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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.
CancerResearch
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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.
TNC Modulates CD47 Loss-Mediated Phagocytosis
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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
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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
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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.
Ma et al.
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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.
TNC Modulates CD47 Loss-Mediated Phagocytosis
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-
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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