CD47 update: a multi-faceted actor in the tumour microenvironment of potential therapeutic interest.
ABSTRACT CD47 is a ubiquitous 50-kDa five-spanning membrane receptor that belongs to the immunoglobulin superfamily. This receptor, also known as IAP (integrin-associated protein), mediates cell-to-cell communication by ligation to transmembrane signal-regulatory proteins SIRPα and SIRPγ and interacts with integrins. CD47 is also implicated in cell-extracellular matrix interactions via ligation with thrombospondins (TSPs). Furthermore, CD47 is involved in many and diverse cellular processes, including apoptosis, proliferation, adhesion and migration. It also plays a key role in many immune and cardiovascular responses. Thus, this multi-faceted receptor might be a central actor in the tumour microenvironment. Solid tumours are composed of not only cancer cells that actively proliferate but also other cell types including immune cells and fibroblasts that make up the tumour microenvironment. Tumour cell proliferation is strongly sustained by continuous sprouting of new vessels, which also represents a gate for metastasis. Moreover, infiltration of inflammatory cells is observed in most neoplasms. Much evidence has accumulated indicating that infiltrating leukocytes promote cancer progression. Given its ubiquitous expression on all the different cell types that compose the tumour microenvironment, targeting CD47 could represent an original therapeutic strategy in the field of oncology. We present a current overview of the biological effects associated with CD47 on cancer cells and stromal cells. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.
- SourceAvailable from: Chinten James Lim[Show abstract] [Hide abstract]
ABSTRACT: 4N1K is a peptide fragment derived from the C-terminal, globular domain of thrombospondin which has been shown to mediate integrin-dependent cell adhesion and promote integrin activation acting via the cell-surface receptor, CD47. However, some studies found that 4N1K could act independently of CD47, putting in question the specificity of 4N1K for CD47. This led us to characterize the cellular and non-cellular effects of 4N1K. We found that 4N1K stimulated a potent increase in binding of a variety of non-specific IgG antibodies to cells in suspension. We also found that these same antibodies, as well as CD47-deficient cells, could bind substrate-immobilized 4N1K significantly better than a control peptide, 4NGG. Furthermore, we found that cells treated with 4N1K at higher concentrations inhibited, while lower concentrations promoted cell adhesion to immobilized fibronectin as an integrin substrate. Importantly, both the stimulatory and the inhibitory activity of 4N1K occurred as efficiently in the CD47-deficient JinB8 cells, as it did in the CD47-expressing parental or in JinB8 cells reconstituted with CD47 expression. Given these results, we suggest that 4N1K interacts non-specifically with epitopes commonly found on the cell surface, and conclude that it is not a suitable peptide for use to study the consequences of CD47 receptor ligation.PLoS ONE 01/2014; 9(5):e98358. · 3.53 Impact Factor
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ABSTRACT: Introduction: CD47 is a ubiquitously expressed cell surface receptor that serves as a counter-receptor for SIRPα in recognition of self by the innate immune system. Independently, CD47 also functions as an important signaling receptor for regulating cell responses to stress. Areas covered: We review the expression, molecular interactions, and pathophysiological functions of CD47 in the cardiovascular and immune systems. CD47 was first identified as a potential tumor marker, and we examine recent evidence that its dysregulation contributes to cancer progression and evasion of anti-tumor immunity. We further discuss therapeutic strategies for enhancing or inhibiting CD47 signaling and applications of such agents in preclinical models of ischemia and ischemia/reperfusion injuries, organ transplantation, pulmonary hypertension, radioprotection, and cancer. Expert opinion: Ongoing studies are revealing a central role of CD47 for conveying signals from the extracellular microenvironment that limit cell and tissue survival upon exposure to various types of stress. Based on this key function, therapeutics targeting CD47 or its ligands thrombospondin-1 and SIRPα could have broad applications spanning reconstructive surgery, engineering of tissues and biocompatible surfaces, vascular diseases, diabetes, organ transplantation, radiation injuries, inflammatory diseases, and cancer.Expert Opinion on Therapeutic Targets 10/2012; · 4.90 Impact Factor
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ABSTRACT: CD47 is a "self marker" that is usually overexpressed on the surface of cancer cells to enable them to escape immunosurveillance. Recognition of CD47 by its receptor, signal regulatory protein α (SIRPα), which is expressed in the macrophages, inhibits phagocytic destruction of cancer cells by the macrophages. In this study, we have first shown that clinical isolates of human melanoma significantly upregulate CD47, possibly as a mechanism to defend themselves against the macrophages. We then exploited RNA interference (RNAi) technology to test the hypothesis that knocking down CD47 in the tumor cells will render them targets for macrophage destruction; hence, creating a novel anti-cancer therapy. Anti-CD47 siRNA was encapsulated in a liposome-protamine-hyaluronic acid (LPH) nanoparticle (NP) formulation to address the challenge of targeted delivery of siRNA-based therapeutics in vivo. Efficient silencing of CD47 in tumor tissues with systemic administration of LPH(CD47) also significantly inhibited the growth of melanoma tumors. In a lung metastasis model, LPH(CD47) efficiently inhibited lung metastasis to about 27% of the untreated control. Moreover, no hematopoietic toxicity was observed in the animals that received multiple doses of LPH(CD47). Our findings indicate CD47 as a potential prognostic marker for melanoma development as well as a target for therapeutic intervention with RNAi-based nanomedicines.Molecular Therapy (2013); doi:10.1038/mt.2013.135.Molecular Therapy 06/2013; · 7.04 Impact Factor
CD47 update: a
multifaceted actor in the
of potential therapeutic
E Sick1,2, A Jeanne1, C Schneider1, S Dedieu1, K Takeda2and L Martiny1
1CNRS, FRE 3481, Matrice Extracellulaire et Dynamique Cellulaire, Université de Reims
Champagne-Ardenne, Faculté des Sciences, Reims, France, and2CNRS, UMR 7213, Laboratoire
de Biophotonique et Pharmacologie, Université de Strasbourg, Faculté de Pharmacie Illkirch,
Dr Emilie Sick, CNRS, FRE 3481,
URCA, Moulin de la Housse, BP
1039, 51687 Reims Cedex 2,
21 July 2011
25 June 2012
28 June 2012
CD47 is a ubiquitous 50 kDa five-spanning membrane receptor that belongs to the immunoglobulin superfamily. This
receptor, also known as integrin-associated protein, mediates cell-to-cell communication by ligation to transmembrane
signal-regulatory proteins SIRPa and SIRPg and interacts with integrins. CD47 is also implicated in cell-extracellular matrix
interactions via ligation with thrombospondins. Furthermore, CD47 is involved in many and diverse cellular processes,
including apoptosis, proliferation, adhesion and migration. It also plays a key role in many immune and cardiovascular
responses. Thus, this multifaceted receptor might be a central actor in the tumour microenvironment. Solid tumours are
composed of not only cancer cells that actively proliferate but also other cell types including immune cells and fibroblasts that
make up the tumour microenvironment. Tumour cell proliferation is strongly sustained by continuous sprouting of new
vessels, which also represents a gate for metastasis. Moreover, infiltration of inflammatory cells is observed in most neoplasms.
Much evidence has accumulated indicating that infiltrating leukocytes promote cancer progression. Given its ubiquitous
expression on all the different cell types that compose the tumour microenvironment, targeting CD47 could represent an
original therapeutic strategy in the field of oncology. We present a current overview of the biological effects associated with
CD47 on cancer cells and stromal cells.
AML, acute myeloid leukaemia; CBD, cell-binding domain; DC, dendritic cell; Drp-1, dynamin-related protein-1; eNOS,
endothelial nitric oxide synthase; FAK, focal adhesion kinase; HIF, hypoxia-inducible factor; HSC, haematopoietic stem
cell; HSPGs, heparan sulfate proteoglycans; IAP, integrin-associated protein; ITIM, immunoreceptor tyrosine-based
inhibitory motif; LSC, leukaemia stem cell; mAb, monoclonal antibody; PCD, programmed cell death; PS,
phosphatidylserine; RBC, red blood cell; ROS, reactive oxygen species; SCID, severe combined immunodeficiency; SIRP,
signal-regulatory protein; Treg, T-regulatory cell; TSP, thrombospondin; VCAM-1, vascular cell adhesion molecule-1
It is now widely accepted that solid tumours are not solely
composed of proliferating aneuploïd cells particularly resist-
ant to cell death. Thus, solid tumours should be considered as
heterogeneous structures containing multiple distinct cell
types, including normal cells recruited into tissues surround-
British Journal of
British Journal of Pharmacology (2012) 167 1415–14301415
© 2012 The Authors
British Journal of Pharmacology © 2012 The British Pharmacological Society
cell-to-cell interactions and secretion of paracrine growth
factors. Together with the vascular network provided by ang-
iogenesis that ensures nutrition and oxygenation of tumours,
the ensemble of these actors defines the tumour microenvi-
ronment. In addition, cancer-associated fibroblasts have been
shown to enhance cancer cell proliferation, angiogenesis,
invasion and metastasis, especially in the context of chronic
inflammation (Tlsty and Coussens, 2006). Finally, many dif-
ferent types of immune cells are also found infiltrated in
tumour masses at early stages. Sustained inflammation is one
of the hallmarks of tumour promotion and correlates with
poor prognosis in many different types of cancer. These infil-
trating cells play key roles in both innate and acquired immu-
nity and include dendritic cells (DCs), macrophages, mast
cells, neutrophils, and T- and B-cells (Hanahan and Weinberg,
CD47 is an ubiquitously expressed membrane receptor
that has been implicated in many normal and pathophysi-
ological processes including apoptosis, proliferation, cell
adhesion, cardiovascular effects, inflammation and immu-
nity. Given this diversity of actions, CD47 appears as a
multifaceted receptor that might represent a key target
in the tumour microenvironment for development of
innovative therapeutic strategies against cancer. In this
review, we update the effects associated with activation or
inhibition of CD47 in both cancer cells and stromal cells,
paying particular attention to vascular cells and immune
CD47 structure and partners
CD47 is an ubiquitous 50 kDa membrane receptor that
belongs to the immunoglobulin superfamily (Frazier et al.,
2010). This receptor was identified through its association
with integrin avb3and was therefore initially called integrin-
associated protein (IAP; Brown et al., 1990). At that time, an
ovarian carcinoma antigen called OA3 was cloned (Campbell
et al., 1992) with a proposed structure having five putative
transmembrane domains, an extracellular N-terminal IgV
domain with five putative N-glycosylation sites and a
C-terminal intracellular tail characterized by alternative splic-
ing (Figure 1). A closely similar structure was concomitantly
proposed for IAP (Lindberg et al., 1993). The ovarian tumour
marker OA3 was subsequently shown to be the same protein
as IAP (Mawby et al., 1994). The OA3 marker was also found
not to be restricted to ovarian carcinoma, rather exhibiting a
larger distribution. CD47 was an orphan receptor until it was
reported that this protein was the receptor for the C-terminal
cell-binding domain (CBD) of thrombospondins (TSPs) (Gao
and Frazier, 1994). Later, structural analysis revealed that a
long-range disulfide bond between the extracellular and the
fourth membrane-spanning domains was required for CD47
ligand binding, signalling and localization into lipid rafts
(Rebres et al., 2001).
Four alternatively spliced forms of CD47 were identified
in both murine and human cells, with variants ranging in
length from 3 to 36 amino acids in the cytoplasmic tail and
Schematic representation of CD47 and its endogenous ligands TSP-1 and SIRP. CD47 is composed of five transmembrane domains, an extracellular
N-terminal IgV domain and an intracytoplasmic C-terminal alternatively spliced domain. TSP-1 comprises several different domains, including the
heparin-binding domain (HBD), the von Willebrand C domain (vWCD), three type 1 properdin repeats, three type 2 EGF-like repeats, seven type
3 calcium-binding repeats and the C-terminal CBD. Several domains of TSP-1 interact with different extracellular matrix components or membrane
receptors (arrows). Key amino acid sequences responsible for TSP-1 ligation to integrins (RGD) and CD47 (RFYVVMWK) are indicated in italics.
SIRPa and g are single transmembrane spanning proteins with 3 Ig-like motifs, including an extracellular IgV domain that interacts with the IgV
domain of CD47.
E Sick et al.
1416British Journal of Pharmacology (2012) 167 1415–1430
having a very high interspecies conservation with respect to
both peptide sequence of the alternatively spliced regions
and gene structure (Reinhold et al., 1995). The second-
shortest form 2 is the most abundantly expressed isoform
while the second most abundant isoform, form 4 (the
longest), is predominantly expressed in the brain and in the
peripheral nervous system; only keratinocytes expressed sig-
nificant amounts of form 1 (Reinhold et al., 1995). Little is
known about the functional significance of this alternative
splicing. Indeed, only a single study has focused on the
expression of these alternatively spliced isoforms: during
memory consolidation in rats, isoforms 3 and 4 are thought
to be closely associated with memory consolidation, while
isoform 2 appears to be the principal signal transducer in
astrocytes (Lee et al., 2000). Using a yeast model, post-
translational modifications such as glycosylation were shown
to benecessary formembrane
(Parthasarathy et al., 2006). However, it is still unknown to
what extent glycosylation influences CD47 ligand-binding
properties and subsequent signalling. Recently, a high
molecular weight isoform of CD47 was identified in Jurkat
cells (Kaur et al., 2011). In these cells, very little CD47 at its
expected molecular weight (55 kDa) was found. Rather, most
immunoreactivity in Western blots migrated with an appar-
ent molecular weight >250 kDa, consistent with CD47 also
being expressed as a proteoglycan, because this isoform was
sensitive to chondroitinase and heparitinase (Kaur et al.,
2011). The glycosaminoglycan residues at Ser64are involved
in CD47-mediated CD69 induction in T-cells. This high
molecular weight isoform is also apparently widely expressed
on vascular cells (Kaur et al., 2011).
The first identified endogenous ligand for CD47 was TSP-1
(Gao and Frazier, 1994). Using synthetic overlapping peptides
from the C-terminal CBD of TSP-1, a consensus sequence
containing the motif VVM was proposed to be essential for
binding to CD47. This resulted in the development of several
CD47 peptide agonists (Gao et al., 1996a), among which pep-
tides 7N3 (1102FIRVVMYEGKK1112), 4N1 (1016RFYVVMWK1024)
and the extended 4N1K (K-1016RFYVVMWK1024-K; i.e. 4N1
flanked by two Lys residues) are still the most widely used
experimentally. Nevertheless, it should be noted that, in at
least two studies, the CD47-binding peptides 4N1 and 4N1K
were reported to induce aggregation of platelets and to
enhance Jurkat cell adhesion in CD47-deficient cells (Tulasne
et al., 2001; Barazi et al., 2002), raising doubt as to the spe-
cificity of these peptides. Thus, results obtained with these
peptides alone [i.e. without confirmation using native TSP-1,
function-blocking anti-CD47 monoclonal antibodies (mAbs)
or CD47-null cells] should be interpreted cautiously because
it cannot be ruled out that these peptides might also bind to
another yet unidentified receptor. It should also be noted that
such controls have in general not been done in published
work using 4N1 or 4N1K.
TSP-1 belongs to a family of multidomain calcium-
binding glycoproteins that associate as homotrimers (TSP-1
and -2) or homopentamers (TSPs 3–5) in mammals. Most
adult tissues co-express one or more TSPs and major altera-
example, highly expressed levels of TSP-1 and -2 are com-
monly found in stromal fibroblasts and endothelial cells
within tumours (Streit et al., 1999; Hawighorst et al., 2001).
In addition to the C-terminal CD47-binding domain, TSP-1
has several other domains that bind to different cell mem-
brane receptors or extracellular matrix, thereby mediating
cell-cell and cell-extracellular matrix interactions (Figure 1).
The N-terminal domain contains a heparin-binding domain
that binds to heparan sulfate proteoglycans (HSPGs) and
integrin a4b1. TSP-1 also contains three types of repeats: type
1 properdin repeats that bind to CD36, collagen type V,
fibronectin and HSPGs; type 2 EGF-like repeats; and type 3
calcium-binding repeats that contain the arginine-glycine-
aspartate (RGD)-sequence involved in ligation of b3 integrin
subunits (Figure 1; Adams, 2001). To date, the X-ray structure
of the CD47/TSP-1 complex has not been resolved. However,
our molecular modelling study has contributed to under-
standing how the TSP-1 CBD interacts with CD47 (Floquet
et al., 2008). Observation of X-ray structure of the CBD of
TSP-1 shows that the 4N1 sequence is normally hidden
within a hydrophobic pocket, preventing any interaction. By
normal mode analysis and energy minimization, it was pro-
posed that the opening of the 4N1 hydrophobic cleft is
driven by large amplitude motions when in close proximity
to CD47 and the phospholipid bilayer. The main biological
effects induced by ligation of TSP-1 CBD to CD47 are sum-
marized in Table 1. Binding of TSP-1 to CD47 influences
several fundamental cellular functions including cell migra-
tion and adhesion, cell proliferation or apoptosis, and takes
part in the regulation of angiogenesis and inflammation.
The second identified endogenous ligand of CD47 was
SIRPa (Vernon-Wilson et al., 2000). This protein was alterna-
tively called BIT (Sano et al., 1997), p84 (Jiang et al., 1999),
SHPS-1 (Babic et al., 2000) or CD172a. As shown in Figure 1,
N-terminal and four tyrosine phosphorylation sites in the
tyrosine-based inhibitory motifs (ITIMs; Brooke et al., 1998).
Additionally, two other signal-regulatory protein (SIRP)
family members, SIRPb (also known as CD172b) and SIRPg
(also known as SIRPb2 and CD172g), have been characterized
in man (Kharitonenkov et al., 1997; Ichigotani et al., 2000).
The N-terminal IgV domain of SIRPa binds to the IgV domain
of CD47 (Vernon-Wilson et al., 2000) and the structure of the
CD47/SIRPa complex has been recently resolved by X-ray
crystallography (protein database code 2uv3; Hatherley et al.,
2008). In spite of the highly conserved structure for SIRP
family members, only SIRPa and SIRPg bind to CD47. Failure
of SIRPb to bind CD47 has been attributed at least in part to
the replacement of Val27 by Met in the IgV domain of SIRPa
(Hatherley et al., 2008). The structure of their cytoplasmic
regions is quite different. SIRPg has a very short cytoplasmic
tail like SIRPb and lacks a charged amino acid residue in its
transmembrane region preventing the association with the
adaptor protein DAP12. This molecular adaptor contains an
immunoreceptor tyrosine-based activation motif, thought to
be responsible for SIRPb-mediated intracellular signalling.
The ligand for SIRPb is still unknown. CD47 ligation of SIRPa
or SIRPg mediates bidirectional signalling responsible for dif-
ferent cell-to-cell effects including inhibition of phagocytosis,
stimulation of cell-cell fusion or T-cell activation (Table 1).
CD47 was initially characterized as associated with
avb3 (Brown et al., 1990) integrins. Since then, numerous
bears two immunoreceptor
CD47 in the tumour microenvironment
British Journal of Pharmacology (2012) 167 1415–14301417
Partners of CD47 and their associated actions
CD47 partnersMain biological and cellular eventsReferences
Apoptosis, cell proliferation, cell survival, cell adhesion,
inhibition of angiogenesis, pro- and anti-inflammatory
effects, platelet activation and aggregation
Dorahy et al. (1997); Manna and Frazier (2004);
Rath et al. (2006a); Lamy et al. (2007); Isenberg
et al. (2009); Xing et al. (2009); Sick et al. (2011)
Serpin A1 (C-terminal domain)
Cell proliferation Congote and Temmel (2004)
Inhibition of phagocytosis, stimulation of cell-cell fusion,
T-cell activation, neutrophil transepithelial migration
Han et al. (2000); Oldenborg et al. (2000); Seiffert
et al. (2001); Liu et al. (2002)
Leukocyte transendothelial migration, T-cell proliferation Piccio et al. (2005); Stefanidakis et al. (2008)
Direct partners (*demonstrated experimentally by immunoprecipitation or FRET in the case of VEGFR-2)
a2*COX-2 expression and intestinal epithelial cell migration
a2b1* Migration and proliferation of smooth muscle cells
a4b1* Adhesion of sickle reticulocytes, B-cell migration
Glycosaminoglycan synthesis by chondrocytes
Fibrillar beta amyloid-mediated microglia activation and
Neutrophil transepithelial migration
avb3* C32 human melanoma cells spreading,
pro-inflammatory cytokine synthesis in human
monocytes, Coxiella burnetii phagocytosis,
promyelocytic leukaemia cell death
Broom et al. (2009)
Chung et al. (1997); Wang and Frazier (1998)
Chung et al. (1997)
Yoshida et al. (2000); Brittain et al. (2004)
Holledge et al. (2008)
Bamberger et al. (2003); Koenigsknecht and
Hofman et al. (2000)
Gao et al. (1996a); Capo et al. (1999); Hermann
et al. (1999); Saumet et al. (2005)
VEGFR-2* Inhibition of VEGFR-2 downstream signallingKaur et al. (2010)
CD36 Amyloid b-induced inhibition of NO signalling, inhibition
of NO-stimulated vascular cell responses and cGMP
Isenberg et al. (2006); Miller et al. (2010a)
Fas/CD95Stimulation of Fas-mediated apoptosisManna et al. (2005)
Gi proteins* SykPlatelet activation (Lyn and FAK phosphorylation) Chung et al. (1997)
AC/PKAPlatelet aggregation (decrease in cAMP), smooth muscle
cell migration, T-cell death
Frazier et al. (1999); Wang et al. (1999); Manna and
ERKJurkat T-lymphoma cell adhesion (phosphorylation);
inhibition of smooth muscle cell migration
Wang et al. (1999); Wilson et al. (1999)
PI3K C32 melanoma cell spreading, T-cell migration,
astrocytoma cell proliferation
Gao et al. (1996b); Li et al. (2005); Sick et al.
PLIC-1* Jurkat T-cells spreading, regulation of heterotrimeric
Wu et al. (1999); N’Diaye and Brown (2003)
BNIP3*T-cell apoptosis Lamy et al. (2003)
RacNeurite and filopodium formation, neuronal
Miyashita et al. (2004); Murata et al. (2006)
Cdc42B-cell migration, neurite and filopodium formation,
Miyashita et al. (2004); Yoshida et al. (2000);
Murata et al. (2006
Src and MEK kinasesEpithelial cell spreading and migrationShinohara et al. (2006)
Drp1Caspase-independent cell death of normal and
Bras et al. (2007)
Protein 4.2Rh complex integrity on red blood cellsBruce et al. (2002)
GCInhibition of NO signalling (decrease in cGMP)Isenberg et al. (2006)
E Sick et al.
1418British Journal of Pharmacology (2012) 167 1415–1430
interactions between CD47 and many different integrin
subunits have been reported (Table 1). For example, in asso-
ciation with a2b1 integrins, CD47 mediates migration and
proliferation of smooth muscle cells (Wang and Frazier,
1998). In cooperation with avb3, CD47 mediates diverse func-
tions such as cell spreading, cytokine synthesis, phagocytosis
and cell death (Gao et al., 1996b; Capo et al., 1999; Hermann
et al., 1999; Saumet et al., 2005). Interaction between avb3
integrin and subsequent intracellular signalling was depend-
ent on the IgV domain of CD47 but did not require the
transmembrane domain (Lindberg et al., 1996). However,
CD47-mediated cellular effects may occur independently of
integrins, especially in red blood cells (RBCs). This is why the
designation CD47 is now considered more appropriate than
the initial name IAP. CD47 has also been shown to cooperate
with the Fas pathway in mediating apoptosis (Manna et al.,
2005). Moreover, recent advances describing the role of CD47
in the field of cardiovascular diseases (Table 1) have under-
lined strong cooperation of CD47 with CD36 (Isenberg et al.,
2006) and VEGFR-2 (Kaur et al., 2010).
Many of the cellular effects of CD47 are mediated by Gi
proteins (Table 1). For example, apoptosis induced by the
peptide agonist 4N1K involves inhibition of AC and a subse-
quent decrease in intracellular cAMP levels (Manna and
Frazier, 2004). CD47-associated platelet activation requires Gi
proteins with activation of Syk kinases that induce phospho-
rylation of Lyn and focal adhesion kinase (FAK) (Chung et al.,
1997). Other kinases identified in the signalling pathway
downstream of Gi proteins include PI3K and MAPK. Taking
into account such G-protein-dependent signalling associated
with CD47 activation, Brown and Frazier (2001) proposed
that CD47 with five transmembrane domains associated to
an integrin dimer might mimic classical heptahelical GPCRs.
In agreement, CD47-dependent signalling involves not only
Gi proteins, but also direct activation of small G-proteins like
Rac and Cdc42, especially in neurones where CD47 plays an
important role in neurite formation and more generally in
neuronal development (Table 1). Furthermore, CD47 also
associates with protein linking integrin-associated protein to
cytoskeleton-1 protein (PLIC-1) known to regulate G-protein
signalling. Several studies have also identified cytoplasmic
Ca2+as an important mediator of CD47 signalling. TSP-1 was
reported to induce an increase in intracellular Ca2+in fibrob-
lasts via its RGD and C-terminal domain (Tsao and Mousa,
1995). CD47 has been shown to mediate fibronectin-induced
intracellular Ca2+rises in HUVECs (Schwartz et al., 1993).
CD47 cross-linking by mAb 1/1A4 resulted in a strong
increase in intracellular Ca2+in Jurkat cells (Waclavicek et al.,
1997). Peptide 4N1 also induced an increase in intracellular
Ca2+responsible for mast cell exocytosis (Sick et al., 2009). In
Jurkat T-cells, a recombinant TSP-1 C-terminal fragment
(E3CaG1) inhibited sGC via increased intracellular Ca2+(Ram-
anathan et al., 2011). In Jurkat cells, increases in Ca2+were
CD47 dependent but did not involve Gi protein activation,
contrary to what we found in mast cells (Sick et al., 2009). In
contrast, in HUVECs, TSP-1 via CD47 is thought to inhibit
acetylcholine-induced increases in intracellular Ca2+(Bauer
et al., 2010). Because decreased Ca2+concentrations inhibit
endothelial nitric oxide synthase (eNOS) Ca2+-dependent
activation, TSP-1 contributes to regulate blood pressure by
limiting eNOS activation and endothelial-dependent vasore-
laxation (Bauer et al., 2010). Several previous studies have
also confirmed that CD47 acts as a key regulator of the
endothelial cells and platelets (Isenberg et al., 2006; 2008a).
Taken together, these data indicate that CD47 signalling
may differ according to cell type. Given the ubiquitous
associated partners are likely to be crucial in defining a given
specific cellular response both in physiological and also
CD47 and tumour cells
Tumour cell apoptosis
Tumour growth is considered, at least in part, to result from
dysfunctions in the balance between apoptosis and cell pro-
liferation. CD47 actively takes part in apoptosis and therefore
plays a key role in maintaining tissue homeostasis. Exposure
to soluble anti-CD47 mAbs resulted in rapid (within 3 h)
apoptosis of Jurkat E6 cells and activated T-cells (Pettersen
et al., 1999). However, this first report underlines the com-
plexity and ambiguity concerning the functional effects of
anti-CD47 mAbs. Indeed, soluble anti-CD47 mAbs Ad22 and
1F7 induced Jurkat cell apoptosis whereas mAbs B6H12 and
2D3 were without effect. Ad22 was reported to react with the
IgV domain of CD47, in close proximity to epitopes defined
by B6H12 and 1F7, whereas 2D3 reacts with a distant region.
Thus, CD47-induced apoptosis was thought to require liga-
tion of distinct epitopes on the IgV domain. Interestingly,
immobilized Ad22 and 2D3 had no influence on Jurkat cell
proliferation, suggesting that the mode of presentation of
anti-CD47 mAbs is critical with respect to functional
responses. Furthermore, the effects of anti-CD47 mAbs also
depend on the cell line tested and on the activation state of
the cell. For example, Ad22 induced cell death of Jurkat cells
and CD3-activated T-cells but not resting T-cells. In these
cells, CD47-induced apoptosis was thought to be independ-
ent of Fas and the tumour necrosis factor receptor-dependent
et al., 1999).
mediated apoptosis induced by TSP-1 or immobilized (B6H12
and BRIC126) but not soluble anti-CD47 mAbs was reported
in many leukaemic cells, including B-chronic lymphocytic
leukaemia (B-CLL) cells (Mateo et al., 1999), promyelocytic
leukaemia NB4 cells (Saumet et al., 2005) and Jurkat T-cells
(Roué et al., 2003). Activation of CD47 apparently leads to a
particular type of cell death called type-III programmed cell
death (PCD) in normal as well as leukaemia cell lines. Type-III
PCD is caspase independent and is characterized by cytoplas-
mic effects such as cell shrinkage, phosphatidylserine (PS)
exposure and mitochondrial matrix swelling, which result in
disruption of mitochondrial transmembrane potential (Dym)
and subsequent reactive oxygen species (ROS) production, as
described for B-cells cultured on immobilized B6H12 mAbs
(Mateo et al., 2002). Neither cytochrome c and apoptosis-
inducing factor nor release of other mitochondrial inter-
membrane proteins were required for CD47-dependent PCD
(Roué et al., 2003; Saumet et al., 2005). Mitochondrial trans-
membrane potential loss and PS externalization were pre-
vented by cytochalasin D, suggesting that actin cytoskeleton
CD47 in the tumour microenvironment
British Journal of Pharmacology (2012) 167 1415–14301419
rearrangement was necessary in CD47-dependent B-CLL cell
death (Mateo et al., 2002; Barbier et al., 2009). Additionally,
immobilized anti-CD47 mAbs induced morphological and
functional changes in B-cell leukaemia cell lines that involve
Rac1 and Cdc42 GTPases (Yoshida et al., 2000). Furthermore,
CD47 ligation failed to induce characteristic type-III PCD
cytoplasmic events in patients who do not express Wiskott–
Aldrich syndrome protein (WASP), indicating that both
Cdc42 and WASP are key components of the signalling
pathway responsible for actin polymerization in CD47-
induced caspase-independent apoptosis (Mateo et al., 2002).
Nevertheless, the molecular basis of CD47-induced type-
III PCD is still incompletely understood. Recently, dynamin-
related protein-1 (Drp-1) was identified as a key molecular
relay in activating PCD (Bras et al., 2007). Chymotrypsin-like
protease activation after CD47 ligation by immobilized
B6H12 mAbs or immobilized TSP-1 was shown to induce
Drp-1 translocation from cytosol to mitochondria, thus
leading to ROS production and ATP loss independently of its
GTPase activity in primary B-cells (Barbier et al., 2009). Of
note, in addition to induction of type-III PCD, immobilized
anti-CD47 1F7 mAbs have been reported to promote death of
Jurkat T-cells and normal murine T-cells by classical caspase-
dependent apoptosis. Indeed, the extracellular IgV domain of
CD47 might interact with Fas receptors to induce apoptosis
without mobilization of CD47-dependent signalling or locali-
zation of CD47 in rafts (Manna et al., 2005).
Considering the key role of TSP-1/CD47 interactions in
leukaemia, the putative therapeutic potential of anti-CD47
mAbs was investigated. A bivalent single-chain Ab fragment
(scFv) of a murine mAb was first shown to promote apoptosis
of hCD47-transfected L1210 murine leukaemic cells without
causing haemagglutination of RBCs (Kikuchi et al., 2004).
Bivalent single-chain Ab fragments were also effective against
the human myeloma cell line KPMM2 implanted in severe
combined immunodeficiency (SCID) mice (Kikuchi et al.,
2005). Antitumour activity of CD47 mAbs was reported in
xenograft models of acute lymphoblastic leukaemia and
B-cell chronic lymphocytic leukaemia. Interestingly, no apop-
totic side effects on CD34+ haematopoietic progenitor/stem
or human endothelial cells were observed in these models
(Uno et al., 2007). Recently, a disulfide-linked dimer of a
single-chain Ab fragment against CD47 induced prolonged
survival of SCID mice engrafted with JOK-1 leukaemic cells
(Sagawa et al., 2011). In vitro results indicate that this effect
genes BNIP3 and RTP801.
The role of CD47 in cancer development is not restricted
to only leukaemia, but has also been shown for vascularized
solid tumours. Triggering of CD47 by 4N1K induces caspase-
independent apoptosis in several human breast cancer cell
lines. Indeed, CD47 activation stimulates cell death of both
non-invasive (MCF-7 and AU-565) as well as invasive (MDA-
MB-231) cell lines through Gi signalling, leading to subse-
quent decrease in cAMP levels and inhibition of PKA (Manna
and Frazier, 2004).
Interestingly, CD47 has been reported to be involved in
resistance of cancer cells to taxol (Lih et al., 2006). The resist-
ance of human prostate cancer cells to taxanes was correlated
with overexpression of a recently identified protein Txr1,
reported to dramatically down-regulate TSP-1 expression.
Stimulation of CD47 with either TSP-1 or 4N1K in resistant
cells increased taxol-induced apoptosis (Lih et al., 2006).
Whether this mechanism is directly or indirectly mediated by
CD47 is unknown. In addition, primary vascular cells cul-
tured from TSP-1-null mice and CD47-null mice were shown
to be resistant to high-dose radiation injury (Isenberg et al.,
2008b). CD47 suppression in mice resulted in increased radia-
tion sensitivity of implanted melanoma and squamous cell
lung tumours, while conferring radioresistance in normal soft
tissues, bone marrow and tumour-associated leukocytes
(Maxhimer et al., 2009a). Radiation-induced cell death was
similar in HUVECs when treated either by mAbs directed
against TSP-1 (clone A6.1) or CD47 (clone B6H12) or by
antisense CD47 morpholinos, consistent with blockade of the
TSP-1/CD47 interaction being responsible for the decrease in
radiation sensitivity. Radioprotection in normal tissues may
be attributable at least in part to restoration of NO-mediated
cytoprotective activity (Isenberg et al., 2008b; Maxhimer
et al., 2009a).
Tumour cell survival and proliferation
Besides its well-described role in inducing apoptosis, CD47
has been paradoxically shown to modulate cell survival
and proliferation. Indeed, we demonstrated that activation
of CD47 by 4N1 results in protection of thyroid cells
from apoptosis induced by C2-ceramides by stimulating the
cAMP/PKA pathway (Rath et al., 2006a). Moreover, 4N1-
mediated activation of CD47 inhibited camptothecin- and
doxorubicin-induced apoptosis of human follicular thyroid
carcinoma FTC-133 cells (Rath et al., 2006b).
Furthermore, we showed that CD47 stimulated by the
agonist 4N1 induces the proliferation of astrocytoma cells but
not normal astrocytes, with downstream signalling subse-
quent to CD47 activation involving Gbg dimer-dependent
activation of the PI3K/Akt pathway in astrocytoma cells (Sick
et al., 2011). As well, a peptidic fragment resulting from
serpin A1 proteolytic cleavage exhibits CD47-dependent
mitogenic activity in both Hep G2 liver cancer cells and
MCF-7 breast cancer cells (Congote and Temmel, 2004).
The implication of CD47 in cell migration was first demon-
strated for neutrophils, with blocking Abs against CD47
inhibiting transmigration of neutrophils and monocytes
through endothelium (Cooper et al., 1995; de Vries et al.,
2002). Increased levels of CD47 expression were correlated to
improved rates of neutrophil migration during inflammatory
responses (Liu et al., 2001). It was also found that CD47 plays
a major role in smooth muscle cell proliferation and chemo-
taxis towards soluble collagen, in part via association with
a2b1 integrins, subsequent Gi-mediated decrease in cAMP
levels and inhibition of ERK activity (Wang and Frazier, 1998;
Wang et al., 1999). Recent data suggest that CD47 stimulates
intestinal epithelial cell migration on collagen-I through
COX-2 expression (Broom et al., 2009). Moreover, increased
expression of CD47 in fibroblasts promotes intercellular
adhesion and decreases the spontaneous migration of Jurkat
lymphocytes through a fibroblast monolayer. This effect was
dependent on Rho family GTPases and Gi proteins (Rebres
et al., 2005). These data suggest that CD47 may restrict the
E Sick et al.
1420British Journal of Pharmacology (2012) 167 1415–1430
infiltration of cancer cells in surrounding tissues and devel-
opment of metastasis. Paradoxically, CD47 was reported to
induce epithelial cell spreading and migration. Indeed, CD47
co-localizes with E-cadherin at cell-cell adhesion sites and
forced expression of CD47 induces epithelial cell spreading
and leads to a partial disruption of cell-cell adhesion (Shino-
hara et al., 2006).
Surprisingly, very few data are available regarding the role
of CD47 in the regulation of cancer cell migration. CD47 was
shown to stimulate migration of several lymphoma cell lines,
but not myeloma cell lines, involving the Cdc42 GTPase and
a tight molecular cooperation with a4b1 integrins (Yoshida
et al., 2000). In addition, CD47 was demonstrated to decrease
Melan-a cell migration but not that of B16F10 melanoma
cells. Aberrant N-glycosylation of SIRPa was observed in
B16F10 melanoma cells and results in impaired binding to
CD47. Binding of CD47 to SIRPa in Melan-a cells is supposed
to down-regulate SIRPa expression levels on adjacent cells by
stimulation of proteosomal degradation. Increased proteo-
somal degradation of SIRPa resulted in inactivation of SHP-2
phosphatase, inhibition of the dephosphorylation of FAK,
subsequent disassembly of focal adhesions and thereby to
inhibition of Melan-a cell motility (Ogura et al., 2004). Resist-
ance to this molecular mechanism in B16 melanoma is
thought to contribute, at least in part, to the more aggressive
behaviour of these cells (Ogura et al., 2004). Blockade of
CD47 by neutralizing Abs reduced migration and chemotaxis
in response to collagen IV of melanoma, prostate cancer and
ovarian cancer-derived cells. These effects were shown to be
dependent on avb3 integrins and intracellular calcium
(Shahan et al., 2000). Finally, CD47 was reported to stimulate
osteoclastogenesis and its disruption may protect against
metastatic tumours in bone (Uluçkan et al., 2009). Taken
together, these data are consistent with activation of CD47
contributing to cell migration, and it therefore may be that
CD47 antagonism represents a promising therapeutic strat-
egy to limit infiltration at tumour sites and to decrease cancer
cell dissemination and formation of metastases.
Cell adhesion and spreading
CD47 is required for post-adhesive events after neutrophil
transmigration across intestinal epithelial cells (Parkos et al.,
1996). C32 melanoma cell spreading onto vitronectin was
stimulated by CD47 through cooperation with avb3and avb5
integrins and involved activation of Gi proteins, FAK and
PI3K (Gao et al., 1996b). This effect apparently depends on
cholesterol, which was thought to maintain the structural
and functional integrity of the CD47/avb3/G-protein complex
in lipid rafts (Green et al., 1999). However, adhesion may also
be stimulated by CD47 independently of cholesterol content
and G-protein signalling. Indeed, CD47 improves avb3avidity
in human ovarian carcinoma cells after cholesterol depletion
in the cell membrane and similarly, a4b1avidity for vascular
cell adhesion molecule-1 (VCAM-1) and TSP-1 in sickle
reticulocytes (Brittain et al., 2004; McDonald et al., 2004).
Thus, CD47 can also transmit inside-out signals contributing
to the regulation of integrin activation and clustering. Differ-
ent pools of integrins appear to be differentially regulated by
CD47 and this could be related to the localization of CD47 in
specific microdomains at the plasma membrane. CD47 can
also control cell-to-cell adhesion by interaction with SIRPg,
for example, in T-cell adhesion to antigen-presenting cells
(Piccio et al., 2005). Moreover, in CD47-deficient fibroblasts,
CD47 expression induces intercellular adhesion, resulting in
cell aggregation even in the absence of active integrins,
SIRPa1 or TSP-1 binding (Rebres et al., 2005). This effect is
proposed to involve CD47 homophilic interaction, but unlike
endothelial cell adhesion molecule-1 or cadherins), expres-
sion of CD47 is not sufficient to induce aggregation and may
require activation by serum (concentration above 4%) or by
4N1K. It is also interesting to note that anti-CD47 2D3 mAbs,
which inhibit CD47-dependent cell aggregation, enhanced
SIRPa1 binding. This effect was also mediated by Fab frag-
ments, and suggests that even monovalent binding causes
CD47 to undergo conformational or clustering changes that
are relevant for SIRPa binding (Rebres et al., 2005). Even if the
homotypic interaction of CD47 with itself was not further
explored, this study underlines two important features that
may explain some of the reported paradoxical results, namely
the influence of serum concentration on the aggregation state
of cells and differential SIRP binding in the presence of CD47
mAbs. CD47 also influences cell adhesion indirectly. Indeed,
TSP-1 induced the up-regulation of the adhesion molecules
intercellular adhesion molecule-1 and VCAM-1 in human
brain microvascular endothelial cells (Xing et al., 2009). In
view of these results, CD47 modulation by blocking strategies
may have detrimental effects resulting in increased cell
migration depending on the cell type considered. Such effects
have to be taken into account in developing anti-metastatic
strategies where CD47 is the target.
CD47 and stromal cells
Angiogenesis represents a key component of solid tumour
development, allowing both nutrition and oxygenation of
tumour cells, thus subsequently contributing to dissemina-
tion and metastasis. Strategies targeting angiogenesis have
emerged over the past decade based on a better understand-
ing of molecular mechanisms underlying angiogenesis. This
has led to the development of anti-angiogenic approaches for
the treatment of cancer, with notably several vascular
endothelial growth factor (VEGF)-neutralizing mAbs being
approved for clinical use in cancer (for review, see Chames
et al., 2009). However, even if effective in certain cancers,
significant problems have appeared. For example, many
patients with metastatic diseases are refractory or acquire
resistance towards VEGF inhibitors (Bergers and Hanahan,
2008). Tumour vessel abnormalities resulting in heterogene-
ous and tortuous vessel networks are also believed to decrease
the efficiency of anti-VEGF strategies (Carmeliet and Jain,
2011). To date, even if current anti-VEGF agents have proved
to be less than ideally efficacious, targeting angiogenesis still
represents a most promising therapeutic strategy.
The first evidence for CD47 involvement in the vascular
system was described in platelets where 4N1 was shown to
stimulate platelet activation and aggregation (Dorahy et al.,
1997). The signalling pathway responsible for platelet activa-
tion involves a Gi protein-mediated decrease in cAMP
CD47 in the tumour microenvironment
British Journal of Pharmacology (2012) 167 1415–14301421
production (Frazier et al., 1999). More recently, several groups
have focused on the putative role of CD47 in angiogenesis. In
vitro, TSP-1 and 4N1K were reported to induce cytotoxicity of
brain endothelial cells and inhibit endothelial cell migration
and tube formation (Xing et al., 2009). These effects were
confirmed in vivo. Using recombinant fragments, the anti-
angiogenic properties of TSP-1 were demonstrated to be
mediated by the interaction between its C-terminal domain
and CD47 (Isenberg et al., 2006), with this interaction being
responsible for the inhibition of NO-induced GC activation.
Therefore, CD47 can modulate blood flow and tissue per-
fusion by interfering with the NO-dependent balance of the
relative state of constriction and relaxation of arteries.
Although CD36 ligation by TSP-1 inhibits NO-stimulated
vascular cell responses and cGMP signalling, the anti-
angiogenic effects of TSP-1 at picomolar physiological con-
centrations are driven solely by CD47 ligation. Moreover,
angiogenic responses following CD36 ligation are also
dependent on CD47. The NO-driven delay in thrombin-
induced platelet activation is blocked by TSP-1 ligation to
CD47 and/or CD36 as shown using, respectively, TSP-1
C-terminal domain and type 1 repeat recombinant fragments
(Isenberg et al., 2008a).
Restoration of NO signalling consecutive to CD47 antago-
nism that results in a rapid restoration of NO-driven blood
flow would be essential for graft survival. Consistently, in a
murine skin graft model, graft survival was dramatically
increased by both morpholino suppression of CD47 expres-
sion and CD47 blockade by miap 301 mAbs (Isenberg et al.,
2008c). In a comparable murine ischaemic model of full
thickness flaps, perfusion, survival and angiogenesis were
optimized using combined treatment with nitrite and TSP-1/
CD47 blockade via anti-TSP-1 A6.1 mAbs or gene silencing
(Isenberg et al., 2009). In this study, a survival rate of almost
100% was attained, indicating that optimal results were
obtained by combined treatment. The efficiency of such an
approach is especially promising for the treatment of burns
and diverse pathologies requiring skin grafts. In support, an
increase in tissue survival following myocutaneous flap
surgery was observed when using CD47-blocking mAbs
(clone miap 301), antisense morpholino or CD47 silencing in
mice (Isenberg et al., 2007). Moreover, increased survival con-
secutive to CD47-blocking mAb treatment was described in
CD36-null mice, suggesting that this response is CD36 inde-
pendent. Flap survival was also increased by CD47-blocking
OX101 mAbs in a rat model (Maxhimer et al., 2009b). In
addition to flap reperfusion enhancement, CD47 blockade
contributed to a decrease in the inflammatory response in
flap tissues after ischaemia-reperfusion injury. Random cuta-
neous flap survival, blood vessel permeability and blood flow
were also drastically increased by antisense CD47 mor-
pholino and anti-TSP mAbs (A6.1) in a porcine model (Isen-
berg et al., 2008d). Considering the high level of homology
between porcine and human tissues, the potential therapeu-
tic targeting of CD47 is supported by these results. Finally,
TSP-1 and CD47 suppression or pretreatment with a CD47-
antagonist mAb miap 301 was shown to enhance tissue sur-
vival and perfusion after ischaemia-reperfusion injury in a
murine liver model, suggesting the inhibition of CD47 could
not only enhance skin graft but also organ survival after
transplantation surgery (Isenberg et al., 2008e).
CD47 ligation by TSP-1 was reported to inhibit the
NO-dependent and NO-independent activation of sGC in
platelets as well as in smooth muscle cells (Miller et al.,
2010b). Furthermore, CD47 activation by TSP-1 controls
intracellular cAMP levels in smooth muscle, directly via
Gi-dependent inhibition of adenylate cyclase and indirectly
through PDE3 inhibition in a cGMP-dependent manner (Yao
et al., 2011). In addition to the modulation of cyclic nucle-
otide levels, CD47 ligation by native TSP-1 or CBD recom-
binant domain disrupted the constitutive association of
VEGFR-2 and CD47 in endothelial cells. This in turn inhibits
VEGFR-2 phosphorylation and its downstream signalling,
without affecting VEGF binding (Kaur et al., 2010). Finally,
CD47 silencing was recently described to result in enhance-
ment of the pro-angiogenic properties of endothelial colony
forming cells by interfering with the SDF-1 chemokine
pathway (Smadja et al., 2011).
The role of NO is quite ambiguous in tumours. Indeed,
NO is reported to increase tumour perfusion via induction of
local vasodilatation. On the contrary, NO is also thought to
decrease tumour perfusion by preferential relaxation of the
normal vascular network outside the tumour, resulting in
blood redistribution away from the tumour (Isenberg et al.,
2008f). Even if the capacity of tumour vasculature to respond
to vasoactive agents is quite limited, recovery of basal per-
fusion after a vasoactive treatment (vasoconstrictor or vasodi-
latator) is modulated by TSP-1 through CD47 interaction.
Indeed, overexpression of truncated TSP-1 lacking parts of its
CBD domain failed to reproduce this activity (Isenberg et al.,
2008f). These data indicate that TSP-1/CD47 interaction may
regulate long-term vascular responses in the tumour context.
The concept that inflammation sustains cancer development
is not new. Indeed, in 1863, Rudolf Virchow first hypoth-
esized that the origin of cancer was at sites of chronic inflam-
mation (Balkwill and Mantovani, 2001). After a long eclipse,
this hypothesis has been more recently supported by clinical
and experimental studies demonstrating that infiltration of
leukocytes into the tumour microenvironment is a key event
in promoting cancer growth (for review, see Tlsty and Cous-
sens, 2006). In this context, cytokines and chemokines as
well as activation of transcription factors such as NF-kB, Stat3
or HIF play major roles in tumour progression. Moreover,
tumour cells are thought to have altered self-antigens that
impair recognition by the immune system, leading to tumour
escape from immune surveillance (for review, see Dunn et al.,
2004). Accordingly, inflammation and escape of tumoural
cells from surveillance by the immune system are fundamen-
tal steps in tumour promotion and, therefore, targeting
immune cells may represent a promising therapeutic strategy
Among tumour infiltrating leukocytes, macrophages rep-
resent the main actors of tumour promotion (for review, see
Mantovani et al., 2008). Several studies have underlined a
role for CD47 as a marker of ‘self’, initially in RBCs. Thus,
RBCs from CD47-/-mice were rapidly cleared by macrophages
when transfused into wild-type mice, whereas they circulated
normally in CD47-/-mice (Oldenborg et al., 2000). This
occurred independently of complement and antibodies and
was mediated by the interaction between SIRPa and CD47.
E Sick et al.
1422British Journal of Pharmacology (2012) 167 1415–1430
Such a molecular interaction is also responsible for maintain-
ing platelet homeostasis (Olsson et al., 2005). The ITIM
domains of SIRPa become phosphorylated upon ligation with
CD47 and recruit SHP phosphatases, resulting in deactivation
of myosin-II without affecting F-actin (Tsai and Discher,
2008). The absence of self-recognition at the cell surface thus
allows contractile engulfment. Several paired receptors
engaged at the immunological synapse trigger phagocytosis
but CD47/SIRPa is the only identified negative regulator.
Interestingly, the expression level of CD47 is likely to vary
under pathophysiological situations. Indeed, CD47 expres-
sion is up-regulated on normal haematopoietic stem cells
(HSCs) when they are recruited to the periphery upon expo-
sure to pro-inflammatory stimuli like granulocyte colony
stimulating factor or LPS (Jaiswal et al., 2009). Increased
CD47 at the cell surface is therefore thought to protect hae-
matopoietic cells from phagocytosis during their trafficking
from bone marrow to the periphery. On the contrary,
decreased expression of CD47 was observed in apoptotic neu-
trophils and fibroblasts and correlated with the degree of
apoptosis. Apoptotic cells lose their ability to activate SIRPa
and are engulfed, unlike viable cells expressing normal
amounts of CD47. Moreover, on apoptotic cells, CD47 is
redistributed on the plasma membrane to form patches. This
is thought to decrease the ability of CD47 to interact with
SIRPa. Additionally, CD47 becomes segregated away from
calreticulin and PSs, which are ‘eat me’ stimuli triggering
low-density lipoprotein receptor-related protein 1-dependent
efferocytosis (Gardai et al., 2005). Interestingly, CD47 expres-
sion levels were shown to increase in several leukaemic cell
lines (Jaiswal et al., 2009), as well as in bladder tumour-
initiating cells (Chan et al., 2009), non-Hodgkin lymphoma
(Chao et al., 2010a) and acute lymphoblastic leukaemia
(Chao et al., 2011). CD47 is thus considered as an adverse
prognostic factor. Minimally expressed on most normal cells,
calreticulin levels are increased at the cell surface of several
cancer cells and are commonly correlated with enhanced
CD47 expression. This is thought to protect tumour cells
2010a,b). Increased levels of CD47 expression observed in
leukaemic mice were confirmed for dysplastic human hae-
matopoietic cells as compared with normal umbilical cord
blood and peripheral blood stem cells. Indeed, granulocyte-
myeloproliferative disorders such as atypical chronic myeloid
leukaemia, proliferative phase chronic myelomonocytic leu-
kaemia and acute myeloid leukaemia (AML) (Jaiswal et al.,
2009). MOLM-13 cells having low CD47 levels derived from a
patient with AML failed to engraft when implanted in immu-
nocompromised mice. In contrast, implanted MOLM-13 cells
overexpressing CD47 lead to development of tumours, con-
sistent with CD47 overexpression enabling these cells to
escape from phagocytosis by macrophages. Moreover, macro-
phages were shown to exert a selective pressure for high-
expressing CD47 clones in vivo. Thus, leukaemic progenitors
are thought to hijack the protective effect of overexpressing
CD47 used by normal HSCs to escape from macrophage-
dependent killing observed during inflammatory states. Con-
sistently, a blocking antibody directed against CD47 was
reported to trigger phagocytosis of human bladder cancer
cells in vitro (Chan et al., 2009). Finally, anti-CD47-blocking
mAbs (clones BRIC126 and B6H12) were shown to promote
macrophage-mediated phagocytosis of acute lymphoblastic
leukaemia cells and leukaemia stem cells (LSCs) and to inhibit
tumour engraftment in vivo (Majeti et al., 2009; Chao et al.,
The interaction of CD47 with SIRPa contributes to fusion
of rat alveolar macrophages and the generation of multinu-
cleated cells (Han et al., 2000) that can differentiate into
osteoclasts in bone. Formation of such giant multinucleated
cells was strongly inhibited both in vivo and in vitro in the
absence of SIRPa/CD47 interaction (Lundberg et al., 2007).
The distance between two cells interacting via CD47 and
SIRPa is supposed to be around 14 nm, a distance similar to
that observed during the establishment of the immunological
synapse (Hatherley et al., 2009). However, this distance may
be reduced to 5–10 nm in macrophages by interaction of
CD47 with a short form of SIRPa that lacks the C1 domain,
thereby leading to fusion (Han et al., 2000). To date, even if
the mechanism is poorly documented, several receptors
including CD44 (Sterling et al., 1998), DC-STAMP (Yagi et al.,
2006), RANK/RANKL and tetraspanin CD9 (Ishii et al., 2006)
have been reported to take part in the macrophage fusion
process leading to formation of osteoclasts or giant cells. The
fusion of cancer cell with macrophages is believed to repre-
sent a putative mechanism for dissemination and develop-
ment of metastasis, as described for melanoma cells fused
with macrophages (Rachkovsky et al., 1998; Chakraborty
et al., 2000). Nevertheless, to date, this proposed mechanism
remains controversial because of the absence of proof of
concept in vivo. However, fusion between intestinal epithelial
cells and macrophages was very recently shown to occur in
vivo in a cancer context and was related to nuclear reprogram-
ming resulting in a transcriptomic identity similar to the
parental cells but also with a unique subset of transcripts
(Powell et al., 2011). Thus, cancer cell fusion with circulating
bone marrow-derived cells contributes to masking the cancer
phenotype, thereby permitting cancer cells to escape from
immune surveillance and to infiltrate in a distant niche. In
this context, even if poorly characterized at present, thera-
peutic inhibition of CD47/SIRPa interactions might provide
not only enhanced clearance and degradation of cancer cells
but also limited metastatic potential.
DCs represent another key class of cells in the tumour
presenting cells of the immune system and thus playing a
critical role in establishing innate and adaptive immune
responses. Tumour-bearing mice as well as patients with
breast cancer, prostate cancer and glioma present a decreased
number of DCs in lymph nodes spleen and skin (Rabinovich
et al., 2007). Indeed, cancer patients have significantly
decreased numbers of circulating and peripheral DCs whereas
immature DCs accumulate. Immature DCs have a reduced
capacity for capturing antigens and a reduced capacity to
stimulate naïve T-cells (Petersen et al., 2010). Interestingly,
stimulation of CD47 by 4N1K inhibits secretion of pro-
inflammatory cytokines like IL-12, TNFa, IL-6 and GM-CSF
by maturing DCs (Demeure et al., 2000). Furthermore, CD47
activation inhibits maturation (i.e. up-regulation of major
histocompatibility complex class II antigens, co-stimulatory
molecules, acquisition of a potent T-cell stimulatory activity)
of immature DCs. CD47 stimulation by 4N1K also directly
CD47 in the tumour microenvironment
British Journal of Pharmacology (2012) 167 1415–14301423
affects DC populations by inducing apoptosis-like cell death
(Johansson and Londei, 2004). Thus, CD47 may participate
in reducing the efficiency of immune responses against
tumour by inducing T-cell tolerance towards tumours.
Various and sometimes contradictory functional effects
have been reported when CD47 is activated on T-cells. It was
reported that CD47-deficient animals exhibit sustained
inflammation related to decreased local apoptosis of T-cells
(Lamy et al., 2007). SIRPg expressed on T-cells which interacts
with CD47 on endothelial cells was critical for T-cell transen-
dothelial migration and in inducing apoptosis of Jurkat cells,
similar to what has been described for SIRPa ligation (Brooke
et al., 2004; Stefanidakis et al., 2008). Furthermore, TSP-1 may
be a potent inhibitor of T-cell receptor (TCR)-mediated T-cell
activation. Indeed, TSP-1 signalling through CD47 and
HSPGs inhibits TCR signalling including suppression of the
autocrine growth factor IL-2 and early activation markers
such as CD69, Egr-1 and PAC-1 (Li et al., 2001). T-regulatory
cells (Tregs) are critical in the limitation of damage induced
by exacerbated responses to ‘self’ or ‘foreign’ antigens, but in
cancer, they have been shown to proliferate. Because tumour
antigens are derived from the host, tumours are more likely
regarded as self by Tregs that actively promote their tolerance.
Increased infiltration of Tregs in tumours is thus correlated
with poor prognosis in several types of cancers (Elkord et al.,
2010). CD47 stimulation by TSP-1 or 4N1K was demonstrated
to trigger the conversion of naïve or memory CD4+ CD25-
T-cells into Tregs (Grimbert et al., 2006). Furthermore, CD47
cross-linking was shown to induce apoptosis of normal T- and
B-cells when cultured on immobilized B6H12 mAbs (Mateo
et al., 2002) or in the presence of soluble anti-CD47 Ad22
mAbs (Lamy et al., 2003). Interestingly, haematopoietic pro-
genitors CD34+ and immature DCs were described to be
resistant to CD47-induced apoptosis (Mateo et al., 2002).
CD47 was also reported to induce the development of
hyporesponsive or anergic T-cells (Avice et al., 2001).
Finally, levels of CD47 expression in head-and-neck squa-
mous cell carcinoma apparently influence the cytotoxicity of
NK cells, with increased expression of CD47 being related to
a decrease in NK cytotoxicity (Kim et al., 2008). The mecha-
nism remains unknown but may represent a possible supple-
mentary pathway for immune escape in cancer.
Taken together, these data indicate that blockade of CD47
receptors apparently triggers immune system responses
against tumours at many different levels. This may thus rep-
resent a promising therapeutic strategy resulting in increased
tumour cell phagocytosis, enhanced DC maturation and sub-
sequently augmented T-cell-mediated cytotoxicity.
This overview of CD47 indicates that CD47 very likely plays
a central role in the tumour microenvironment. Considering
its ubiquitous expression, its ability to modulate tumour cell
growth and migration and to limit angiogenesis, and the
development of immune responses against tumours, CD47
therefore appears to represent an exciting target candidate for
new therapeutic approaches against cancer (Figure 2).
A number of recent studies using function-blocking anti-
CD47 mAbs are consistent with an eventual therapeutic
application in the field of leukaemia. For example, single-
chain Ab fragments against CD47 were successfully used to
induce apoptosis of malignant lymphoid cells isolated from
patient with B-CLL and to significantly increase survival rate
of mice implanted with human lymphocytic leukaemia cell
lines (Sagawa et al., 2011). In addition to directly inducing
apoptosis, other mechanisms of action for anti-CD47 mAbs
dependent cytotoxicity and the induction of leukaemic stem
cell phagocytosis by disruption of CD47/SIRPa interaction. A
demonstration of this latter effect was the phagocytosis of
human AML LSCs treated with B6H12 mAbs by mouse mac-
rophages in vivo (Majeti et al., 2009). Of note, the mouse
anti-CD47-blocking mAb miap 301 promoted phagocytosis
of mouse AML while not depleting normal HSCs in vivo
(Majeti et al., 2009), consistent with the feasibility of thera-
peutic targeting of CD47. A synergic effect of anti-CD47
mAbs in combination with rituximab, an anti-CD20 mAb, in
promoting phagocytosis was described for the eradication of
non-Hodgkin lymphoma (Chao et al., 2010a). To date, most
of the studies reporting successful eradication of leukaemia
cells by anti-CD47 mAbs were done using human tumour
xenograft models in immunodeficient mice. Because only
human leukaemic cells were targeted by the human anti-
CD47 mAbs used in such experimental models, interpreta-
tion of the observed depletion of leukaemic cells is somewhat
hampered. Notably, characterization of possible side effects
that could occur in immunocompetent mice or in humans is
not accessible with these models.
To our knowledge, very few studies have addressed the
role of CD47 in solid tumours, which are highly vascular-
ized, and for which little is known about pathological
cell-to-cell interactions in tumour stroma. An accurate
description of CD47 function during vascularization at the
site of tumours is still lacking and available data sometimes
appear contradictory or paradoxical. TSP-1, a well-known
inhibitor of angiogenesis, acts physiologically via CD47
through inhibition of NO signalling. However, little is
known about TSP-1 responsiveness in tumour vasculature
where NO could act as a friend or a foe, either by dilatation
of blood vessels in the tumour or dilatation of peripheral
blood vessels resulting in redistribution of blood away from
the tumour. Thus, in this context, it is currently very diffi-
cult to predict outcomes if systemic blocking strategies
against CD47 were to be tested.
Furthermore, as described beforehand, depending on the
cell type considered, CD47 activation is reported either to
induce apoptosis or in contrast to enhance proliferation and
survival. On the other hand, cell migration appears to be
quite universally stimulated by CD47 ligation and activation
(Figure 2). Thus, antagonism of CD47 should reduce cell
migration, thereby representing a plausible strategy to fight
against cancer cell dissemination. Blockade of SIRPa/CD47
interaction that subsequently promotes immune system
responses against tumours (Figure 2), especially in cases
where CD47 is highly overexpressed, likewise appears to be a
Given the ubiquitous expression of CD47, specific target-
ing will be a critical issue to resolve. This might be addressed
using mAbs directed against both CD47 and a specific tumour
cell marker. For example, because high levels of CD47 are
E Sick et al.
1424 British Journal of Pharmacology (2012) 167 1415–1430
found in prostate cancer (Vallbo and Damber, 2005), devel-
opment of blocking CD47 mAbs that also target the prostate-
specific antigen could be of particular therapeutic interest. It
should nevertheless be pointed out that strategies using anti-
CD47 mAbs may also mask binding sites for different endog-
enous ligands or partners, which might prevent some of their
beneficial effects against tumours. Therefore, the develop-
ment of alternative strategies using synthetic competitive
antagonists of CD47 is likely required. In this context,
detailed X-ray analysis and molecular modelling studies of
the TSP-1/CD47 complex would be helpful. Indeed, this
might lead to the characterization of non-evident binding
sites or other sites of interaction, thereby perhaps contribut-
ing to the development of new highly specific and efficacious
small molecule antagonists of CD47.
In conclusion, it thus may be that CD47 represents an
interesting therapeutic target for the treatment of cancer.
However, evaluation is clearly hampered, given the current
lack of candidate molecules and accordingly, the absence of
E. S. was a recipient of a Fellowship from the Région
Champagne-Ardenne. A. J. was a recipient of a Fellowship
from the Ministère de la Recherche.
Conflict of interest
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