Identiﬁcation of the Novel Role of CD24 as an Oncogenesis ...Oncogenesis Regulator and Therapeutic Target for Triple-Negative Breast Cancer Shih-Hsuan Chan1,2,3, Kuo-Wang Tsai4,5,

Cancer Biology and Translational Studies

Identification of the Novel Role of CD24 as anOncogenesis Regulator and Therapeutic Targetfor Triple-Negative Breast CancerShih-Hsuan Chan1,2,3, Kuo-Wang Tsai4,5, Shu-Yi Chiu3,Wen-Hung Kuo6,Heng-Yi Chen7, Shih Sheng Jiang8, King-Jen Chang9,Wen-Chun Hung10,and Lu-Hai Wang1,2,3,11

Abstract

Triple-negative breast cancer (TNBC) is the most aggres-sive breast cancer subtype, with unfavorable prognosis and5-year survival. The purpose of this study was to investigatethe underlying mechanisms involved in TNBC progression.We determined that CD24 expression was elevated in highlylung and lymph node metastatic TNBC cells. CD24 deple-tion inhibited primary tumor growth and lymph node andlung metastasis and reduced the number of blood andlymphatic vessels in the tumor microenvironment. CD24knockdown impaired EGFR/Met-mediated signaling andreduced lymphangiogenesis- and angiogenesis-related

molecules, including vascular endothelial growth factorsA and C, by promoting EGFR and Met protein instabilityvia the lysosomal degradation pathway. CD24 monoclonalantibody treatment reduced lung metastasis and prolongedthe survival in a lung metastasis mouse model. Clinicalanalyses revealed that the CD24high/METhigh "double-posi-tive" signature identified a subset of TNBC patients withworst outcomes. We conclude that CD24 could be a ther-apeutic target by itself and in combination with the Metexpression could be a good prognostic biomarker for TNBCpatients.

IntroductionBreast cancer is the most frequently diagnosed cancer among

women worldwide with over 1 million cases and nearly400,000 deaths per year (1, 2). Breast cancer is considered aheterogeneous disease that is classified into four subtypesbased on the presence of hormone receptors for estrogen (ER)and progesterone (PR) as well as the expression and geneamplification status of HER2/ErbB2. These four subtypes areluminal A (ERþ or PRþ/HER2�), luminal B (ERþ or PRþ/HER2þ),HER2 (ER� or PR�/HER2þ), and basal-like triple-negative

breast cancer (TNBC; ER�/PR�/HER2�; refs. 3, 4). The substan-tial differences in receptor status in the breast cancer subtypesoften serve as a guideline for different therapeutic interventions(3–6). However, the therapeutic choices for patients withTNBC are limited because of the lack of targeted therapeuticapproaches (7). Cohort studies have indicated that patientswith TNBC are associated with shorter relapse intervalsand poorer overall survival within first 5 years after theirinitial diagnosis than patients with other breast cancer subtypes(3, 8).

EGFR and Met are primary surface receptors for EGF andhepatocyte growth factor (HGF), respectively. Ligand-mediatedEGFR/Met activation promotes tumor angiogenesis and pro-gression through a common downstream Stat3/Src/Akt signal-ing pathway (9–11). In recent years, high frequency of EGFRand Met expression has been observed in patients with TNBCand is strongly associated with poor prognosis and overallsurvival (12, 13). TNBC patients coexpressing Met and EGFRhave shorter disease-free survival than those expressing onlyEGFR (14). These findings suggest that Met and EGFR play animportant role in TNBC progression.

CD24, a heavily glycosylated mucin-type glycosylphospha-tidylinostol-anchored cell surface molecule, has been identifiedas being highly expressed in several human cancers including,breast, lung, and hepatocellular carcinoma. In breast and lungcancers, CD24 has been identified as a key surface receptor inP-selectin binding (15, 16), a cell adhesion molecule expressedon activated endothelial cell and platelets (17), that promotestumor growth by activating Src kinase through the lipid raft(18–20). Clinically, a high CD24 expression is associated withpoor outcomes for cancer patients; however, the role of CD24in TNBC progression is still poorly understood.

1Graduate Institute of Integrated Medicine, China Medical University,Taichung, Taiwan. 2Institute of Molecular and Medicine, National Tsing HuaUniversity, Hsinchu, Taiwan. 3Institute of Molecular and Genomic Medicine,National Health Research Institutes, Miaoli, Taiwan. 4Department of MedicalEducation and Research, Kaohsiung Veterans General Hospital, Kaohsiung,Taiwan. 5Department of Chemical Biology, National Pingtung University ofEducation, Pingtung, Taiwan. 6Department of Surgery, National TaiwanUniversity Hospital, Taipei, Taiwan. 7Graduate Institute of Life Sciences,National Defense Medical Center, Taipei, Taiwan. 8National Institute of CancerResearch, National Health Research Institutes, Miaoli, Taiwan. 9Department ofSurgery, Taiwan Adventist Hospital, Taipei, Taiwan. 10National Instituteof Cancer Research, National Health Research Institutes, Tainan, Taiwan.11Chinese Medicine Research Center, China Medical University, Taichung,Taiwan.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Lu-Hai Wang, China Medical University, Taichung40402, Taiwan. Phone: 886-4-22057153; Fax: 886-4-22060248; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-18-0292

�2018 American Association for Cancer Research.

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In this study, we used an orthotopic xenograft mouse model tostudy the metastatic lung and lymph node (LN) colonization ofTNBC. Our study revealed a novel mechanism in which CD24positively regulates lymphangiogenesis and angiogenesis throughEGF-induced EGFR/Stat3/Src and HGF-induced Met/Stat3/Srcsignaling cascades to promote primary tumor growth as well asTNBC LN and lung metastasis. Our clinical analyses identified anew prognostic marker for TNBC in that patients coexpressingCD24 and Met had poorest outcomes. Our study suggested thatCD24 could be a treatment target for TNBC, and likely for otherbreast cancer subtypes with CD24 overexpression.

Materials and MethodsCell culture

MDA-MB-231, MCF-7, BT549, Hs578T, and human umbilicalvein endothelial cells (HUVEC) were obtained from the ATCC.Human dermal lymphatic epithelial cells (LEC) were obtainedfrom PromoCell. Human breast cancer cell lines were cultured inDMEM supplemented with 10% FBS (Invitrogen). MDA-MB-231–derived sublines LC and IV2 (21) were also cultured in theabove condition. The detailed information was provided inthe Supplementary File. LECs were cultured in endothelial cellgrowth medium MV2 (EGM-MV2) according to the manufac-turer's instruction. HUVECs were cultured in medium 199 with25 U/mL heparin (Sigma), 30 mg/mL endothelial cell growthsupplement (Millipore), 2 mmol/L L-glutamine, and 10% FBS.All cell lines were cultured in a humidified incubator at 37�Cwith5% CO2. All cell lines were verified as mycoplasma-free onDecember 20, 2017, by DAPI staining and are routinely checkedby DNA short tandem repeat (STR) for verification.

Flow cytometryCells were incubated with 5 mmol/L ETDA for 10 minutes to

detach cells from the petri dish. Cells were then washed with5 mL PBS twice to remove EDTA. Note that 5 � 105 cells werestained with FITC-conjugated mouse anti- human CD44 andPhycoerythrin-conjugated mouse anti- human CD24 (BDBioscience) at 1/20 dilution at 4�C for 40 minutes and werekept away from light. Calibur CellQuest Pro (BD) software wasapplied for data acquisition and analysis.

Western blot analysisCells were lysed in RIPA buffer (1% TritonX-100, 50 mmol/L,

pH 7.4, Tris-HCl, 150 mmol/L NaCl2, 0.1% SDS, 1% cholerate),and cell lysates were subjected to SDS-PAGE electrophoresis.Protein was transferred onto PVDF membrane overnight at35 V. PVDF membrane was blocked in 5% non-fat milk andhybridized with the specific primary antibodies followed by thehorseradish peroxidase–conjugated secondary antibody.

In vivo selection of lung-dormant breast cancer cells andhighly metastatic cells

The procedure of establishing lung-dormant breast cancer cellswas as follows: One million MDA-MB-231 parental cells wereinjected orthotopically in SCID mouse at second mammary fatpad. After 3 months, mice were sacrificed and tumor nodules inthe lung were isolated and subsequently cultured in DMEMsupplemented with 10% FBS. The lung-dormant cells isolatedfrom three lung nodules were named as LC-1, LC-2, and LC-3sublines. The resulting LC sublines were authenticated using STR

analysis before carrying out the subsequent functional assays.The highly metastatic IV2 cells were generated from the in vivotail vein injection protocol, and the detailed procedure waspreviously described (21). The SCID mice were provided by theNational Laboratory Animal Center (Taiwan). All animal studieswere approved by Institutional Animal Care and Use Committeeof National Health Research Institute.

RNA extraction and quantitative PCR (qRT-PCR)Detailed procedures of RNA extraction and qRT-PCR are

described elsewhere (21). Specific primers used in qRT-PCR arelisted in Supplementary Table S4.

Immunofluorescence staining and confocal microscopyanalysis

Cells were grown on poly-L-lysine–coated coverslips in 6-wellplates and were incubated overnight. The control siRNA andCD24 siRNA were transfected into cells respectively usingRNAiMAX (Invitrogen) for 8 hours. The transfected cells werefixed and permeabilized with 3.75% formaldehyde and 0.1%saponin, respectively. The permeabilized cells were blocked in 1%BSA for 1 hour and stained with the specific primary antibodiesat 4�C overnight followed by staining with fluorescent dye–conjugated secondary antibodies for 1 hour. Cells were washedwith PBS andmounted in Prolong Antifade reagents (Invitrogen).Cells were observed and photographed using the Leica SP5 IIscanning confocal microscope with 60x objective HCX PLAPO(NA¼ 1.25, oil immersion; Leica). Colocalization of internalizedreceptors and lysosomes was analyzed by using the Colocaliza-tion module of the Leica LASX software according to the softwareinstruction.

Lentivirus preparation and generation of CD24 knockdownlines

Lentivirus-based CD24 shRNA virus was obtained by transientcotransfection of pLKO vector containing shRNA of CD24 withpCMVDR8.91 and pMD.G, into a human 293T cell line. Thesupernatant that contains virus particles was concentrated bymixing with 1/4 volume of Lenti-X solution (Clontech) at 4�Covernight and then was centrifuged at 3,500� g for 50minutes toobtain the concentrated virus particles. Cells were infectedwith thecontrol virus or virus containing CD24 shRNA for 48 hours, andthe infected cellswere selectedwith5mg/mLpuromycin for 1week.The knockdown efficiency of CD24 was examined by qRT-PCRusing the specific human CD24 primers. For the In Vivo ImagingSystems (IVIS) study, cells were further labeled with a luciferasereporter by transfection with the pLVX-GFP-2A-Luc plasmid.

Protein degradation assayCells were treated with 20 mg/mL cycloheximide (CHX) for the

indicated time points and then harvested in RIPA buffer contain-ing protease inhibitors (Roche) for the subsequent Western blotanalysis.

Transwell invasion assayTranswell invasion assayswere carriedout using8.0mmBiocoat

culture insert (BD Biosciences) as described previously (21).

Transwell migration assay of LECs and HUVECsOne million control and CD24-depleted cells were seeded in

6-cm plates respectively 24 hours prior to assay. The conditional

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medium (CM) was collected from the culture medium of thecontrol and CD24-depleted cells. LECs and HUVECs were seededin the 0.2 mm Transwell insert sitting on the 6-well plate, and CMwas added to the lower chamber. After 24-hour incubation, themigrated LEC and HUVEC cells were stained with 0.5% crystalviolet staining solution for 20 minutes. The migrated cells wereanalyzed using ImageJ software (NIH, USA).

Endothelial cell tube formation assayTen thousand HUVECs cells labeled with CellTracker green

(Thermo Fisher Inc.) were mixed with CM collected from theculture medium of the control and CD24-depleted cells and wereseeded in the Matrigel-coated 24-well plate for 4 hours followedby fixation with 3.75% formaldehyde. Tube formation wasobserved and photographed by fluorescent microscope.

Time-lapse imagingNote that 1 � 104 cells were plated in 6-well plate overnight.

The plate was then placed on the stage of the live imagingmicroscope Leica AF 6000 LX (Leica) to perform time-lapserecording. The migration distance and velocity of cells wereanalyzed using MetaMorph software (Molecular Devices).

Antibodies and reagentsDetailed information of antibodies and reagents used in this

study is listed in Supplementary Table S3.

In vivo lungmetastasis assay.Note that 1�106 cellswith indicatedtreatments were suspended in 100 mL PBS and injected individ-ually into the tail veins of C.B -17 SCIDmice. Mice were sacrificedat the indicated timepoint, and lung tissues were dissected andsubjected to histologic examination.

Histologic analysisXenograft tumors and mouse lungs were fixed in 4% formal-

dehyde overnight and were embedded in paraffin. Sections (6 mmthick) were prepared from tissue blocks and were subjected tohematoxylin–eosin (HE) stain.

In vivo lung targeting assayNote that 1 � 106 cells were suspended in 100 mL PBS and

subsequently injected into the tail veins of SCID mice. After 16hours, mice were then anesthetized with isoflurane and weresubjected to lung perfusion. In brief, mouse chest was openedup, and an incision wasmade in the left ventricle of the heart witha scalpel. A butterfly needle attached to a 50-mL syringe fill withPBS was inserted to the right ventricle of the heart, and PBS wasgently pushed into the pulmonary circulation until the mouselungs were white and the fluid coming out of the left ventriclebecame clear.

In vivo monoclonal antibody treatmentCD24 monoclonal antibody was purchased from Beckman

Coulter, Inc. (IM0118). This antibody was adapted from priortrials in mice (22) and humans (23, 24). Administration ofCD24 monoclonal antibody was as the following: 7 days aftercancer cells' i.v. injection, mice were injected with CD24monoclonal antibody every 2 days (0.01 mg in 100 mL PBS;0.03 mg/kg) with six injections in total. The control group wassimilarly injected with mouse IgG1 control antibody. For the

survival assay, higher concentration of CD24 mAb was used(0.04 mg/100 mL; 0.12 mg/kg).

The orthotopic xenograft mouse modelLuciferase-labeled cells with different treatments were injected

into fourth fat pad of mice. Tumor growth was followed untilthe endpoint of the experiment where tumors reached 2 cm indiameter.

Oligonucleotide transfectionCells were transfected with CD24 siRNA purchased from

Thermo-Fisher using Lipofectamine RNAiMAX (Invitrogen) for8 hours. The transfected cells were further incubated for 40 hoursin 10% FBS DMEM and harvested at 48 hours posttransfectionfor subsequent qRT-PCR and Western blot analysis.

IHC stainingIHC staining was carried out as the published procedure (25)

using anti-CD31, anti-LYVE1, anti-Ki67, anti-CD24, anti-EGFR,and anti-Met. The results of IHC score of CD24 were determinedas follows: 0 score: No observed membrane staining; 1þ score:Incomplete membrane staining that is faint in >10% tumorcells; 2þ score: Circumferential membrane staining that isincomplete in >10% tumor cells or complete circumferentialmembrane staining in210% tumor cells; 3þ score: Dark, homo-genous, chicken wire pattern in >10% tumor cells. The H-scoreof CD24, EGFR, and Met in 133 TNBC cases was calculated asthe staining intensity of cancer cells (0 ¼ none; 1 ¼ weak; 2 ¼moderate; and 3¼ strong) multiplied by the percentage of tumorsections being stained (0%–100%). All IHC samples were inde-pendently scored by two investigators in a double-blindedmanner and were reviewed by a pathologist.

Gene set enrichment analysisWe applied gene set enrichment analysis (GSEA; ref. 26) to

identify the functional gene sets or biological pathwaysenriched in the differentially expressed genes between theparental 231 cells and LC cells. In the analysis, log2R was usedas ranking metric, where R was the ratio of normalized geneexpression level of a gene in LC cells to that in parental 231cells; C2 (CP and CGP) was the gene set collection used. GSEADesktop Application (http://software.broadinstitute.org/gsea/downloads.jsp) was used for GSEA implementation.

Clinical dataset analysisThe mRNA levels of CD24, MET, EGFR, and patients' survival

status in public datasets (27, 28)were fetched from theOncominedatabase (https://www.oncomine.org). Breast cancer samples inCurtis dataset were categorized into luminal A, luminal B, HER2,and triple negative for the subsequent Kaplan–Meir survivalanalysis. The normalized RNA-seq data of CD24 were retrievedfrom The Cancer Genome Atlas (TCGA) dataset and categorizedinto LN positive and negative for the subsequent Kaplan–Meiersurvival analysis. For the association study of CD24 andMET combination with survival status, TNBC samples in Curtisdataset were further grouped as "CD24high/MET high," "CD24 high/MET low," "CD24 low/MET high," and "CD24 low/MET low." Forthe association study of CD24 and EGFR combination withsurvival status, TNBC samples were grouped as "CD24 high/EGFR high," "CD24 high/EGFRlow," "CD24 low/EGFR high," and"CD24 low/EGFR low." The Kaplan–Meir survival analysis was

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performed based on the median value of mRNA levels of therespective gene analyzed. A gene expression level lower than themedian value was grouped as "low expression," whereas a geneexpression higher than the median value was defined as "highexpression." The median values of the analyzed datasets weredescribed in the figure legend of Fig. 6.

Clinical samplesFifteen paired primary and LN metastatic tumors were

collected according to National Taiwan University Hospital'sInstitutional Review Board–approved guideline. One hundredand thirty-three TNBC tumor specimens were obtainedthrough the archives of the Department of Pathology, Kaoh-siung Veterans General Hospital (KSVGH), between 1991 and1999 and were approved by the Institutional Review Board at(KSVGH). Written-informed consents were obtained from thepatients. This study was performed in accordance with theDeclaration of Helsinki.

Statistical analysisStatistical analysis was conducted using the SPSS 18.0

statistical software package (SPSS Inc.). The Student t test wasused for the comparison between the control and the exper-imental group. One-way ANOVA was applied to comparethe multiple groups of more than two. Differences wereconsidered significant at P < 0.05 (�, P < 0.05; ��, P < 0.01;and ��, P < 0.001). N.S. represented no significance. TheKaplan–Meier survival curve was analyzed with the Cox pro-portional hazards regression model. Data were presented asthe mean � SEM.

ResultsEstablishment and characterization of TNBC derived fromlong-term lungmetastatic nodules in a xenograft mouse system

To identify the crucial molecular changes of metastatic TNBCcells that colonize in distant organs and how they react and adaptto a foreign environment, we isolated and analyzed metastaticcancer cells that colonized in mouse lungs. As shown in Supple-mentary Fig. S1A, mice were sacrificed 3 months after the cancercells were injected, and metastatic nodules were detected in themouse lung. Three nodules were dissected individually for in vitroculturing to establish the three sublines, which we referred toas the "lung colonization (LC)" sublines, LC-1, LC-2, and LC-3(Supplementary Fig. S1B–S1D).

First, we compared the cellular behaviors of the resulting LCcells with the original MDA-MB-231 parental cells andMDA-MB-231-IV2 cells, which were established from the early lung metas-tases of our previous study (21). The representative data werecollected from LC-1, IV2-1, and 231 cells. Time-lapse imagingrevealed that the migratory distance and velocity of the LC-1 cellswere the shortest and slowest, respectively, among the threecompared isogenic lines, whereas the IV2-1 cells outperformedthe 231 and LC-1 cells for these aspects (Fig. 1A and B). Transwellinvasion assay showed that the LC-1 cells had the poorest invasiveability among three isogenic lines (Fig. 1C). In addition, we foundthe LC cells, which exhibited decreased migration/invasion abil-ity, could form the highest number of colonies in the soft agarcompared with the 231 and IV2 cells, suggesting that the LC cellsgained an increased ability for anchorage-independent growth(AIG; Fig. 1D).

Screening of epithelial–mesenchymal transition/mesenchymal–epithelial transition and breast cancer–initiating cell-related gene profiles revealed the moleculartraits of MET in LC sublines

Because the LC cells exhibited epithelial-like morphologyand poor in vitro migration/invasion (Supplementary Fig. S1D;Fig. 1A–D), we subsequently examined whether the levels ofepithelial–mesenchymal transition/mesenchymal–epithelialtransition (EMT/MET)- and cancer-initiating cell (CIC)–relatedgenes had been altered. The results of qRT-PCR showed thatthe EMT/MET-related genes were not significantly changed(>2-fold change) in the LC cells, except that the expression ofCLND1, a gene encodes for a tight junction protein claudin-1,exclusively expressed in epithelial cells, was increased by at least8-fold compared with that in parental cells (Fig. 1E). Screeningof the expression of breast cancer–initiating cells (BCIC)makers revealed that CD24 expression was increased approx-imately 35- and 5-fold in the LC-1 and IV2-1 cells, respectively,compared with that in the parental cells (Fig. 1E). This resultwas confirmed through flow cytometry analysis; that is, approx-imately 20% and 7% of the LC-1 cells and IV2-1 cells exhibitedCD24 positivity, respectively, compared with the parental cells,of which less than 2% of cells expressed surface CD24 (Fig. 1F).Western blotting analysis showed that the LC cells expressedhigh levels of CD24, claudin-1, ZO-1, which is another tightjunction protein, and fibronectin (Fig. 1G), which werereported to be important for cancer cells to grow in the lungmicroenvironment (29). Immunofluorescent staining con-firmed that claudin-1 was localized to the membrane and thecell–cell junction region in LC-1 cells, and no claudin-1 expres-sion was detected in both 231 and IV2-1 cells (Fig. 1H). Tomore effectively characterize the intrinsic property of the LCcells, we subsequently examined whether the activity of smallGTPase Rac1/Cdc42, which is important for cell motility andinvasiveness, was changed in the MET-like LC cells. UsingGTPase pull-down assay, we determined that the LC cellsexhibited a low level of GTP-bound Rac1 and Cdc42, whichcould account for their decreased migration and invasionability (Fig. 1I).

LC cells display strong in vivo tumorigenicity and LNmetastasisNext, we examined the in vivo behavior of the LC cells. One

million luciferase-labeled LC and 231 cells were separatelyinjected into the mammary fat pad no 4 of each SCID mouse toobserve the tumor growth and the metastatic spreading. An IVISimaging system was used to examine the metastatic spreading oftumor cells 7 weeks after injection.

The IVIS results showed that the LC cells grew faster andresulted in bigger tumors that did the parental 231 cells (Sup-plementary Fig. S2A and S2B) and exhibited increased LNmetastasis ability compared with the parental 231 cells (Sup-plementary Fig. S2B). The IHC staining of cell proliferationmarker Ki-67 revealed that LC-derived tumors showed increasedKi-67 expression in the nucleus as compared with 231-derivedtumors, suggesting that LC cells were more proliferative than231 cells in the in vivo setting (Supplementary Fig. S2C).Furthermore, lung and LN metastasis analyses were performedusing ex vivo IVIS imaging (Supplementary Fig. S2D). Thephoton signals (the presence of metastatic cells) in the mouselung and LNs were normalized to primary tumor size. Thequantitative results of ex vivo IVIS imaging showed that more

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Figure 1.

Functional and molecular characterization of breast cancer cells derived from the long-term lung metastatic nodules in an orthotopic xenograft mouse model.A, The representative images of the cell tracking assay. The result showed that LC subline displayed reduced motility as compared with its isogenic231-P and IV2 lines. B, Analysis of cell movement in three categories (distance of migration, distance to origin, and velocity) generated from the celltracking assay showed that LC was inferior to its isogenic lines in all three criteria. Data are mean � SEM (n ¼ 5). � , P < 0.05. C, Transwell invasion assayindicated that LC cells displayed the poorest invasiveness among isogenic lines. Data are mean � SEM (n ¼ 3). � , P < 0.01. D, Soft-agar assay revealedthat LC cells showed the strongest ability of AIG as compared with the parental MDA-MB-231 and IV2 lines. Data are mean � SEM (n ¼ 3). � , P < 0.01.E, EMT/MET- and BCIC-related genes were analyzed with qRT-PCR in LC sublines in comparison with other two isogenic lines, the parental MDA-MB-231 andIV2 lines. � , Changes over 2-fold and P < 0.01. F, Flow cytometry analysis of the surface CD44 and CD24 expression in three isogenic lines. G, EMT/MET-relatedproteins were analyzed for the three isogenic lines by Western blot. H, Expression and localization of claudin-1 among the parental MDA-MB-231, IV2-1,and LC-1 cells. Scale bars, 15 mm. I, Analysis of Rac1/Cdc42 activity in the three isogenic lines using GST-PAK pull-down assay. Each experiment was performedin triplicates and was repeated at least 3 times.






photons were detected in the LNs and lungs of mice injectedwith the LC cells than in those of mice injected with the parental231 cells (Supplementary Fig. S2E). Notably, although LCcells had decreased in vitro cell motility and invasiveness(Fig. 1C and D), they could metastasize to the LNs and lung,and efficiently colonize there.

CD24 inhibition impairs in vitro AIG and in vivo lungmetastaticcolonization

Screening the EMT/BCIC makers revealed that the LC cellsexpressed a high level of CD24 (Fig. 1E and F); this promptedus to examine whether CD24 is associated with LC cell tumor-igenicity and metastasis (Supplementary Fig. S2). Furthermore,the role of CD24 in TNBC LN and lung metastasis has not beenaddressed. Therefore, we investigated the contribution of CD24 toTNBC metastatic colonization and progression.

The effect of CD24 on a metastatic colonization-related prop-erty (i.e., AIG) was assessed. Soft-agar assay showed that CD24depletion in CD24-expressing LC cells considerably impairedtheir colony-forming ability (Supplementary Fig. S3A and S3B).This phenomenon was also observed in other CD24-expressingTNBC lines MDA-MB-468 and Hs578T (Supplementary Fig. S3Aand S3B). In addition, CD24 inhibition also reduced the cellproliferation of CD24-expressing TNBC cells (SupplementaryFig. S3C). Similar results were observed in the CD24-expressingluminal breast cancer cell line MCF-7 (Supplementary Fig. S4).

To explore the potential role of CD24 in metastasis, we gen-erated stable CD24 knockdown (KD) IV2 cells that also featureCD24 expression. One million luciferase-labeled CD24 KD IV2cells or the control IV2 cellswere injected through the tail vein intoSCID mice, respectively, and lung colonization status was exam-ined 3 weeks later using the IVIS imaging system. The resultsshowed that theCD24KD IV2 cells exhibited substantiallyweakerlung colonization signals than did the control cells (Supplemen-tary Fig. S5A and S5B). HE staining ofmouse lung sections furtherconfirmed this result (Supplementary Fig. S5C and S5D).

Because extravasation is the crucial step for the cancer cells tocolonize the distant organs (30), we examined whether the earlyextravasation event was the defining factor for the decreased lungcolonization of the CD24 KD cells. Lung perfusion prior to lungtissue collection was performed to examine the early lung target-ing event. Mice i.v. injected with CD24 KD cells or control cellswere perfused with 1X PBS 20 hours after injection to removecancer cells inside the lung blood vessels. Cancer cells in themouse lung (indicating that the cells had completed extravasa-tion) were detected by qRT-PCR using human-specific HPRT1gene primers. The result showed no significant difference in theearly lung targetingbetween the groupofmice injectedwithCD24KD and that injected with control cells (Supplementary Fig. S6).Collectively, these results indicated that CD24 was less importantfor targeting but was crucial for metastatic lung colonization.

CD24 depletion greatly reduces tumorigenicity, the metastaticability of IV2 and LC cells in the orthotopic mouse model

To further assess the role of CD24 in TNBC metastasis, theorthotopic mouse model was used. One million cells each fromthe two luciferase-labeled CD24 KD IV2 lines (CD24 KD andCD24 KD-2) and the IV2 control cells were injected into thefourth mammary fat pad of SCID mice individually to observetumor growth and metastatic progression. As the tumor pro-gressed, the CD24 KD tumors not only grew more slowly than

those of the control group (Fig. 2A–D) but also failed to meta-stasize to the neighboring LNs (inguinal and axillary LN; Fig. 2Eand F). This was confirmed through ex vivo IVIS imaging whichrevealed that CD24 depletion led to decreased LN as well aslung metastasis (Fig. 2G). The qRT-PCR detection of metastaticcells in mouse lungs and LNs exhibited the similar results(Fig. 2H). The metastasis signals were normalized to primarytumor size. The effect of CD24 depletion on the in vivo propertyof the LC line, which had strong LN metastatic ability (Sup-plementary Fig. S2), was also examined. As expected, the CD24KD LC cells exhibited decreased tumorigenicity (Fig. 2I–L) andfailed to metastasize to LNs and lung compared with thecontrol LC cells (Fig. 2M and N). Conversely, ectopic expres-sion of CD24 in the CD24-low 231 cells considerably promot-ed primary tumor growth compared with the control cells(Supplementary Fig. S7A–S7D).

Taken together, both tail vein and orthotopic metastasis mod-els indicated that CD24 has a key role in TNBC tumor progressionas well as LN and lung colonization.

CD24 depletion impairs lymphangiogenesis and angiogenesisthrough VEGF-C and VEGF-A downregulation

Because tumor lymphangiogenesis and angiogenesis arecrucial in the growth and metastatic progression of tumors,we next examined the effect of CD24 KD on these events inxenograft tumors.

IHC staining of the lymphatic vessel marker LYVE-1 and theblood vessel marker CD31 revealed that IV2 and LC tumors withCD24 KD had considerably decreased lymphatic and bloodvessels compared with the control IV2 and LC tumors (Supple-mentary Fig. S8A and S8B). Conversely, 231 tumors derived fromCD24-transfected 231 cells had increased lymphatic and bloodvessels compared with the control 231 cells–derived tumors(Supplementary Fig. S7E and S7F). This result motivated us tofurther examine the association between CD24 and lymphaticvessel and blood vessel formation. Tumor-secreted vascular endo-thelial growth factors C, D, and A (VEGF-C, VEGF-D, and VEGF-A,respectively) are the well-known growth factors that recruitnearby lymphatic endothelial cells and endothelial cells to thetumor site to form supportive lymphatic and blood vessels fortumor growth (31, 32). First, we examinedwhether LC or IV2 cellsexhibited increased VEGF-A, -C, and -D expression. The qRT-PCRresult indicated that the LC subline expressed the highest level ofVEGF-A, -C, and -Dwith a 2- to 6-fold increase compared with the231 cells (Supplementary Fig. S8C). In addition, the IV2 cellsexhibited significantly increased VEGF-A and -C expression (Sup-plementary Fig. S8C). Knockdown of CD24 in the LC or IV2 cellsreduced the VEGF-A and -C mRNA expression level, whereas theexpression level of VEGF-D was unaffected (SupplementaryFig. S8D). These results were confirmed through ELISA of thesefactors in the culture media (Supplementary Fig. S8E).

On the basis of these findings, we subsequently examinedwhether CD24 depletion could affect endothelial cell migrationand tube formation. Coculture Transwell assay showed thatknockdown of CD24 in the IV2 and LC cells significantly reducedIV2 or LC cells' CM-induced migration of LECs and HUVECs(Supplementary Fig. S8F). Treating HUVEC cells with the CMcollected from the culture of CD24-depleted IV2 or LC cellsreduced the tube formation (Supplementary Fig. S8G).

These results combined with the aforementioned in vivoobservations suggested that CD24 plays a central role in

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regulating tumor-derived VEGF-A and -C expression to estab-lish lymphatic and blood vessel networks at primary tumorsite to facilitate growth, metastatic spread, and subsequentmetastatic colonization.

CD24 depletion attenuates EGF/EGFR/Stat3/Src and HGF/Met/Stat3/Src signaling cascades

Because of the lack of intracellular kinase domain in CD24(19), we hypothesized that membrane-bound CD24might affect

Figure 2.

Depletion of CD24 greatly reduces tumor growth, LN, and lung metastatic abilities of IV2 and LC cells in a xenograft orthotopic mouse model. A, Primarytumor growth of mice injected with CD24-depleted IV2 cells or the control cells was examined in the orthotopic mouse model. B and C, CD24 KD IV2cells grew slower and formed smaller tumors than the control cells. Data are mean � SEM. � , P < 0.05. D, The qRT-PCR analysis of CD24 levels in thecontrol, CD24 KD, and CD24 KD-2 tumors. Data are mean � SEM. � , P < 0.01. E, Long exposure (60 seconds) of IVIS imaging revealed that CD24 KDtumor–bearing SCID mice showed no sign of LN metastasis compared with the control tumor-bearing SCID mice whose LN metastases were detected.F, The quantification of luminescent photon signals of axillary LN in the control tumor- and CD24 KD tumor–bearing mice. The photon signals werenormalized to the respective primary tumor size. Data are mean � SEM. � , P < 0.01. G, The representative IVIS image of ex vivo examination of LNmetastasis of the control tumor- and CD24 KD tumor–bearing mice. H, Detection of metastatic cells in mouse lung and LNs with qRT-PCR using human-specificprimers. Data are mean � SEM. � , P < 0.01. I–K, CD24 KD LC cells (n ¼ 5) grew slower and formed smaller tumors than the control LC cells (n ¼ 5) inthe orthotopic mouse model. L, The qRT-PCR analysis of CD24 levels in the control and CD24 KD LC tumors. M and N, The presence of metastaticcells in mouse lung and LNs was determined with qRT-PCR using human-specific primers. The photon signals were normalized to the respective primary tumorsize. Data are mean � SEM. � , P < 0.01.






other membrane receptors that mediate VEGF expression toregulate lymphangiogenesis and angiogenesis. This speculationwas supported by our GSEA of differential gene expression profilebetween CD24high LC cells and the parental 231 cells, whichrevealed that the EGFR- and Met-mediated pathways (9, 33, 34),two key receptor tyrosine kinase (RTK) signaling pathwaysknown to activate VEGF expression in cancers, were significantlyenriched (Fig. 3A).

These findings prompted us to investigate the role of CD24in EGFR- and Met-driven downstream signaling as well asVEGF expression in TNBC cells. We first confirmed that treat-ment of TNBC cells with 50 ng/mL of HGF or 100 ng/mL ofEGF could induce VEGF-A and -C expression (SupplementaryFig. S9A and S9B). The effect of CD24 depletion on HGF- andEGF-induced signaling cascade was subsequently examined.CD24 expression in the IV2, LC, and two other TNBC lines,BT-549 and Hs578T, was depleted, and the cells were treatedwith HGF at a concentration of 50 ng/mL for 20 minutes. Theresults showed that as compared with the control cells, CD24depletion significantly reduced HGF-induced Met phosphory-lation and downstream Src/Stat3/Akt phosphorylation, whichare known to drive VEGF production (Fig. 3B). This phenom-enon was observed for all four TNBC cell lines tested. Inaddition, we noticed that the protein level of Met was signif-

icantly decreased in CD24-depleted TNBC cells (Fig. 3B). Thismight explain in part the inhibition of HGF-induced Met/Stat3/Src/Akt phosphorylation in the CD24-depleted cells.

To analyze EGFR-mediated signaling, TNBC cells were treatedwith EGF at a concentration of 100 ng/mL. The EGF-inducedEGFR/Stat3/Src/Akt phosphorylation cascade was subsequentlyanalyzed through Western blotting. Similarly, the EGF-inducedEGFR/Stat3/Src and Akt/MEK1/2 phosphorylation was signifi-cantly decreased in CD24-depleted TNBC cells compared withthe control cells (Fig. 3B). Notably, we observed EGFR proteinwas significantly decreased in the absence of CD24 in the TNBCcells (Fig. 3B). Overall, these findings above revealed the new roleof CD24 in EGFRhigh and Methigh TNBC in that CD24 losssignificantly impaired EGF- and HGF-induced signaling.

CD24 depletion increases protein instability and lysosome-mediated degradation of EGFR and Met

Because CD24 loss caused the downregulation of EGFRand Met at the protein level, we evaluated whether CD24could affect EGFR and Met protein stability. CD24-depletedcells were treated with CHX for 24 hours, and EGFR and Metprotein levels were analyzed over time. Western blottingshowed that EGFR and Met protein levels were significantlydecreased at a faster rate over 24 hours in the CD24-depleted

Figure 3.

Depletion of CD24 attenuates EGF/EGFR/Stat3/Src and HGF/Met/Stat3/Src signaling cascades by destabilizing EGFR and Met. A, GSEA was appliedto analyze the potential enriched pathway(s) in LC cells. Two angiogenesis-related RTK signaling pathways, Met and EGFR, were shown to be enrichedin LC cells. B, The effect of CD24 depletion on HGF- and EGF-driven VEGF signaling cascades in CD24-expressing TNBC lines. Western blot analysisshowed that knockdown of CD24 led to the reduction of HGF- and EGF-induced Met/Stat3/Src/Akt and EGFR/Stat3/Src/Akt signaling cascades. Eachexperiment was performed in triplicates and was repeated at least 3 times. C, The effect of CD24 KD on protein stability of EGFR and Met. Cellstransfected with the control siRNA or CD24 siRNA were both treated with CHX (20 mg/mL) over the course of 24 hours. Loss of CD24 greatlydecreased protein stability of EGFR and Met. D, The effect of bafilomycin A1 (BA) treatment in CD24-depleted cells in the absence or presence of HGF(50 ng/mL) and EGF (100 mg/mL).

Chan et al.





cells compared with the control cells (Fig. 3C), indicating thatCD24 loss increases the protein instability of EGFR and Met.The protein degradation of membrane EGFR and Met primarilyoccurs through receptor internalization followed by lysosome-dependent degradation (35). Here, we demonstrated thatbafilomycin A1 treatment, a known V-ATPase inhibitor, couldnot restore the activation of EGFR and Met upon ligand stimu-lation but the total protein level in CD24-depleted BT549 cells,implying that the receptor internalization may occur uponCD24 depletion (Fig. 3D). We subsequently explored the effectof CD24 depletion on ligand-induced EGFR/Met internaliza-tion and degradation through immunofluorescence staining.Eight hours after CD24 siRNA transfection, the control andCD24 siRNA-transfected cells were serum-starved overnightfollowed by 2 hours of EGF or HGF stimulation, and immu-nofluorescent staining was performed. The results indicatedthat at 0 hours before ligand stimulation, the internalizedEGFR and Met (indicated by red fluorescence) were observedin CD24-depleted LC cells compared with the control cells, inwhich EGFR/Met could be found on the membrane (Fig. 4Aand C, top plots). We found that some internalized EGFR/Metwere colocalized with lysosomes (indicated by green fluores-cence; LAMP-1–positive vesicles). These results were supportedby Z stack analysis and fluorescence intensity score (Fig. 4Aand C). Quantitative colocalization analysis using Mander'scoefficient analysis (ImageJ plug-in) suggested that the CD24-depleted cells without EGF/HGF stimulation underwent anapproximately 7-fold increase in EGFR/Met and LAMP-1 dou-ble-positive vesicles compared with the control (Fig. 4A and C;bottom right). Next, ligand-induced internalization and deg-radation of EGFR and Met were examined in CD24-depletedcells. After 2-hour EGF/HGF treatment, we determined thatthe internalized EGFR/Met more frequently colocalized withlysosomes in the CD24-depleted cells compared with thecontrol cells (Fig. 4B and D, top plot). Moreover, the Z stackanalysis and fluorescence intensity score provided similarresults (Fig. 4A and C, bottom plot). Quantitative colocali-zation analysis indicated that CD24-depleted cells withEGF/HGF stimulation exhibited an approximately 48% and31% increase in EGFR/LAMP-1 and Met/LAMP-1 double-positive vesicles, respectively, compared with the control (Fig.4B and D, bottom right).

Overall, confocal microscopy analysis demonstrated that lossof CD24 expression increased EGFR/Met internalization andsubsequent lysosome-mediated degradation in both steady-stateand ligand-stimulated conditions.

CD24 mAb treatment reduces the lung metastatic burden andsignificantly prolongs the survival time in a lung metastasismouse model

We subsequently assessed whether CD24, a membrane-boundmolecule vital for primary tumor growth and metastasis,could be a therapeutic target for cancer therapy. A previousstudy reported on the use of the ALB9 mAb against CD24 forbladder cancer therapy in a mouse model (36). In the presentstudy, we evaluated the therapeutic potential of ALB9 mAb onTNBC metastatic colonization. First, we examined the effect ofCD24 mAb on AIG of LC cells. The result revealed that incu-bating of 15,000 LC cells with CD24 mAb ALB9 at a concen-tration of 2 mg/mL for 30 minutes prior to the soft-agar colonyassay significantly impaired the AIG of LC cells as compared

with the cells treated with the control antibody (Fig. 5A).Subsequently, we used this mAb for the in vivo experiment tostudy the effect of CD24 mAb on lung metastatic colonizationof TNBC. Mice were i.v. injected with 0.5 million luciferase-labeled LC cells followed by ALB9 mAb treatment (Fig. 5B). Themice were treated with ALB9 mAb or IgG1 control antibody at adosage of 10 mg in 100 mL 1X PBS once every 2 days over 6 times(Fig. 5B). The lung metastatic burden of the mice was moni-tored before and after antibody treatment using the IVIS sys-tem. The IVIS result showed that the two groups of miceexhibited no significant differences in lung metastatic burdenbefore antibody injection (Fig. 5B). By week four, the mousegroup that received ALB9 mAb treatment exhibited a reducedlung metastatic burden as indicated by luminescent signalscompared with the mouse group treated with the IgG1 control.We also observed the similar inhibitory effect of CD24 mAbusing another CD24-expressing TNBC cell line MDA-MB-468(Supplementary Fig. S10). Moreover, we assessed whetherCD24 mAb treatment could prolong the survival of micewith metastatic lung burden. Ten mice were i.v. injected with2 � 105 LC cells and randomized into two groups with eachgroup containing 5 mice. One week later, mice were i.v. injectedwith either the control IgG1 Ab (n ¼ 5) or CD24 mAb (n ¼ 5)every 2 days at a dose of 0.12 mg/kg each injection for a totalof six injections (Fig. 5C, left). By 20 days, the body weight ofmice without CD24 mAb treatment began to drop significantlyas compared with those with CD24 mAb treatment (Fig. 5C,right). The Kaplan–Meier survival analysis showed that micewith CD24 mAb treatment significantly survived longer thanthe control mice (Fig. 5C, Log-rank: P ¼ 0.02). Our dataindicated that CD24 mAb treatment could significantly pro-long the survival time of mice bearing lung metastatic burden,suggesting the therapeutic potential of CD24 mAb in CD24-positive breast cancer patients.

CD24high/METhigh "double-positive" signature predicts pooroverall survival and a short interval of metastatic recurrencein patients with TNBC

We subsequently explored the clinical relevance of CD24 inTNBC patients. The Kaplan–Meier analysis indicated that highexpression of CD24 was significantly correlated with poor5-year survival of breast cancer patients in public Curtis datasets(Fig. 6A). When stratified into different subtypes, a high level ofCD24 was significantly associated with poorer 5-year survival inpatients with luminal A and TNBC (Fig. 6A; SupplementaryTable S1). By contrast, although CD24 expression status didnot statistically correlate with 5-year survival of patients withHER2 and luminal B breast cancer (Fig. 6A; SupplementaryTable S1), we nevertheless observed a trend of relatively poorsurvival in the CD24high population. Moreover, a high level ofCD24 was associated with higher 5-year metastatic recurrence,although it did not reach the level of statistical significance(Fig. 6B; Supplementary Table S2). In addition, we found thesimilar result in TCGA dataset and further showed that highCD24 expression was significantly correlated with poorer over-all survival in breast cancer patients with LN metastasis ascompared with those without (Fig. 6C). We also collected pair-ed primary tumor and metastatic LN from 15 TNBC patients toexamine the expression of CD24. Among those 15 patients,7 (47%) showed higher CD24 expression in metastatic LN ascompared with the corresponding primary tumors. The






representative CD24 IHC images from 5 patients were shownin Fig. 6D. Five patients were CD24 negative both in primaryand metastatic LN, and CD24 was similarly expressed in pri-mary and metastatic LN in 3 patients (Fig. 6D). Together, theseresults suggested that breast cancer patients exhibiting highCD24 expression presented poor clinical outcomes, particularly

patients with TNBC and luminal A, and were correlated with LNmetastasis.

Because the presence of CD24 is important for mediating theEGF- and HGF-driven pathways in TNBC cells (Figs. 3 and 4), weexamined the correlation of CD24 expression levels in combina-tion with MET/EGFR levels with the survival status of TNBC

Figure 4.

Depletion of CD24 facilitates EGFR/Met internalization to promote protein degradation via lysosome-dependent pathway. A and C, Representativeimages of immunofluorescence staining of EGFR/Met and LAMP-1 before EGF/HGF stimulation in the control and CD24-depleted LC cells. B andD, Representative images of immunofluorescence staining of EGFR/Met and lysosome after EGF/HGF stimulation in the control- and CD24-depleted LC cells.The internalized receptor and its colocalization with lysosome were measured by confocal microscopy. Z stack (y–z axis) and colocalization intensityanalysis were shown at the right and beneath the immunofluorescence images, respectively. Cells treated with or without EGF/HGF for 2 hours wereimmunostained with antibodies against EGFR/Met and LAMP-1, a lysosome marker, and imaged by 3D confocal microscopy. The y–z cross-sectional images ofthe cell are shown. The overlapped region of red and green fluorescence signals indicated by black arrows was measured by LAS AF software. It showed thecolocalization of EGFR/Met and LAMP-1. Quantification of colocalization of EGFR with LAMP-1 was measured using the Mander's coefficient (JACoPplugin, ImageJ). 4.8% � 2.5% (n ¼ 10 cells; mean � SD) and 38.1% � 2.7% (n ¼ 10 cells; mean � SD) of EGFR were observed to colocalize withLAMP-1 in the control- and CD24-depleted LC cells, respectively, without EGF stimulation. 49.9% � 4.4% (n ¼ 10 cells; mean � SD) and 74.2% � 4.0%(n ¼ 10 cells; mean � SD) of EGFR were observed to colocalize with LAMP-1 in the control- and CD24-depleted LC cell, respectively, with EGFstimulation for 2 hours. 5.1% � 0.5% (n ¼ 10 cells; mean � SD) and 35.1% � 3.7% (n ¼ 10 cells; mean � SD) of Met were observed to colocalize withLAMP-1 in the control- and CD24-depleted LC cells, respectively, without HGF stimulation. 53.11% � 2.8% (n ¼ 10 cells; mean � SD) and 69.9% � 1.7%(n ¼ 10 cells; mean � SD) of Met were observed to colocalize with LAMP-1 in the control- and CD24-depleted LC cells, respectively, with HGF stimulationfor 2 hours. Scale bars, 15 mm.

Chan et al.





patients. As shown in Fig. 6B, we found that TNBC patientscoexpressing CD24 andMET (CD24highMEThigh signature) exhib-ited the poorest 5-year survival among patients stratified byCD24/MET expression status (Fig. 6B). In addition, CD24high-

METhigh signature also predicted faster metastatic recurrence inTNBC patients (P¼ 0.004, Fig. 6C). In terms of the association ofcoexpression status of CD24 and EGFR in TNBC, we determinedthatEGFR expression is significantly correlatedwith themetastaticevents in patients with TNBC regardless of CD24 expression(Fig. 6C). By contrast, TNBC patients coexpressing CD24 andEGFR, unlike CD24high/METhigh signature, did not show poorer5-year survival (Fig. 6B). In addition, we found CD24 wassignificantly correlated with EGFR (Spearman correlation:0.224, P ¼ 0.009) and Met (Spearman correlation: 0.410,P < 0.0001) in terms of protein expression (SupplementaryFig. S11). Taken together, our clinical analyses revealed a novel"CD24high/METhigh double-positive signature" of TNBC patients,which can predict a poor outcome of patients with TNBC.

DiscussionIn previous studies, enrichment of the CD44þ/CD24�/low

and CD44�/CD24þ cell populations has been observed inbasal-like and luminal breast cancer cell lines, respectively(37, 38), and CD44þ/CD24�/low cells were determined to bemore invasive than CD44�/CD24high cells (38). Moreover,several studies have reported that CD24 is a disfavored markerfor BCICs, whose commonly known marker signature isCD44þ/CD24�/ALDH1þ (39, 40). In this study, we deter-mined that MDA-MB-231–derived LC cells, which were iso-lated from long-term metastatic lung nodules, exhibited highCD24 level and several epithelial-like phenotypic and molec-ular features (Fig. 1). Notably, we found that despite theirdecreased in vitromotility and invasiveness, CD44þ/CD24þ LCcells were more potent in primary tumor growth, as well asLN and lung metastasis than the CD44þ/CD24� parentalMDA-MB-231 cells (Supplementary Fig. S2). Although thesefindings seemingly contradict the established knowledge

Figure 5.

CD24 mAb treatment reduces the lung metastatic burden and significantly prolongs the survival time in a lung metastasis mouse model. A, The effectof CD24 mAb on anchorage-independent growth of LC cells. Incubation of LC cells with 2 mg/mL CD24 mAb ALB9 30 minutes prior to the soft-agarassay impaired the colony-forming ability of LC cells as compared with the cells treated with the control antibody mouse IgG1. B, The schematics ofCD24 mAb treatment. Note that 0.5 million luciferase-labeled LC cells were injected intravenously via tail vein into SCID mice. Seven days later, CD24 mAbALB9 (n ¼ 5) or mouse IgG1 control (n ¼ 5) was administered through tail vein injection (10 mg in 100 mL PBS; 0.03 mg/kg) every 2 days for 6 injections intotal. The IVIS images of mice after intravenous injection after 1 week and 4 weeks later were shown. The histogram is the quantification result ofmouse lung ROI signals 10 minutes, 10 days, and 30 days after intravenous injection. � , P < 0.05. C, Ten mice were intravenously injected with 2 � 105 LC cellsand randomized into two groups prior to CD24 mAb administration. Mice were injected with CD24 mAb (n ¼ 5) or mouse IgG1 Ab (n ¼ 5) at a dose of40 mg (0.12 mg/kg). Body weight of mice during the course of treatment. P < 0.05. D, The Kaplan–Meier survival plot of mice received mouse IgG1 Ab (n¼ 5) orCD24 mAb ALB9. Log-rank: P ¼ 0.03.






Figure 6.

CD24high and METhigh "double-positive" signature predicts poorer overall survival and shorter time of metastatic recurrence in TNBC patients. A, Kaplan–Meiersurvival analysis of breast cancer patients in Curtis dataset. The median value of CD24 for the cohort of total BC, TNBC, HER2, luminal A, and luminal B inCurtis dataset is 7.572, 7.891, 8.059, 7.393, and 7.233, respectively. B, Kaplan–Meier survival analysis of metastasis recurrence in Ma dataset. The medium value ofCD24 in Ma dataset is 3.678. C, Kaplan–Meier survival analysis of breast cancer patients and patients with or without LN metastasis according to CD24 level inTCGA RNA-seq dataset. D, The representative images of CD24 IHC staining from paired primary tumor and metastatic LN (left). CD24 IHC scores in 15patients (right). E, Kaplan–Meier survival analysis of TNBC patients with "CD24 highMET high," "CD24 highMET low," "CD24 lowMET high," or "CD24 lowMET low."Themedian values ofCD24 andMET are 7.981 and 3.31. F,Kaplan–Meier survival analysis on TNBCpatientswith "CD24 highEGFR high," "CD24 high EGFR low," "CD24low EGFR high," or "CD24 low EGFR low." The median values of CD24 and EGFR are 7.981 and 2.155. G, The schematic of CD24's role in TNBC progression.

Chan et al.





that CD24þ breast cancer cells exhibit a more differentiatedand less tumorigenic phenotype, several reports have associ-ated CD24 expression with tumor progression and metastaticbehavior (41, 42). Furthermore, a comprehensive study ofmolecular and phenotypic analysis of CD24þ and CD44þ cellsfrom breast carcinomas revealed that the number of CD24þ

cells was dramatically and consistently increased in distantmetastases regardless of the type of the primary tumor and sitesof the distant metastasis (43).

In the CD24 KD xenograft mouse model, CD24 depletiongreatly reduced the primary tumor growth, as well as LN andlung metastasis in both the IV2- and LC cells–derived tumormodel (Fig. 2). We observed that LC/IV2 cells could expresshigh levels of VEGF-A and VEGF-C to attract endothelial cellsand lymphatic endothelial cells to the proximity of the primarytumor to form vascular and lymphovascular networks in thetumor microenvironment (Supplementary Fig. S8A–S8C).

Metastasizing process consists of multiple steps includinglocal invasiveness, intravasation into blood or lymphatic ves-sels and extravasation at target organs, as well as survival andoutgrowth at those organs, which are all very important for themetastasis. With respect to the role of CD24 in metastasis, wethink it contributes to initial survival and subsequent out-growth of the disseminated cancer cells in target organs likelyvia enhancing the angiogenesis and lymphoangiogenesis sim-ilar to that in the primary tumor. Our lung targeting assayrevealed that CD24KD did not significantly affect the initiallung targeting (Supplementary Fig. S6) but greatly suppressedthe later macroscopic lung metastases (Supplementary Fig. S5),suggesting that CD24 plays a more important role in lungcolonization and outgrowth than in initial invasiveness andextravasation during dissemination of primary cancer cells.Collectively, out data suggested that CD24 plays an importantrole in primary tumor growth, as well as in outgrowth ofdisseminated cancer cells in the metastasizing organ such aslung during TNBC metastasis.

Another important role of CD24 in TNBC cell metastasis islikely its effect on EGF/HGF-induced angiogenesis- and lymphan-giogenesis-related signaling pathways. We found that CD24depletion in CD24-expressing TNBC cells attenuated theligand-induced EGFR and Met downstream phosphorylationcascades (Fig. 3A and B). We determined that CD24 exerted itsfunctions by stabilizing EGFR and Met at the protein level tosustain the EGF- and HGF-induced Src/Stat3 signaling, which isimportant for tumor angiogenesis (Fig. 3C and D; refs. 44, 45).Immunostaining analysis supported the results of Western blot-ting that depletion of CD24 could promote EGFR and Metinternalization and lysosome-mediated degradation in theabsence of a ligand (i.e., EGF and HGF, respectively), comparedwith the control cells (Fig. 4A and C). Furthermore, loss of CD24expression accelerated EGFR and Met degradation upon ligandstimulation, as indicated by the increased colocalization of EGFR/Met and lysosomes (Fig. 4B and D).

Previous studies have reported that tumor-promotingcancer-associated fibroblasts and tumor-associated macro-phages express high levels of HGF and EGF to enhancetumorigenesis (46, 47). Thus, the CD24-mediated enhance-ment of Met and EGFR pathways may play an important rolein the context of the tumor-promoting microenvironment,and it may account for a large part of the biological functionsaffected by CD24. We propose that the presence of CD24 on

the cell membrane may positively regulate EGFR/Met stabilityin TNBC (Fig. 6G).

Clinically, high levels of CD24 are most significantly asso-ciated with the shorter survival of TNBC patients comparedwith the patients with the HER2-positive and luminal subtypes(TNBC: P ¼ 0.02; Luminal A: P ¼ 0.03; Luminal B: P ¼ 0.1212;HER2: P ¼ 0.24, Fig. 6A). Analysis of the clinical relevance ofcoexpressing CD24 and EGFR/MET in TNBC patients showedthat CD24highMEThigh was associated with poor 5-year survival(P ¼ 0.193, Fig. 6E) and significantly increased the risk ofrecurrence within the first 5 years after initial treatment (P ¼0.0043, Fig. 6E). In addition, Deng and colleagues reported thatCD24 expression is significantly associated with docetaxelresistance in TNBC patients, adding further clinical relevanceof CD24 to TNBC (48). Taken together, our clinical analysesindicate that CD24 alone is a good prognostic factor forTNBC patients, and in combination with MET expression, theCD24highMEThigh signature is an effective prognosis marker forTNBC patients.

Much effort has been devoted to identifying and validatingpotential markers for developing targeted therapy for TNBCpatients. In this study, we explored the possibility of usingCD24 mAb to treat the metastatic disease of CD24-positiveTNBC in a tail vein xenograft mouse model. We demonstratedthat CD24 mAb treatment significantly reduced lung tumorgrowth (Fig. 5B; Supplementary Fig. S10) and greatly pro-longed the survival of mice (Fig. 5C and D). Our findingprovides the first evidence to suggest the use of CD24 mAb asa targeted therapy for CD24-positive TNBC patients. In addi-tion, luminal A patients with high CD24 expression alsoexhibited poor prognoses (Fig. 6A); therefore, the therapeuticefficacy of CD24 mAb could be tested for other CD24-positivebreast cancer subtypes.

Overall, our study unveiled the important role of CD24 inTNBC progression and metastasis via a novel mechanism ofCD24/Met/EGFR-mediated lymphangiogenesis and angiogen-esis in the tumor microenvironment. CD24 may be a potentialtherapeutic target for CD24-positive TNBC and other breastcancer types.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S.-H. Chan, L.-H. WangDevelopment of methodology: S.-H. Chan, S.-Y. Chiu, L.-H. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.-H. Chan, S.-Y. Chiu, W.-H. Kuo, H.-Y. Chen,K.-J. ChangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.-H. Chan, K.-W. Tsai, S.-Y. Chiu, S.S. JiangWriting, review, and/or revision of the manuscript: S.-H. Chan, L.-H. WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.-H. Chan, W.-C. HungStudy supervision: L.-H. Wang

AcknowledgmentsWe thank Taiwan Bioinformatics Institute Core Facility for assistance in

using Oncomine and NHRI Optical Biology Core Laboratory (NOBC) forconfocal microscopy analysis. This work was financially supported by the"Chinese Medicine Research Center, China Medical University" from theFeatured Areas Research Center Program within the framework of the HigherEducation Sprout Project by the Ministry of Education (to L.-H. Wang) in






Taiwan (CMRC-CHM-7). This study was also supported by the grants MOST-105-2320-B-039-067 and MOST-104-2320-B-039-055-MY3 awarded by theMinistry of Science and Technology (to L.-H. Wang).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 19, 2018; revised July 12, 2018; accepted October 23, 2018;published first October 31, 2018.

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2019;18:147-161. Published OnlineFirst October 31, 2018.Mol Cancer Ther Shih-Hsuan Chan, Kuo-Wang Tsai, Shu-Yi Chiu, et al.

CancerRegulator and Therapeutic Target for Triple-Negative Breast Identification of the Novel Role of CD24 as an Oncogenesis

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Identiﬁcation of the Novel Role of CD24 as an Oncogenesis ...Oncogenesis Regulator and Therapeutic Target for Triple-Negative Breast Cancer Shih-Hsuan Chan1,2,3, Kuo-Wang Tsai4,5,