours and then tested forchemotactic cell migration in response toSDF-1. Data are presented as the percentagemigration (± s.e.m.) compared withuntreated control cells. (B) SDF-1-triggeredERK-phosphorylation in CD24–/– (N232.18and 18H18–) and CD24+/+ (18H18+) cells.Cells were exposed for the indicated lengthof time (in minutes) to 100 ng/ml SDF-1then lysed. An equal volume of lysate wasmixed with sample buffer and analysed bySDS-PAGE and western blotting using aphospho-ERK-specific antibody and ECLdetection. For a loading control, blots werereprobed with an ERK-1-specific antibody.(C) CD24–/– (18H18–) cells were treated withfluvastatin for 72 hours and then ERKphosphorylation in response to SDF-1 wastested. Note that ERK phosphorylation in18H18– cells was abolished after fluvastatintreatment.

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also display low levels of cholesterol de novo synthesis, it islikely that the regulation is mostly via the export of cholesterol.Further experiments are needed to more closely define thismechanism.

Previous studies have shown that the function of the CXCR4receptor is sensitive to cholesterol manipulation (Nguyen andTaub, 2002 and Nguyen and Taub, 2003). These authorsclaimed that normal cholesterol is crucial to the conformationalintegrity and function of the chemokine receptors CXCR4 andCCR5. In our study, we investigated the possibility of a linkbetween cholesterol levels and CD24 expression and usedSDF-1-triggered cell migration as a sensitive readout. Indeed,

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the influence of CD24 on cell motility was evident in pre-B-cell lines and also in freshly isolated CD19+ pre-B cells fromthe bone marrow but not from the spleen. Interestingly, thephenotype of CD24–/– mice displays a slight block in thedifferentiation of B cells and a reduction in cell number of pre-B cells in the bone marrow compared with CD24+/+ mice(Nielsen et al., 1997). However, numbers of mature B-cellswere normal in the spleen of CD24–/– mice. The resultspresented in this report can offer an explanation for thesefindings. It is possible that SDF-1 presented by the bonemarrow stroma of CD24–/– animals may lead to hyperactivationof the CXCR4 receptor owing to the lack of CD24. This could

MDA-MB-231CD24low

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Fig. 5. CD24high and CD24low variants of MDA-MB-231 breastcarcinoma cells. (A) Phenotype of MDA-MB-231 sub-lines.CD24high-expressing cells were generated by stable overexpression ofCD24 and FACS selection. Both sub-lines were transduced with aretroviral CXCR4-GFP expression construct. The transduced cellswere sorted for CXCR4 expression. (B) Migration of MDA-MB-231CD24high versus CD24low breast carcinoma cells (either non-transduced or CXCR4-GFP tranduced) in response to SDF-1. Notethe overall elevated level of cell migration in CXCR4-GFP-overexpressing cells (**P�0.033; *P�0.05; ***P�0.02). (C) Migration of MDA-MB-231 CD24high CXCR4-GFP cells after transfection with CD24-specific or control siRNA. FACS analysis indicates reduced CD24expression 72 hours after CD24 siRNA transfection compared with the control siRNA (*P�0.013).

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result in aberrant motility and guidance within the bonemarrow of CD24–/– cells. It is also possible that the effectscaused by the absence of CD24 are less prominent in matureB cells in the periphery compared with the bone marrow whichcould be explained by an antagonistic effect of other GPI-anchored molecules.

CD24 also modulated the responsiveness of the CXCR4receptor in MDA-MB-231 breast carcinoma cells. In thiscellular system of high- or low-expressing variants, the totalcellular cholesterol content was not altered by CD24. Thiscould be due to inherent differences between lymphoid andepithelial cells or to the presence of caveolins as suggestedpreviously for CHO cells (Abrami et al., 2001). We observedthat CD24low cells migrated better in response to SDF-1,showed increased proliferation in the presence of SDF-1 andexhibit enhanced tumour growth in NOD/SCID mice.Interestingly, we did not observe elevated growth in CXCR4-overexpressing cells in our NOD/SCID mice experiments (Fig.6A,B). This is in contrast to earlier reports which showed thatoverexpression of CXCR4 in MDA-MB-231 breast cancercells led to increased tumour volume in NOD/SCID mice(Darash-Yahana et al., 2004). The reason for this discrepancy

could be that breast cancer cells used in this previous reportpossess no endogenous CXCR4 expression whereas our MDA-MB-231 cells show moderate levels of functional CXCR4receptor endogenously on the cell surface (Fig. 5A,B). Thismay lead to saturation, because the endogenous CXCR4receptor is already able to promote tumour growth in sucha way that cannot be further augmented by additionaloverexpression of CXCR4.

Importantly, in the pre-B cell and in the breast cancer cell-line system we observed that the presence of CD24 caused adepletion of the CXCR4 receptor from membrane rafts. It iswell established that lipid rafts represent important platformsto couple receptors to signalling machinery (Harder andEngelhardt, 2004). Owing to the presence of kinases, G-proteins and other signalling components, lipid rafts play apivotal role in signal transduction (Brown and London, 1998;Kurzchalia and Parton, 1999). Cholesterol is an integralcomponent of membrane rafts and GPI-anchored proteins suchas CD24 are exclusively present in rafts.

The importance of the incorporation of the CXCR4 receptorinto membrane lipid rafts has recently been analysed(Wysoczynski et al., 2005). Molecules such as fibrinogen

and fibronectin have a priming effect onhematopoietic stem/progenitor cells. Theinteraction between these molecules, receptorsand other adhesion molecules on the cell surfaceseems to be crucial for increasing theincorporation of CXCR4 into membrane lipidrafts. The described raft residence of CXCR4 isin line with other reports which show that theinability of SDF-1 to bind to non-raft-associatedCXCR4 may play a regulatory role inmaintaining receptor activity and the migratorypotential of cells (Nguyen and Taub, 2002). Inthe context of �4 integrin activation inlymphocytes, it has been described that CXCR4has to be embedded within lipid rafts. CXCR4,as a G-protein coupled receptor, requires intactmembrane rafts to convert chemokine signallinginto productive �4 integrin avidity stimulation(Shamri et al., 2002). Our data are in agreementwith these earlier findings. We observed in pre-B cells that owing to the depletion of CXCR4from rafts there was also an uncoupling ofthe receptor from downstream signallingcomponents. In CD24+/+ 18H18+ cells, nophosphorylation of ERK in response to SDF-1was observed. As summarized in the modelpresented in Fig. 8, we propose that CD24affects CXCR4 function by modulating the raftresidence of the receptor.

Previous studies have shown that proteintyrosine phosphatases (SHIP1 and SHIP2), aswell as membrane-expressed hematopoieticphosphatase CD45 are also involved in themodulation of CXCR4 signalling (Fernandiset al., 2003). SDF-1-triggered migration isimpaired in SHIP1/2-knockout mice (Chernocket al., 2001) and lymphocytes negative for CD45show reduced chemotaxis towards SDF-1(Fernandis et al., 2003). Interestingly, following

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Fig. 6. CD24 prevents growth of MDA-MB-231 tumours in NOD/SCID mice.(A) Tumour volume of subcutaneously injected MDA-MB-231 CD24high andCD24low cells after 37 days (n=7; *P�0.0039). (B) Tumour volume ofsubcutaneously injected MDA-MB-231 CD24high CXCR4-GFP and MDA-MB-231CD24low CXCR4-GFP cells after 37 days (n=8; * P�0.0019). (C) Cell numbers ofMDA-MB-231 CD24high and CD24low cells after incubation with different SDF-1concentrations for 24 hours (*P�0.019; **P�0.007).

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SDF-1 stimulation, CD45 was found to colocalizewith CXCR4 in lipid rafts (Fernandis et al., 2003).Other molecules have been identified that mayincrease the sensitivity/responsiveness of CXCR4-positive cells to SDF-1 such as complement cleavagefragments, platelet-derived microvesicles, hyaluronicacid, fibronectin or soluble uPAR (Kucia et al., 2004;Wysoczynski et al., 2005). Factors such as LPS,heparin or other chemokines (MIP1� or RANTES)can desensitize the CXCR4 receptor whereasthe action of proteinases such as CD26(dipetidylpeptidase IV) under inflammatoryconditions can silence CXCR4/SDF-1 signalling bycleavage of the ligand or receptor (Kucia et al., 2004;Epstein, 2004). The mechanism of CXCR4 silencingby CD24 is novel as it does not interfere with proteinexpression but is mediated by uncoupling of thereceptor.

The SDF-1/CXCR4 axis was initially studied forits importance in initiating cell trafficking. Thebiological effects of this interaction were studied inthe context of cell motility during inflammation(Gerard and Rollins, 2001; Moser et al., 1998), tissueregeneration (Kucia et al., 2004), HIV infection(Berger et al., 1999) and lately also concerning theability to promote cell growth and survival ofprogenitor and tumour cells (Smith et al., 2004;Muller et al., 2001; Kucia et al., 2004; Epstein, 2004).Many tumour cells express functional CXCR4receptor and several recent reports have shown thatCXCR4 receptor expression predicts a poor outcomefor cancers of the breast (Muller et al., 2001), skin(Scala et al., 2005), ovary (Scotton et al., 2002) andprostate (Darash-Yahana et al., 2004). It is believedthat CXCR4 can direct tumour cells into SDF-1-richtissues or organs of the body such as bone marrowand lymph nodes (Muller et al., 2001). SDF-1 is alsosecreted by the liver and kidneys and the centralnervous system (Stumm et al., 2002) and secretioncan be enhanced by tissue damage. Tumour cells, inparticular so called ‘tumour stem cells’ may lodgeand grow in SDF-1-rich niches (Epstein, 2004).

In particular, when present in the cytoplasm oftumour cells, CD24 expression has also beenassociated with poor prognosis in breast (Kristiansenet al., 2003), ovarian (Kristiansen et al., 2002; Choiet al., 2005) and prostate cancer (Kristiansen et al.,2004a). Interestingly, a recent study on primary breastcancer cells isolated from pleural infusions of tumourpatients has shown that the ability to form tumoursin NOD/SCID mice is far greater in theCD44+CD24low/– fraction compared with theCD44+CD24high fraction (Al-Hajj et al., 2003;Abraham et al., 2005). Xenografted tumoursappearing in mice injected with CD44+CD24low/negative

cells were again heterogeneous in expression andranged from CD24high to CD24low expression (Al-Hajj et al., 2003). Owing to its enhanced tumour-forming ability, the CD44+CD24low/– fraction wasconsidered to represent ‘breast tumour stem cells’ (Al-Hajj etal., 2003). A recent study has challenged this view by

Journal of Cell Science 119 (2)

demonstrating that the percentage of CD44+CD24low/– cells inbreast carcinoma tissue samples is highly variable (between 0-

Fig. 7. CD24 affects the localization of CXCR4 in lipid rafts. (A) Distributionof CD24 in membrane rafts isolated from CD24+/+ 18H18+ cells. Gradientfractions were probed with mAb M1.69 to mouse CD24 followed byperoxidase-conjugated secondary antibody and ECL detection. (B)Distribution of CXCR4 in raft and non-raft fractions of the indicated pre-B-cell lines (upper panels). Note the presence of CXCR4 only in raft fraction 2of CD24–/– cells (*). Distribution of Fyn in raft and non-raft fractions of theindicated pre-B cell lines (lower panels). Note the presence of Fyn in raftfraction 2 of all cell lines. (C) Distribution of CXCR4 in raft and non-raftfractions of CD24high and CD24low MDA-MB-231 CXCR4-GFP cells. Notethe presence of CXCR4 in raft fraction 2 of only CD24low cells as indicatedwith (*) and the equal presence of Fyn in both cell lines.

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80%) (Abraham et al., 2005). The size of this sub-fraction hadno influence on overall survival time but was favourable fordistant metastasis. Our study suggests that low CD24expression levels can promote the growth ability of breasttumour cells by augmenting CXCR4 responsiveness.

In summary, our results demonstrate for the first time a novelfunction of CD24 as a regulator of CXCR4 responsiveness inlymphoid cells and in carcinoma cells. This effect is due to theunique localization of CD24 in rafts. The mechanism by whichCD24 uncouples CXCR4 signalling and the possibility thatCD24 can regulate other raft-associated cell functions warrantsfurther investigation.

Materials and MethodsMaterials and antibodiesThe following antibodies were used: PE-conjugated monoclonal antibody (mAb)M1/69 to mouse CD24 and the corresponding PE conjugated isotype controlantibody from Becton Dickinson (Heidelberg, Germany), mAb 79 to mouse CD24(Kadmon et al., 1994) and mAb SWA11 to human CD24 (Weber et al., 1993);biotinylated mAb to mouse CXCR4 from Becton Dickinson (clone 2B11), mAb tomouse CXCR4 from Capralogics (Hardwick, MA) (CI00116) and ProSci (Poway,CA) (1012), and to human CXCR4 from R&D Systems (Wiesbaden, Germany)(MAB 172, clone 44716) and Chemicon (Hofheim, Germany) (AB1847); mAb tophospho-specific ERK1/2 (clone 20A) and ERK 1 (clone MK12) from BectonDickinson; polyclonal Ab to Calnexin from Stressgen (Victoria, Canada) (# SPA-860); polyclonal Ab to Fyn from Santa Cruz Biotechnologies (sc-016); PE-conjugated mAb to mouse CD45 (CD45R/B220) from Becton Dickinson (553090).SDF-1�, biotinylated SDF-1�, streptavidin-FITC, IP-10, MIP-3� and BLC were allobtained from R&D Systems.

Retroviral constructionThe LZRS-CXCR4-GFP-IRES-Zeocin construct (kind gift from Dr Hordijk,University of Amsterdam, The Netherlands) was transfected into amphotrophicPhoenix packaging cells via calcium phosphate transfection to produce retroviruses.

MDA-MB-231 CD24high or CD24low were infected with retrovirus-containingsupernatant in the presence of polybrene (Sigma, Taufkirchen, Germany) andstably transduced cells were sorted for CXCR4 expression by FACSVantage.

Cell culture and animalsThe production and characterization of the CD24–/– pre-B lymphocytic cellline N232.18 and the CD24 retransfected sub-line 18H18+ have beenpreviously described (Hahne et al., 1994). 18H18– cells were obtained from18H18+ cells by selecting CD24-loss variants using FACS. Pre-B cells werecultured in RPMI 1640 containing 10% fetal calf serum, glutamine andpenicillin/streptomycin. CD24–/– mice (C57BL/6) were initially obtainedfrom Peter Nielsen (Max-Planck Institute for Immunobiology, Freiburg,Germany) and bred at the DKFZ. Bone marrow and spleen cells werecollected from 6- to 8-week-old female mice and CD19+ B lymphocyteswere isolated using a mouse B-cell isolation kit (Miltenyi, Bergisch-Gladbach, Germany). MDA-MB-231 CD24high cells were established bytransfection with a CD24-pcDNA3.1 expression plasmid with SuperFectfrom Qiagen (Hilden, Germany). MDA-MB-231 CD24high and MDA-MB-231 CD24low breast cancer cell lines were maintained in DMEM with 10%fetal calf serum, glutamine and penicillin/streptomycin.

Fluorescence-activated cell sortingCells were washed, resuspended in cold PBS containing 5% fetal calf serum,and then incubated with mAb to CD24 or CXCR4 for 30 minutes followedby washing and incubation for 20 minutes with PE-conjugated IgG secondaryantibodies (Jackson ImmunoResearch). Mouse CXCR4 was detected usingbiotinylated CXCR4 mAb and Streptavidin conjugated PE (JacksonImmunoResearch). Cells were analysed with a FACScan or sorted withFACSVantage (Becton Dickinson, Heidelberg, Germany). For data analysis,Cellquest or FlowJow software was used.

Filipin stainingCells were fixed with 1% formaldehyde/PBS and then stained with FilipinIII (Sigma F4767; Taufkirchen, Germany) at a concentration of 0.05 mg/mlin 10% FCS/PBS for 1 hour at room temperature in the dark. Filipin wasmeasured with FACSVantage or FACSDiva instruments using a UV laser(Becton Dickinson).

Cholesterol determinationFor cholesterol quantification, lipids were extracted by incubation in

hexane/isopropanol (3:2) for 30 minutes on ice. Following overnight evaporation ofthe solvent, the dried lipids were resolved in absolute ethanol and the relativecholesterol content was measured with an Amplex Red cholesterol assay fromMolecular Probes (PoortGebouw, Netherlands). The fluorometric method was usedaccording to the manufacturer’s instruction.

Cholesterol manipulationTo inhibit cholesterol uptake as well as cholesterol biosynthesis, cells were culturedin RPMI medium containing 10% lipoprotein-deficient serum (Sigma) in thepresence of the HMG-CoA-reductase inhibitor fluvastatin (kind gift from JörgKreuzer, University of Heidelberg, Germany) at 0.5 �M for 72 hours. Forcholesterol loading, 18H18+ cells were incubated with soluble cholesterol (Sigma)at 30 �g/ml for 30 minutes at 37°C.

Chemotactic transmigration assayAround 5�105 pre-B cells in 100 �l RPMI/0.5% BSA were added to Transwellinserts (Costar) with a 5 �m pore size. Serially diluted recombinant SDF-1� or otherchemokines were added to the lower chamber and pre-B cells were allowed tomigrate up to 16 hours. Transwell inserts were removed and the migrated cells inthe lower chamber were counted with a Coulter Counter Z2 (Beckman Coulter;Krefeld, Germany). MDA-MB-231 cells were serum-starved overnight beforeapplication to Transwell inserts with an 8 �m pore-size. To facilitate migration, theunderside of the filters were coated with fibronectin (7.5 �g/ml). Approximately 3-5�105 cells in 100 �l RPMI/0.5% BSA were suspended in the upper chamber.Recombinant SDF-1� was added to the lower chamber, then after 4-6 hours ofincubation, the cells on the upper surface of the filters were removed by wiping withcotton swabs. Migrated cells on the lower surface of the filter were detached using100 mM EDTA in PBS and counted with a Coulter Counter Z2.

CD24 siRNA transfectionControl siRNA and CD24 siRNA duplex (sense, 5�-ACA ACA ACU GGA ACUUCA A dTdT-3�) were purchased from MWG Biotech (Ebersberg, Germany). CD24and control siRNAs were transfected into MDA-MB-231 CXCR4-GFP CD24high

cells at a final concentration of 100 nM with Oligofectamine transfection reagents(Invitrogen, Karlsruhe, Germany).

Lipid raft preparation and western blot analysisCells were lysed in ice-cold lysis buffer in 25 mM Tris-HCl pH 8.0 containing 1%

Alteredphosphorylation, reduced migration and

attenuated tumour growthdue to changes in cholesterol/CXCR4 raft residence

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Fig. 8. Model of CD24 activity and CXCR4 function. In CD24–/– cells(N232.18 and 18H18– as well as in MDA-MB-231 CD24low cells) theCXCR4 receptor is more abundant in membrane rafts. As a result of thisresidence, CXCR4 signalling in response to SDF-1 triggers cell motilityvia ERK phosphorylation. In CD24+/+ cells (18H18+ and MDA-MB-231CD24high cells), CXCR4 is excluded from lipid rafts and cannot transmitsignals in response to SDF-1. This prevents ERK phosphorylation, blockscell motility and attenuates tumour growth.

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Triton X-100 (Roche), 1 mM PMSF, 10 �g/ml aprotinin, pepstatin and leupeptin,100 mM NaF and 10 mM Na3VO4 for 30 minutes on ice. The lysate was mixed withan equal volume of 85% sucrose (w/v in TBS), and a step gradient was preparedby overlaying with 35% sucrose in TBS followed by a final layer of 5% sucrose.The gradient was centrifuged for 20-22 hours at 200,000 g using a Beckman SW60rotor. Fractions of 0.5 ml were collected from the top of the gradient and precipitatedwith chloroform/methanol as described previously (Wessel and Flügge, 1984). ForCD24 detection, fractions were precipitated with a tenfold volume of acetone andthen washed with a fivefold volume of 50% acetone/H2O. Fractions were then boiledin the presence of SDS sample buffer under reducing conditions. Equal proteinloading of the cell lysates was standardized with the Biorad protein determinationkit. Samples were separated on 10% SDS-PAGE gels and transferred to Immobilonmembranes using semi-dry blotting. After blocking with 5% skimmed milk in TBS,membranes were probed with primary antibodies followed by horseradishperoxidase-conjugated anti-mouse or anti-rabbit secondary antibodies and ECLdetection (Amersham-Pharmacia, Freiburg, Germany).

Tumour growth in vivoApproximately 1�107 cells were injected subcutaneously into the left and rightflank, respectively, of female NOD/SCID mice. Tumour growth was monitoredevery 3-4 days over 40 days at which point the experiment was terminated andtumours were collected for histological evaluation. At different time points thetumour was measured and the volume was calculated using the formula:V=(L�W2)�/6.

In vitro growthAround 5�104 cells were grown in triplicate on 24-well plates in DMEM/10% FCS.After 24 hours, medium was removed and replaced with serum-free medium withor without 10, 20 or 30 nM SDF-1. Cells were cultured for additional 24 hours andthen harvested and counted with a Coulter Counter Z2.

Data analysisStatistical significance was assessed using a Student’s t-test. The correlationcoefficient was calculated using Microsoft Excel software.

We thank Verena Gschwend for help with isolating bone-marrow-derived B cells; Klaus Hexel for FACS sorting and Filipinmeasurement and Michael Sanderson for helpful discussions.

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