extracellular matrix protein tenascin c increases ...tumor biology and immunology extracellular...

Tumor Biology and Immunology

Extracellular Matrix Protein Tenascin C IncreasesPhagocytosis Mediated by CD47 Loss of Functionin GlioblastomaDing Ma1,2, Senquan Liu3, Bachchu Lal1,2, Shuang Wei1,2, Shuyan Wang1,2, Daqian Zhan1,2,Hao Zhang4, Richard S. Lee5, Peisong Gao6, Hernando Lopez-Bertoni1,2, Mingyao Ying1,2,Jian Jian Li7, John Laterra1,2,8,9, Mary Ann Wilson1,2, and Shuli Xia1,2

Abstract

Glioblastomas (GBM) are highly infiltrated by myeloid-derived innate immune cells that contribute to the immuno-suppressive nature of the brain tumor microenvironment(TME). CD47 has been shown to mediate immune evasion,as the CD47–SIRPa axis prevents phagocytosis of tumor cellsby macrophages and other myeloid cells. In this study, weestablished CD47 homozygous deletion (CD47�/�) inhuman and mouse GBM cells and investigated the impact ofeliminating the "don't eat me" signal on tumor growth andtumor–TME interactions. CD47 knockout (KO) did not sig-nificantly alter tumor cell proliferation in vitro but significantlyincreased phagocytosis of tumor cells by macrophages incocultures. Compared with CD47wild-type xenografts, ortho-topic xenografts derived from CD47�/� tumor cells grewsignificantly slower with enhanced tumor cell phagocytosisand increased recruitment ofM2-like tumor-associatedmicro-glia/macrophages (TAM). CD47 KO increased tumor-associ-ated extracellular matrix protein tenascin C (TNC) in xeno-

grafts, which was further examined in vitro. CD47 loss offunction upregulated TNC expression in tumor cells via aNotch pathway–mediated mechanism. Depletion of TNC intumor cells enhanced the growth of CD47�/� xenografts in vivoand decreased the number of TAM. TNC knockdown alsoinhibited phagocytosis of CD47�/� tumor cells in cocultures.Furthermore, TNC stimulated release of proinflammatoryfactors including TNFa via a Toll-like receptor 4 and STAT3-dependent mechanism in human macrophage cells. Theseresults reveal a vital role for TNC in immunomodulation inbrain tumor biology and demonstrate the prominence of theTME extracellularmatrix in affecting the antitumor function ofbrain innate immune cells.

Significance: These findings link TNC to CD47-drivenphagocytosis and demonstrate that TNC affects the antitumorfunction of brain TAM, facilitating the development of novelinnate immune system–based therapies for brain tumors.

IntroductionGliomas account for 70% of all adult brain tumors. Grade IV

astrocytoma, glioblastoma (GBM), is the most common andaggressive primary brain tumor, accounting for approximately50% of all glial tumor types. Because of tumor heterogeneity

and the blood–brain barrier, GBM remains refractory to currenttreatment modalities including surgery, radiotherapy, and che-motherapy; the median survival time of patients with GBM isapproximately 15 to 20 months (1). Recent progress in immu-notherapy-based treatment options in other tumor types hasencouraged interest in developing similar approaches that mightbe effective for this devastating malignancy (2). However, currentimmunotherapies have not yet improved the survival of GBMpatients (3). Understanding how tumor interacts with brainimmune systems, including the innate immune system, anddeveloping improved therapeutic options for GBM are urgentlyneeded.

Innate immune cells, such as microglia, macrophages, andmyeloid-derived suppressor cells, are known to be present withinGBM. Tumor-associated microglia/macrophages (TAM) contrib-ute to 30% to 50%of brain tumormass (4, 5) and are educated bytumor cells to acquire a tumor-promotingM2-like phenotype thatis able to produce anti-inflammatory and immune-suppressivefactors in the tumor microenvironment (TME). In GBMs, TAMswith different phenotypes coexist, including an antitumor, proin-flammatory M1-like phenotype. The activation status rather thanthe abundance of TAMs present in the TME has been shown tohave prognostic value (6).

TME can influence the properties of TAMs (7). Extracellularmatrix (ECM) is an important component of the TME and is

1Hugo W. Moser Research Institute at Kennedy Krieger, Baltimore, Maryland.2Department of Neurology, Bloomberg School of Public Health, Johns HopkinsSchool of Medicine, Baltimore, Maryland. 3Department of Medicine, BloombergSchool of Public Health, Johns Hopkins School of Medicine, Baltimore, Maryland.4Department of Molecular Microbiology and Immunology, Bloomberg School ofPublic Health, Johns Hopkins School of Medicine, Baltimore, Maryland. 5Depart-ment of Psychiatry and Behavioral Sciences, Johns Hopkins School of Medicine,Baltimore, Maryland. 6Asthma and Allergy Center, Johns Hopkins School ofMedicine, Baltimore, Maryland. 7Department of Radiation Oncology, Universityof California Davis, Sacramento, California. 8Department of Neurosurgery, JohnsHopkins School of Medicine, Baltimore, Maryland. 9Department of Oncology,Johns Hopkins School of Medicine, Baltimore, Maryland.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Shuli Xia, HugoW.Moser Research Institute at KennedyKrieger/Johns Hopkins School of Medicine, 707 N. Broadway, Room 400K,Baltimore, MD 21205. Phone: 443-923-9498; Fax: 443-923-2695; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-18-3125

�2019 American Association for Cancer Research.

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composed of a complex mixture of macromolecules includingglycoproteins, proteoglycans, and polysaccharides. A body ofevidence indicates that in addition to their canonical role inmaintaining and regulating tissue organization, ECM compo-nents function as signaling molecules by interacting with mem-brane-bound receptors to regulate cell growth, motility, andimmune response (8). Although tightly controlled during embry-onic development and organ homeostasis, these ECM-mediatedpathways are commonly deregulated and disorganized in cancer.How ECM regulates immune response of brain tumors is largelyunknown.

In this study, we usedCD47 knockout (KO) human andmouseglioma models to investigate how ECM modulates the interac-tions between brain tumor cells and the innate immune system.CD47, also known as integrin-associated protein, is a ubiquitous50 kDa membrane-bound protein consisting of a single N-termi-nal IgV extracellular domain, five membrane-spanning segments,and a short C-terminal cytoplasmic tail (9). CD47 has beenreported to mediate immune evasion by interacting with thesignal regulatory protein-alpha (SIRPa) expressed on macro-phages and other myeloid cells (10). CD47 binding to SIRPacauses phosphorylation of the SIRPa cytoplasmic immunorecep-tor tyrosine-based inhibitionmotifs, leading to the recruitment ofSrc homology 2 domain–containing tyrosine phosphatases,which prevents myosin-IIA accumulation at the phagocytic syn-apse and consequently inhibits phagocytosis (11). Thus, theCD47–SIRPa axis, also known as a "don't eat me" signal, func-tions as a negative checkpoint for innate immunity. CD47 expres-sion is elevated in many cancers; The Cancer Genome Atlas dataanalysis indicated that high CD47 expression in GBM correlateswith poor patient survival (12). Blocking the CD47/SIRPa inter-action facilitates phagocytosis and inhibits tumor growth inmanypreclinical cancer models (12, 13); for example, the administra-tion of anti-CD47 mAbs reduced tumor growth and preventedlung cancer progression (14).

Abundant evidence supports that the signaling functions ofCD47 gowell beyond this passive antiphagocytic role, with CD47acting as a sensor for cell–microenvironment signals. As anexample,CD47 interactswithother signalingmolecules includingthematricellular glycoprotein thrombospondin-1 (TSP-1; refs. 15,16) to regulate various cellular functions including cellmigration,axonextension, cytokineproduction, andT-cell activation(17–19).Therefore, it is critical to dissect the signaling network of CD47 intumor cells and tumor cell–immune cell interactions. The currentstudy in GBMmodels is aimed to understand how TME influenceshost response to tumor cells carrying CD47 KO. We found thatCD47 KO dramatically increased tumor-associated ECM proteintenascin C (TNC) in vitro and in vivo. Our results demonstrate theimportance of the ECM protein in the antitumor function of braininnate immune cells.

Materials and MethodsReagents and cell cultures

All reagents were purchased from MilliporeSigma unless oth-erwise stated.HumanGBMcellsU87,mouse glioma cellsGL-261,and human monocyte cells THP-1 were original purchased fromthe ATCC. All cell lines are free from Mycoplasma and authenti-cated with short tandem repeat profiling by Johns HopkinsGenetic Resources Core facility using Promega GenePrint 10system.

Generation of CD47 KO cell lines by CRISPR-Cas9 systemCas9-GFP plasmid was purchased from Addgene. For targeting

CD47, two gRNAs targeting CD47 were cloned into pX330M(Addgene) according to the addgene cloning protocol. HumanCD47 gRNA targeting sequences were: 50-CGACCGCCGCCGCGCGTCACAGG (intron) and 50-CAGCAACAGCGCCGCTAC-CAGGG (first exon). Mouse CD47 gRNA targeting sequenceswere: 50-cccttgcatcgtccgtaatgtgg (intron) and 50-cagtagttttctttacgt-taagg (first exon). Glioma cells (2 � 105 per well in 6-well plate)were cotransfected with the two gRNA plasmids and Cas9-GFPusing lipofectamine 3000. After 2 days, transfected cells weresorted by flow cytometry (GFPþ) and subcloned. Genomic DNAwas extracted from clonal cells and amplified using the primer setas follows: for human: forward: 50-GTCTGGAGCCTGCGACTG;reverse: 50-GTGTGTGCATTTGGAGATGG; for mouse: forward: 50-gtctactggctggtgtgcaa; reverse: 50-catcgcgcttatccattttc. Sangersequencing of the PCR products was performed to screen CD47genomic KO.

Preparationof cell lineswithTNCknockdownusing shRNAandlentivirus system

To knock down TNC expression, lentivirus containing a controlnonsilencing (NS) sequence or TNC shRNA in a GIPZ viral vector(Thermo Fisher Scientific) containing the GFP coding frame wasintroduced into cells (20).

Cell migration assayMigration was quantified by Boyden chamber transwell assays

(8-mm pore size; Corning Costar) following our publishedwork (21, 22).

Quantitative real-time PCRTotal RNA was extracted using the RNeasy Mini Kit (Qiagen).

After reverse transcription using cDNA reverse transcriptase(Applied Biosystems) and Oligo(dT) primer, quantitative real-time PCR (qRT-PCR) was performed using SYBR Green PCR Mix(Applied Biosystems) and IQ5 detection system (Bio-Rad). Prim-er sequences used in this studywere listed in Supplementary TableS1. Relative gene expression was normalized to GAPDH.

ImmunoblotProteins were detected and quantified using the Odyssey Infra-

red Imager (LI-COR Biosciences) with secondary antibodieslabeled by IRDye infrared dyes (LI-COR Biosciences) and nor-malized toGAPDHor b-actin following our publishedwork (20).The antibodies used for this studywere listed below,most of themfrom Cell Signaling Technology unless otherwise stated: CD47(Santa Cruz Biotechnology); TNC (mouse and human, Millipor-eSigma); TNC (human only, Santa Cruz Biotechnology); STAT-3;phospho-STAT-3; Akt; phosphor-Akt; Jagged-1; NOTCH1; NICD;b-actin (MilliporeSigma); GAPDH (MilliporeSigma).

ELISA of TNFaHumanmonocyte cells THP-1 were seeded onto 12-well plates

(1.5 � 105/well) and incubated with phorbol 12-myristate13-acetate (PMA, 25 ng/mL) for 48 hours to introduce differen-tiation. Cells were treated with TNC protein at 1, 3, and 10 mg/mLfor another 8 hours in serum-free medium, and the supernatantwas collected for TNFa measurement using a TNFa ELISA kit(R&D Systems). TNC was purchased from Millipore and purifiedfrom the conditioned medium of human U251 GBM cell line by

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chromatography. All measurements were conducted according tothe manufacturer's protocol using a microplate spectrophotom-eter (Molecular Devices). For some experiments, cells were incu-bated with STAT3 inhibitor (Stattic, 5 mmol/L) or TLR-4 inhibitor(TAK242, 10 mmol/L) for 1 hour prior to TNC treatment.

In vitro phagocytosis assayTHP-1 cells were seeded onto 12-well plates (1.5 � 105/well)

and incubated with PMA (25 ng/mL) for 48 hours to inducedifferentiation. Cancer cells were labeled with carboxyfluoresceinsuccinimidyl ester (CFSE; Thermo Fisher Scientific) following themanufacturer's protocol. For each experiment, CFSE-labeledtumor cells (3� 105) were added to macrophages and incubatedin a final volume of 1mL serum-free medium at 37�C for 2 hours.Macrophages were stained with CD11c-APC (Thermo FisherScientific) for 30 minutes. Phagocytosis was assessed by flowcytometry (BD). Nonstained and CD11c-APC–stained THP-1cells were used for proper gating of flow cytometry analysis.

Tumor xenografts and immunofluorescent imagesFor intracranial xenografts, 8-week-old female SCID (NCI)

received 100,000 viable CD47 WT or CD47�/� U87 cells in 2 mLof PBS by stereotactic injection into the right caudate/putamen.Mice were sacrificed approximately 3 to 4 weeks after implanta-tion, and tumor volumes were estimated based on the formula:

vol ¼ (sq. root of maximum cross-sectional area)3 (23). Allanimal protocols used in this study were approved by the JohnsHopkins School of Medicine Animal Care and Use Committee.

Immunofluorescent staining of tumor sections was performedfollowing the protocol in Wu and colleagues (24). The primaryantibodies used for immunofluorescent staining were as follow-ing: Iba-1 (Wako, ThermoFisher); iNOS (ThermoFisher); TGM2(Cell Signaling Technology); Arginase-1 (Cell Signaling Technol-ogy). Immunofluorescent images were taken under fluorescentmicroscopy and analyzed using Axiovision software (Zeiss).Fluorescent microphotographs were taken, and positive stainingswere manually counted or quantified by ImageJ (NIH).

Statistical analysisStatistical analysis was performed using Prism software

(GraphPad). Post hoc tests included the Student t test and Tukeymultiple comparison tests as appropriate. Data are representedas mean value � SEM, and significance was set at P < 0.05.

ResultsCD47 KO increases glioma cell phagocytosis

We employed the CRISPR-Cas9 technique to completelyknockout CD47 expression in human GBM cells to investigatethe effect of CD47 loss of function on phagocytosis and tumor

Figure 1.

Establish CD47 KO human GBM cells bygenome editing. A, Schematic graph ofgenome editing strategy with two gRNAsto knockout CD47. B, PCR product fromgenomic DNA of selected clones showingheterozygous and homozygous deletionof CD47. C, Sanger sequencing of clone21 showing deletion of part of theCD47 coding sequence. D, Flowcytometry analysis with a CD47 antibodyindicated no CD47 expression on cellmembrane in CD47�/� cells.Representative data of threemeasurements. E, Immunocytostaining ofCD47 in control and CD47�/� cells. Bar,20 mm. F, CD47 KOminimally affected cellproliferation in vitro.G, CD47 KOdecreased cell migration of clone 7, buthad no effect on clone 21 (G), n¼ 3.H, Phagocytosis analysis of THP-1 cellscocultured with control and CD47�/�

cells. I,Quantification of phagocytosis rateof THP-1 cells against CD47WT andCD47�/� tumor cells. � , P < 0.05 and�� , P < 0.001; n¼ 6.

TNC Modulates CD47 Loss-Mediated Phagocytosis

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growth. Two guide RNAs (gRNA) separated by 108 bp anddesigned to target the first exon of CD47 were transfected intoU87 GBM cells (Fig. 1A). In all, approximately 20 clones wereselected and analyzed via PCR for CD47 KO using a primer pairflanking the two gRNA target sites. Clones with heterozygousCD47deletion produced twoPCRproducts of 588 bp and480 bp,and homozygous deletion generated one 480 bp PCR product(Fig. 1B). Sanger sequencing confirmed the homozygous deletionof the 108 bp targetedCD47 genomic sequence in cells from clone7 and clone 21 (CD47�/�, Fig. 1C). Cell surface CD47 expressionwas absent in these twoCD47�/� clonal lines asmeasuredbybothflow cytometry (Fig. 1D) and immunofluorescence (Fig. 1E).

Cell monolayer proliferation assays showed no significantdifference in cell growth between CD47�/� cells and CD47wild-type (WT) control cells (Fig. 1F). Transwell migration assaysrevealed that CD47 KO did not have a consistent effect on tumor

cell migration, with clone 7 CD47 KO cells migrating slower thanCD47 WT cells, but clone 21 similar to that of control (Fig. 1G).

Human monocyte cells THP-1 were differentiated into macro-phages by PMA (25ng/mL, 48–72hours) andused to evaluate theeffect of CD47 KO on phagocytosis of U87 cell. CD47 WT andCD47�/� cells were labeled with CFSE, which covalently reactswith amine-containing residues of intracellular proteins. LabeledU87 cells were cocultured with differentiated THP-1 cells for 2hours. The mixture was harvested and stained with the macro-phage-specific antibody CD11c conjugated with allophycocyanin(APC). Flow cytometry analysis was employed to detect CD11cþ/CFSEþ macrophages, indicative of U87 phagocytosis. The phago-cytosis index was calculated as the percentage of CD11cþ THP-1cells that were also CFSEþ (red boxes in Fig. 1H and I). Cocultur-ing U87 WT cells with THP-1 cells revealed a baseline phagocy-tosis index of approximately 9.5%. CD47 KO increased the

Figure 2.

Effect of CD47 KO on tumor growth. A,Representative H&E staining of xenografts derivedfrom control and CD47�/� cells. Bar, 500 mm.B,Quantification of the size ofWT and CD47�/�

xenografts. C, Immunofluorescent staining confirmedthat CD47 expression was eliminated in CD47�/�

xenografts. Bar, 20 mm. D and E, Ki67 staining andquantification inWT and CD47�/� xenografts.Bar, 20 mm. F, Representative microphotographs ofH&E staining of well-demarcated margins in WTtumors (left) and irregular CD47�/� tumor margins(right). Bar, 100 mm. G, Xenografts wereimmunostained with an antibody against humannuclear–specific antigen to show tumor margins. Bar,200 mm. H and I, Laminin staining to show bloodvessels in control and CD47�/� xenografts.Bar, 100 mm. �� , P < 0.001. In vivo experiments wererepeated once.N¼ 8 in total.

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phagocytosis index by 3–4 fold to 39% and 31% in clones 7 and21, respectively (Fig. 1I, n¼ 6, P < 0.001). This result is consistentwith an antiphagocytosis function of CD47 expression on tumorcells.

CD47 loss of function decreases GBM xenograft growthWe examined how CD47 KO affected GBM xenograft growth.

U87WT or CD47�/� cells (100,000) were separately injected intothe caudate/putamen of Balb/c immunodeficient (SCID) mice(experiments were repeated once, n ¼ 8 total); animals weresacrificed approximately 4 weeks after implantation, and brainsections were stained with hematoxylin and eosin (H&E; Fig. 2A).Control tumors were substantially larger with an average estimat-ed volume of 40 mm3 compared with xenografts from CD47�/�

clones 21 and 7 that were approximately 5.7 mm3 and 1.3 mm3

after 4 weeks, respectively (Fig. 2B, P < 0.001). CD47 expressionwas confirmed to be low in tumors derived from CD47�/� cells(Fig. 2C). The proliferation rate of tumor cells was examined byKi67 staining, and no significant difference between WT andCD47�/� tumors was found (48% vs. 40%, Fig. 2D and E). Closerexamination of the xenografts revealed histopathologic differ-ences between the control and CD47�/� tumors. Control tumorsdisplayed well-demarcated tumor margins as revealed by H&Estaining (Fig. 2F, left), and immunofluorescence staining with ananti-human nuclear–specific antigen antibody (Fig. 2G, left). Incomparison, CD47�/� xenografts had very irregular margins(Fig. 2F and G, right plots). Furthermore, when similar-sizedcontrol and CD47�/� xenografts were compared, we found fewerblood vessels in CD47�/� xenografts (Fig. 2H and I, P < 0.001),whichmay limit tumor growth. IHC staining for cleaved caspase 3revealed no differences in tumor cell apoptosis between controland CD47�/� xenografts (Supplementary Fig. S1A and S1B).

These findings, in conjunction with our results showing thatCD47 KO minimally affected the growth rate and migration ofU87 cells while increasingmacrophage phagocytosis of tumor cellin vitro, led us to hypothesize that the substantial differences inWTand CD47�/� in vivo growth patterns resulted from differences ininteractions between tumor cells and innate immune cells.

CD47 loss of function recruits more TAMsThe glioma-associated innate immune system, the main con-

stituents of which are TAMs, remains intact in SCID mice. Weexamined TAMs in CD47 WT and CD47�/� xenografts usingspecific markers. Immunofluorescence staining of the generalmicroglial/macrophage marker Iba-1 revealed that consistentwith reports from others (25), TAMs formed a dense bandsurrounding the WT tumors and appeared sparsely within thetumors (Supplementary Fig. S2). Themorphology of TAMs in thetumor core resembled amoeboid-like microglia/macrophageswith stout processes (Fig. 3A, top plots). In CD47�/� xenografts,the morphology of TAMs in the tumor core was similar to that ofcontrols, but we observed an increase in the density of TAMs inCD47KO xenografts (Fig. 3A, bottomplots). The average numberof Iba-1þ cells per microscopic field was 105 in CD47�/� xeno-grafts, almost 2-fold higher than that of control (Fig. 3B, P < 0.05).

CD47 antibodyhas been shown todriveM2 toM1polarizationof macrophages in vitro (26). We asked if CD47 KO induces anincrease in M1-like TAMs in GBM orthotopic xenografts. Expres-sion of M1 marker iNOS (27) as well as M2 markers arginase 1(Arg-1; ref. 28) and transglutaminase 2 (Tgm2; ref. 29) wasexamined by immunofluorescence to evaluate relative numbersof M1 and M2 macrophages in orthotopic WT and CD47�/�

xenografts. We found very few cells expressing the M1 markeriNOS in the control andCD47�/� xenografts (Supplementary Fig.

Figure 3.

Distribution of TAMs in xenografts.A,Microglial/macrophage marker Iba-1 staining indicated higher density of TAMs in CD47�/� xenografts. B,Quantification ofIbaþ cells per microscopic field in control and CD47�/� xenografts. C and D, Costaining of the M2marker Arg-1 (red) and Iba-1 (green) in xenografts andquantification of Arg-1þ cells per field. E, Double staining of TAMs (Iba-1þ, green) and tumor cells (HuNuþ, red) showing host immune cells with human tumornuclei (arrows) in CD47�/� xenografts. F, Confocal microscopic imaging of the double staining of Iba-1 and HuNu in a CD47�/� xenograft showing the twomarkers were from the same cells (arrows).G,Quantification of Iba-1þ cells with HuNuþ staining per microscopic field. � , P < 0.05; n¼ 8; bar, 20 mm.






S3A). On the other hand, the xenografts were infiltrated with M2-like TAMs as evidenced by Arg-1, which costained with Iba-1(Fig. 3C). The average number of Arg-1þ TAMs per microscopicfield was significantly increased by CD47 KO, from 22 in WTtumors to 48 in CD47�/� tumors (Fig. 3D, P < 0.05). Another M2marker TGM2also increased by approximately 3-fold inCD47�/�

xenografts (Supplementary Fig. S3B).To determine if CD47 KO stimulated phagocytosis in vivo, we

costained brain sections with Iba-1 (Fig. 3E, green) and humannuclear–specific antigen (Fig. 3E, HuNu, red). Confocal micro-scopic analysis demonstrated the engulfment of tumor cells bymicroglia/macrophages (Fig. 3F, arrows).We counted total Iba-1þ

cells and cells double labeled with Iba-1 and HuNu. In CD47�/�

xenografts, an average of approximately 5.1% Iba-1þ cells werealso positive for HuNu in the nuclei; in contrast, Iba-1 and HuNudouble-stained cellswere rare inWTxenografts (Fig. 3G,P

NICD, were increased 1.9- to 4.7-fold in CD47�/� cells in com-parison with WT cells (Fig. 4F). Jagged-1 and NOTCH1 mRNAlevels were also upregulated in CD47�/� cells (Fig. 4G). Treatingcells with the specific Notch pathway inhibitor DAPT (N-[N-(3,5-Diflurophenaacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester)decreased TNC protein level in CD47�/� cells but not in WT cells(Fig. 4H), suggesting that Notch signaling was a driver of TNCupregulation in the CD47�/� cells.

TNC is also upregulated in mouse glioma cells with CD47 KOTo determine whether our major finding in SCID mice also

applies to immunocompetent animal models, we knocked outCd47 in mouse glioma GL261 cells using genome editing andgRNAs targeting mouse Cd47 exon 2 (Supplementary Fig. S4A).After subcloning and genotyping, we obtained GL261 cells withCD47 KO. Shown in Supplementary Fig. S4B is the Sangersequencing of the PCR product from genomic DNA of GL261clone 29with a deletion of 161 bp at theCd47 exon 2. Cell surfaceCD47 expression was absent in clone 29 as measured by flowcytometry (Fig. 5A). CD47 KO did not alter GL261 proliferationand migration. Phagocytosis analysis with THP-1 cells confirmedthat CD47KO increased the phagocytosis index by approximately2-fold from 19% to 40.1% in mouse glioma cells (Fig. 5B,P

cytoskeleton regulation (31, 32), we hypothesized that this func-tion of TNC reflected changes in tumor cell phagocytosis by TAMs.Indeed, we found TNCKD inhibited phagocytosis triggered byCD47 KO in vitro (Fig. 7A). As shown before, CD47 KO increasedphagocytosis of control U87 cells by approximately 3- to 4-fold,from 15.2% to 49.1%. TNCKD significantly decreased phagocy-tosis induced by CD47KO from49.1% to 28% (Fig. 7B, P < 0.05).We also noticed that compared with U87 CD47WT cells, TNCKDdecreased the baseline phagocytosis from 15.2% to 8.1%, whichmay partially explain the increased tumor size from CD47 WTþTNCKD cells. Our findings suggest that elevated expression ofTNC in CD47�/� cells promoted phagocytosis, and TNC expres-

sion in tumor cells could contribute to baseline phagocytosisindependent of CD47.

Because TNC is upregulated in response to inflammation (33),we hypothesized that TNC may elicit other immunomodulationfunctions to facilitate tumor cell–immune cell interactions. Tothis end, we studied TNC gain of function using exogenous TNC.THP-1 cells were treated with human TNC (1–10 mg/mL) for 8hours followed by RT-PCR to measure the expression level ofseveral proinflammatory factors including IL1b, IL6, and TNFa.Cells treated with lipopolysaccharides (LPS) were used as apositive control. Real-time quantitative RT-PCR revealed a con-centration-dependent upregulation of these proinflammatory

Figure 6.

The effect of TNC loss of function on the antitumorfunction of CD47 KO.A, Knocking down TNCexpression inWT cells and clone 21 CD47�/� cellsusing TNC-specific shRNA (TNCKD). NS shRNAwas used as a control. B,Growth curve of TNCKDcells in comparison with their counterparts. C, H&Estaining of xenografts derived from CD47WT andCD47�/� cells harboring TNCKD. Bar, 500 mm.D,Quantification of tumor size. E, Staining ofhuman-specific TNC confirmed TNCdownregulation in xenografts derived from cellsreceiving TNC shRNA. Bar, 100 mm. F, Staining ofthe microglial/macrophage marker Iba-1 in TNCKDxenografts. Bar, 20 mm. G,Quantification of thenumber of Iba-1þ cells in control and CD47�/�

xenografts with and without TNCKD. TNCKDdecreased Iba1þ cells in CD47�/� xenografts.� , P < 0.05; n¼ 5.

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factors (Fig. 7C, P < 0.05). ELISA of THP-1 conditioned mediumrevealed concentration-dependent increase in TNFa productiondriven by TNC (Fig. 7D, P < 0.05). We performed IHC staining of

TNFa in CD47 WT � NS and CD47�/� � TNCKD xenografts.Consistent with our in vitro studies, our in vivo staining indicatedthat CD47 KO increased TNFa staining by 63%, which was

Figure 7.

Effect of TNC on phagocytosis and macrophagecells. A, TNCKD in tumor cells decreasedphagocytosis of WT and CD47�/� cells bymacrophages. B,Quantification of phagocytosis,n¼ 3. C, RT-PCR indicated that exogenous TNCinduced expression of proinflammatory genesincluding IL1b, IL6, and TNFa in THP-1 cells in adose-dependent manner. LPS was used as apositive control, n¼ 6. D, ELISA showed TNCincreased TNFa secretion in THP-1 cells in a dose-dependent manner. n¼ 3. E, IHC staining of TNFain CD47WT� NS and CD47�/� � TNCKDxenografts. Methyl green (green) was used tocounterstain nuclei. Bar, 20 mm. F,Quantification ofpercentage of cells with TNFa staining in thexenografts. n¼ 5. G, Immunoblot analysis indicatedthat TNC treatment activated STAT3 in THP-1 cells,which was blocked by the STAT3 inhibitor stattic.H, Stattic prevented TNFa production in THP-1 cellstreated with TNC. n¼ 3. I, TLR4 inhibitor TAK242blocked STAT3 activation in THP-1 cells in responseto TNC. J, TAK242 decreased TNC-induced TNFaproduction in THP-1 cells. n¼ 3. �, P < 0.05.






reversed when TNC was knocked down (Fig. 7E and F, P < 0.05).This result indicated that TNCupregulation driven byCD47KO isinvolved in the increased cytokine production.

STAT3 was activated in THP-1 cells following TNC treatment(Fig. 7G). The STAT3-specific inhibitor stattic (5 mmol/L) pre-vented STAT3 activation in TNC-treated THP-1 cells and inhibitedTNFa production induced by TNC (Fig. 7H, P < 0.05). Preincu-bating THP-1 cells with the Toll-like receptor 4 (TLR4) inhibitorTAK242 (10 mmol/L) also inhibited TNC-induced STAT3 activa-tion andTNFaproduction (Fig. 7I and J,P

phagocytosis, it is appealing to think that instead of being apromoter of tumormalignancy, the elevated TNC in brain tumorsmay be a defensive mechanism by the innate system to try toeliminate malignant cells.

Over thepast years, the useof immune-checkpoint inhibitors totarget the adaptive immune systems has been one of the mostsignificant advances in antitumor treatment. However, theincreased use of these agents has led to an augmented apprecia-tion that theywerenot suitable for all patients. Recent studies haveindicated that the innate immune system checkpoint such asCD47 may be an interesting therapeutic target (44). A bettermechanistic understanding of CD47 signaling network will aidin the development of novel antitumor reagent. Our study pro-vides the first evidence demonstrating that microenvironmentalTNC is involved in phagocytosis of tumor cell by TAMs. Futurestudies of the precise mechanism by which TNC regulates phago-cytosis will shed light on how to exploit the innate immunesystem to target brain tumors.

Disclosure of Potential Conflicts of InterestJ. Laterra has done expert testimony for Alston & Bird. No potential conflicts

of interest were disclosed by the other authors.

Authors' ContributionsConception and design: S. Xia, S. Liu, P. Gao, J. LaterraDevelopment of methodology: D. Ma, S. Liu, B. Lal, S. Wang, J.J. Li,M.A. Wilson, S. XiaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Ma, S. Liu, H. Zhang, R.S. Lee, M. Ying,Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Ma, M. Ying, J.J. Li, S. XiaWriting, review, and/or revision of the manuscript: D. Ma, S. Liu, H. Lopez-Bertoni, M. Ying, J. Laterra, M.A. Wilson, S. XiaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D. Ma, D. Zhan, S. XiaStudy supervision: P. Gao, S. Xia

AcknowledgmentsThis work was supported by grants from NIH/NINDS R01 NS091165

(S. Xia), R01 NS099460 (M. Ying), R01 NS096754 (J. Laterra), and R01NS076759 (J. Laterra).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedOctober 4, 2018; revised January 30, 2019; acceptedMarch18, 2019;published first March 21, 2019.

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Ma et al.




2019;79:2697-2708. Published OnlineFirst March 21, 2019.Cancer Res Ding Ma, Senquan Liu, Bachchu Lal, et al. Mediated by CD47 Loss of Function in GlioblastomaExtracellular Matrix Protein Tenascin C Increases Phagocytosis

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