About this issue
Cells in the human body are constantly under mechanical
force – they are being pushed, pulled, squeezed, pressed,
and stretched. While these forces are essential for
development and homeostasis, they can also cause
cancer. But little is understood how forces drive disease,
and how they can be manipulated to provide patient
benefit. This theme issue brings together cell and systems
biologists, clinicians, and bioengineers to discuss the
latest developments in research in the exciting field of
cancer mechanobiology.
This issue is based on a Royal Society discussion
meeting held in June 2018.
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Introduction The forces of cancer
Chris Bakal and Julia Sero
Mechanotransduction: from the cell surface to the nucleus
via RhoA
Keith Burridge, Elizabeth Monaghan-Benson and David M
Graham
The plasma membrane as a mechanochemical transducer
Anabel-Lise Le Roux, Xarxa Quiroga, Nikhil Walani, Marino
Arroyo and Pere Roca-Cusachs
Connections between the cell cycle, cell adhesion and the
cytoskeleton
Matthew C Jones, Junzhe Zha and Martin J Humphries
Forces and constraints controlling podosome assembly and
disassembly
Nisha Bte Mohd Rafiq et al.
Cooperativity between stromal cytokines drives the invasive
migration of human breast cancer cells
Yair Elisha, Yael Sagi, Georg Klein, Ravid Straussman and
Benjamin Geiger
Is cell migration a selectable trait in the natural evolution of
cancer development?
Andrea Disanza et al.
Engineering approaches to studying cancer cell migration in
three-dimensional environments
Noam Zuela-Sopilniak and Jan Lammerding
Cell migration through three-dimensional confining pores:
speed accelerations by deformation and recoil of the
nucleus
Marina Krause et al.
Tissue mechanics, an important regulator of development
and disease
Nadia ME Ayad, Shelly Kaushik and Valerie M Weaver
Applicability of drug response metrics for cancer studies
using biomaterials
Elizabeth A Brooks et al.
Immunotherapy: breaching the barriers for cancer treatment
Victor G Martinez, Danielle Park, and Sophie E Acton
The macrophage checkpoint CD47:SIRPa for recognition of
‘self’ cells: from clinical trials of blocking antibodies to
mechanobiological fundamentals
Jason C Andrechak, Lawrence J Dooling and Dennis E
Discher Front image Melanoma cell spreading on a collagen matrix and imaged using Total
Internal Reflection Fluorescence (TIRF) microscopy. Tubulin is labelled in blue, F-
actin in green, and Paxillin in red. Image was provided by Chris Bakal (Institute of
Cancer Research).
Contents
royalsocietypublishing.org/journal/rstb
ReviewCite this article: Andrechak JC, Dooling LJ,
Discher DE. 2019 The macrophage checkpoint
CD47 : SIRPa for recognition of ‘self ’ cells:
from clinical trials of blocking antibodies to
mechanobiological fundamentals. Phil.
Trans. R. Soc. B 374: 20180217.
http://dx.doi.org/10.1098/rstb.2018.0217
Accepted: 15 April 2019
One contribution of 12 to a discussion meeting
issue ‘Forces in cancer: interdisciplinary
approaches in tumour mechanobiology’.
Subject Areas:bioengineering, biophysics, cellular biology,
immunology
Keywords:adhesion, signalling, blockade, plasticity
Author for correspondence:Dennis E. Discher
e-mail: [email protected]
& 2019 The Author(s) Published by the Royal Society. All rights reserved.
Electronic supplementary material is available
online at rs.figshare.com.
The macrophage checkpoint CD47 : SIRPafor recognition of ‘self ’ cells: from clinicaltrials of blocking antibodies tomechanobiological fundamentals
Jason C. Andrechak1,2, Lawrence J. Dooling1 and Dennis E. Discher1
1Biophysical Engineering Labs and 2Bioengineering Graduate Group, University of Pennsylvania, Philadelphia,PA, USA
JCA, 0000-0002-6659-9488; LJD, 0000-0002-1688-2066; DED, 0000-0001-6163-2229
Immunotherapies against some solid tumour types have recently shown
unprecedented, durable cures in the clinic, and the most successful thus
far involves blocking inhibitory receptor ‘checkpoints’ on T cells. A similar
approach with macrophages is emerging by blocking the ubiquitously
expressed ‘marker of self’ CD47 from binding the inhibitory receptor
SIRPa on macrophages. Here, we first summarize available information on
the safety and efficacy of CD47 blockade, which raises some safety concerns
with the clearance of ‘self’ cells but also suggests some success against hae-
matological (liquid) and solid cancers. Checkpoint blockade generally
benefits from parallel activation of the immune cell, which can occur for
macrophages in multiple ways, such as by combination with a second,
tumour-opsonizing antibody and perhaps also via rigidity sensing. Cyto-
skeletal forces in phagocytosis and inhibitory ‘self’-signalling are thus
reviewed together with macrophage mechanosensing, which extends to reg-
ulating levels of SIRPa and the nuclear protein lamin A, which affects
phenotype and cell trafficking. Considerations of such physical factors in
cancer and the immune system can inform the design of new immunothera-
pies and help to refine existing therapies to improve safety and efficacy.
This article is part of a discussion meeting issue ‘Forces in cancer: inter-
disciplinary approaches in tumour mechanobiology’.
1. IntroductionSpecific molecular interactions between two cells or a cell and extracellular
matrix are often viewed as pro-adhesive and ultimately favouring attachment.
However, specific interactions can also be inhibitory, as is the case for several tar-
gets for cancer therapy in the clinic. Both pro-adhesive and inhibitory
interactions can also involve important mechanobiological factors. Immune
cells provide particularly illustrative examples as they frequently contact cells
that either belong to ‘self’ (the same organism) or are ‘foreign’ (e.g. microbes
that breach epithelia). Specific molecular interactions at immune cell surfaces
lead to recognition of ‘self’ or else result in forceful attack and elimination of
‘foreign’. An important example with T cells is the protein PD-1, which interacts
with PD-L1 on multiple ‘self’ cells in parallel with T cell receptor interactions; if
PD-L1 activates the T cell to attack, PD-1 can effectively passivate it. In cancer,
blocking this PD-1 : PD-L1 checkpoint by systemic injections of antibodies to
either of these two proteins leads to T cell elimination of tumours in a minor frac-
tion (approx. 10–30%) of otherwise untreatable patients, and the patients that
respond best are those with the most mutated (i.e. ‘foreign’) tumours [1–4]. In
simplest molecular terms, the T cell receptor activates kinases that signal acti-
vation while PD-1 : PD-L1 activates a phosphatase (e.g. SHP isoform) that
dominates in its inhibition—although there remains much to learn. Mechano-
biology is involved at least via the kinases and/or phosphatases that regulate
macrophage target cancer cell(a)
royalsociety
2
local membrane mechanics on the small scale and/or cyto-skeletal function at a larger scale [5,6]. Importantly, this
paradigm of activation-dominated-by-inhibition applies not
only to other lymphocytes (e.g. natural killer (NK) cells [7])
but also to macrophages, which are the focus of this review.
– no opsonizing Ab– SIRPa : CD47 engaged– ‘self’ signalling
– opsonizing Ab– CD47 blocked– target engulfment
Fc receptor
opsonizing Ab
SIRPa
CD47
anti-CD47 Ab
(b)
Figure 1. ‘Self ’-signalling and opsonization in phagocytosis. Binding of CD47expressed by a target cancer cell to SIRPa on the macrophage surface signals‘self ’ to the immune cell and inhibits phagocytic clearance (a). Inhibition of‘self ’-signalling with an anti-CD47 antibody (Ab) in combination with atumour-opsonizing Ab that engages macrophage Fc receptors leads tophagocytosis of the target (b).
publishing.org/journal/rstbPhil.Trans.R.Soc.B
374:20180217
2. CD47 : SIRPa as a macrophage checkpointin cancer
‘Marker of self’ membrane protein CD47 is normally
expressed on all cells and binds with weak, sub-micromolar
affinity to ‘signal regulatory protein’ SIRPa on macrophages,
including precursor monocytes. CD47 : SIRPa binding leads
to local accumulation of SIRPa at phagocytic synapses and
ultimately to inhibition of engulfment of ‘self’ cells
(figure 1) [8,9]. This inhibitory interaction occurs in parallel
with various activating interactions, only some of which are
well characterized. The clearest example of activation is
through immunoglobulin G (IgG) antibodies which bind to
a target cell and which also engage activating Fc receptors
(FcRs) on macrophages. Some of the key FcRs signal via
kinases in very similar ways to integrins activated by extra-
cellular matrix, with a downstream accumulation of focal
adhesion proteins such as phospho-paxillin and talin as
well as sensitivity to whether the adhesive substrate—i.e.
target for phagocytosis—is soft or stiff [10,11]. However,
adhesion and phagocytosis of stiff targets more than soft is
just one aspect of the mechanosensitivity of macrophages.
CD47 : SIRPa blockade strategies have revitalized decades
of interest in macrophages as effector cells for cancer therapy.
CD47 is expressed on cancer cells [12,13] and was originally
described as the OA3 antigen, which is highly upregulated on
ovarian cancer cells [14]; CD47 should in principle shield
cancer cells from immune surveillance and removal by phagocy-
tic cells. However, solid tumours possess mechanical properties
that can influence cancer phenotypes [15] and that might also
influence immune cells, including macrophages. Studies in
mice, for example, suggest high collagen microenvironments
(associated with stiffness) cause macrophages to upregulate
SIRPa expression and also cause macrophages to switch off a
pro-phagocytic phenotype [16]. These factors motivate a
renewed focus on the mechanobiology of phagocytic cells
which certainly include macrophages but also neutrophils [17]
and dendritic cells [18], which both express SIRPa.
Patient safety is always a concern in therapy, and the
main goal of any Phase 1 clinical trial in the US is to establish
a safety window for dosing. Because CD47 is expressed on all
cells, anti-CD47 antibodies injected intravenously are readily
predicted to bind blood cells. Indeed, the pioneering studies
that first described ‘marker of self’ function showed that
CD47-deficient mouse red blood cells (RBCs) are engulfed
within hours of injection into normal mice by macrophages
in the spleen [9], with CD47-deficient platelets exhibiting
similar clearance [19]. CD47 knockout in at least one strain
of mouse (non-obese diabetic, NOD) leads not only to anae-
mia (RBC loss) but also to premature death of mice with
autoimmunity against RBCs [20]. None of these mouse
studies clearly determined what factors on a CD47-deficient
RBC or platelet activate the engulfment by normal splenic
macrophages, but anti-CD47 IgG will engage FcRs and
tend to activate phagocytosis. As with integrin-based
adhesion, however, a high density of adhesive ligands (IgG
in this case) favours focal adhesion complex formation and
activated adhesion, and CD47 is more of a low-density
signalling molecule on RBCs [8]. Regardless, the various find-
ings in mice underscore the importance of safety studies, and
the report of autoimmunity also has intriguing implications
for the development of anti-cancer immunity.
3. Clinical blockade of the anti-adhesiveCD47-SIRPa interaction—a current snapshot
According to the National Institutes of Health (NIH) database
of clinical trials (clinicaltrials.gov), there are presently 15
ongoing anti-CD47 interventional clinical trials, with all but
two in Phase 1 [21–35]. Although tumour cells are unlikely
to display sufficiently strong ‘eat me’ factors to activate macro-
phages [16], and modest upregulation of CD47 on tumours
(e.g. ovarian cancer [14]) is unlikely to present sufficient anti-
CD47 to activate phagocytosis, clinical trials all must start
with injections of just anti-CD47 to test safety without interfer-
ence. All current clinical trials targeting CD47 consist of
monoclonal antibodies or engineered fusion proteins and are
being driven by companies—no doubt because of the costs
involved. Designations for these agents are Hu5F9-G4 (Forty-
Seven Inc., Menlo Park, CA), CC-90002 (Celgene Corporation,
Summit, NJ), TTI-621 and TTI-622 (Trillium Therapeutics Inc.,
Mississauga, Ontario, Canada), SRF231 (Surface Oncology,
Cambridge, MA), ALX148 (ALX Oncology Inc., Burlingame,
CA) and IBI188 (Innovent Biologics, Suzhou, PR China). Clini-
cal trial details are summarized in table 1 (complemented by a
thorough review of preclinical information in [36]). Reporting
Tabl
e1.
Sum
mar
yof
ongo
ing
anti-
CD47
clini
calt
rials.
drug
nam
eco
nditi
ons
com
pany
tria
l
iden
tifier
a
stud
y
phas
est
atus
inte
rven
tion
type
Hu5F
9-G4
acut
em
yelo
idleu
kaem
iaFo
rty-S
even
,Inc
.NC
T026
7833
8Ph
ase
1co
mpl
eted
mon
othe
rapy
mye
lody
splas
ticsy
ndro
me
acut
em
yelo
idleu
kaem
ia,NC
T032
4847
9Ph
ase
1re
cruiti
ngco
mbi
natio
nw
ithaz
aciti
dine
mye
lody
splas
ticsy
ndro
me
colo
recta
lneo
plas
ms,
solid
tum
ours
NCT0
2953
782
Phas
e1b
/
2
recru
iting
com
bina
tion
with
cetu
ximab
solid
tum
ours
NCT0
2216
409
Phas
e1
recru
iting
mon
othe
rapy
non-
Hodg
kinlym
phom
a,in
dolen
tlym
phom
a,di
ffuse
large
B-ce
lllym
phom
a
NCT0
2953
509
Phas
e1b
/
2
recru
iting
com
bina
tion
with
ritux
imab
solid
tum
ours,
ovar
ianca
ncer
Forty
-Sev
en,I
nc.,
with
Mer
ck
Celg
ene
NCT0
3558
139
Phas
e1
recru
iting
com
bina
tion
with
avelu
mab
CC-9
0002
acut
em
yelo
idleu
kaem
ia,
mye
lody
splas
ticsy
ndro
me
NCT0
2641
002
Phas
e1
term
inat
ed
(Oct.
2018
)
mon
othe
rapy
haem
atol
ogic
neop
lasm
sNC
T023
6719
6Ph
ase
1re
cruiti
ngco
mbi
natio
nw
ithrit
uxim
ab
TTI-6
21ha
emat
olog
icm
align
ancie
s,so
lidtu
mou
rsTr
illium
Ther
apeu
tics,
Inc.
NCT0
2663
518
Phas
e1
recru
iting
mon
othe
rapy
,com
bina
tion
with
ritux
imab
,com
bina
tion
with
nivo
lum
ab
solid
tum
ours,
myc
osis
fung
oides
,
mela
nom
a,M
erke
l-cell
carci
nom
a,
squa
mou
sce
llca
rcino
ma,
HPV-
relat
ed
mali
gnan
tneo
plas
m,s
oftt
issue
sarco
ma
NCT0
2890
36Ph
ase
1re
cruiti
ngm
onot
hera
pyvs
vario
usco
mbi
natio
nth
erap
ies(P
D-1
:PD-
L1in
hibi
tor,
pegy
lated
inte
rfero
n-a
2a,T
-Vec
,rad
iation
)
TTI-6
22lym
phom
a,m
yelo
ma
NCT0
3530
683
Phas
e1
recru
iting
mon
othe
rapy
vsva
rious
com
bina
tion
ther
apies
(PD-
1:P
D-L1
inhi
bito
r,rit
uxim
ab,p
rote
asom
e-in
hibi
torr
egim
en)
SRF2
31ad
vanc
edso
lidca
ncer
s,ha
emat
olog
icca
ncer
sSu
rface
Onco
logy
NCT0
3512
340
Phas
e1
recru
iting
mon
othe
rapy
ALX1
48m
etas
tatic
canc
er,s
olid
tum
our,
adva
nced
canc
erno
n-Ho
dgkin
lymph
oma
ALX
Onco
logy
,Inc
.NC
T030
1321
8Ph
ase
1re
cruiti
ngco
mbi
natio
nw
ithpe
mbr
olizu
mab
,tra
stuzu
mab
,orr
ituxim
ab
IBI1
88ad
vanc
edm
align
ancie
sIn
nove
ntBi
olog
icsNC
T037
6314
9Ph
ase
1re
cruiti
ngm
onot
hera
py
adva
nced
mali
gnan
cies
NCT0
3717
103
Phas
e1
recru
iting
mon
othe
rapy
vsco
mbi
natio
nw
ithrit
uxim
ab
a NCT
num
berf
rom
clini
caltr
ials.g
ovda
taba
se.T
able
data
curre
ntas
ofM
arch
2019
.
royalsocietypublishing.org/journal/rstbPhil.Trans.R.Soc.B
374:20180217
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royalsocietypublishing.org/journal/rstbPhil.Trans.R.Soc.B
374:20180217
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on these trials remains sparse, but details are emerging in meet-ing abstracts and on company websites. Such information is
public but should be treated with caution as it is not clear that
companies will publish in peer-reviewed forums, though pub-
lications are now beginning to appear. Nonetheless, the
information provides some initial insight into the potential
safety and efficacy of CD47 blockade therapies in humans.
Hu5F9-G4 is a humanized anti-CD47 monoclonal IgG4
antibody and is the most advanced therapeutic in this class to
progress through the clinic [37,38]. The Fc end of an IgG4
engages FcRs only weakly; one might therefore expect minimal
interactions with FcRs on splenic macrophages and perhaps
other macrophages. In one of the most advanced trials of
Hu5F9-G4, the Phase 1b/2 study for relapsed/refractory non-
Hodgkin lymphoma (r/r NHL), patients combined anti-CD47
with rituximab (anti-CD20) to provide an ‘eat me’ signal to acti-
vate macrophages; the reported objective response rate (ORR)
was 50% and the complete response rate (CRR) was 32% [39].
These numbers were revised in late 2018 to 40% ORR and 33%
CRR in 15 diffuse large B-cell lymphoma patients and 71%
ORR and 43% CRR in seven follicular lymphoma patients [40].
The investigators also noted that they were able to limit anaemia
by the administration of a priming dose to clear older RBCs prior
to maintenance doses, though to what extent is unclear.
In Phase 1a of the trial for relapsed/refractory acute
myeloid leukaemia (r/r AML), 15 patients tolerated
Hu5F9-G4 well with no maximum tolerated dose (MTD)
reported. However, just two patients exhibited biological
activity in response, which the authors defined as ‘significant
reduction in marrow cellularity observed similarly in pre-
clinical models’. The majority of the studied patients (79%)
showed grade 3 anaemia prior to dosing in the study and
also received transfusions of RBCs [41]. In prior reported
safety results of 13 patients [42], the maximum observed hae-
moglobin (Hb) drop was 5.2 g dl21, representing a loss of
about 40% of RBCs, though the median decrease was
1.2 g dl21. Antibodies are of course bivalent, and some anti-
RBC antibodies have long been known to drive cross-linking
of RBCs (haemagglutination); such adhesive clusters of cells
can be expected to have impaired circulation, especially
through the narrow slits of the spleen—which would tend
to favour macrophage interactions and anaemia. Forty-
Seven, Inc. has also initiated additional trials against other
malignancies to test Hu5F9-G4 in combination with the che-
motherapeutic azacitidine, the anti-epidermal growth factor
receptor (EGFR) monoclonal antibody cetuximab, and the
PD-L1 targeting antibody avelumab [22,25,28]. Later reports
in the first-in-human trial with advanced solid tumours
indicated a priming dosing led to 57 and 36% of patients
showing transient anaemia and haemagglutination, respect-
ively, mostly grade 2 [43,44]. Whether the loss of RBCs can
ever contribute to an autoimmune response to RBCs or
other cell types has not been addressed.
TTI-621 is a fully human recombinant SIRPa-Fc fusion
protein developed by Trillium Therapeutics, Inc. with preclini-
cal evidence for enhanced phagocytosis in AML and B
lymphoma xenograft models [45]. It consists of the N-terminal
V-type immunoglobulin-like domain of human SIRPa as a
fusion with the Fc portion of human IgG1. IgG1 is expected
to bind FcRs more strongly than IgG4 and may increase
both antibody-dependent cell-mediated cytotoxicity and
complement-dependent cytotoxicity effects. It is being admi-
nistered intravenously for haematologic malignancies and
intratumorally for solid tumours and mycosis fungoides
[46]. Early reports in late 2016 showed two patients with
dose-limiting toxicities in grade 3 elevated liver enzymes
and grade 4 thrombocytopenia (loss of platelets). This result
raises questions of the drug safety profile, although later the
company stated that this thrombocytopenia was transient
and reduced after multiple infusions. The company also
reported that 9 out of 10 patients in the intratumorally admi-
nistered trial saw a reduction of lesions [46], and later, 15 out
of 17 r/r mycosis fungoides and Sezary syndrome patients
were described as having measurable improvement in lesion
severity [47]. They have expanded their study to new cohorts
receiving TTI-621 in combination with approaches such as a
PD-1 : PD-L1 inhibitor or radiation therapy. A second candi-
date, linked to an IgG4 domain instead of IgG1, TTI-622,
was initiated in a Phase 1a/1b clinical trial in mid-2018 [33].
SRF231 is a fully human anti-CD47 antibody being evalu-
ated against advanced solid tumours and lymphoma/chronic
lymphocytic leukaemia [48]. CC-90002 is a humanized
anti-CD47 monoclonal antibody of the IgG4 subclass in
early clinical development, with preclinical work presented
at the 2017 ASCO Annual Meeting [49]. Both CC-90002 and
SRF231 seem to avoid haemagglutination, but how this is
achieved is unclear [50]. Notably, despite the expansion of
anti-CD47 efforts by pharmaceutical companies, Celgene
terminated its monotherapy trial of CC-90002 in October
2018 while continuing to recruit in its combination trial with
rituximab [34,35]. This further highlights the importance
of combining an activating signal for phagocytosis with
blockade of the macrophage checkpoint.
ALX148 is a fusion protein with two CD47 binding
domains derived from the SIRPa N-terminal D1 domain
and an inactive Fc region designed to mitigate haemaggluti-
nation and anaemia [51]. The presence of an inert Fc region
also indicates its design as a combination therapeutic with
tumour-opsonizing antibodies, unlike agents discussed thus
far which can directly engage FcRs on the macrophage
surface to mediate effector function [52]. Reports at the 2018
ASCO Annual Meeting showed data from the first 30 patients
enrolled, 25 of whom received only ALX148, with the remain-
ing five patients receiving combination regimens with
pembrolizumab (three patients), trastuzumab (one patient)
or rituximab (one patient). ALX148 was generally tolerated,
with four combination patients achieving stable disease
states though two patients exhibited grade 3 thrombocytopenia
[53].
IBI188 is a fully human anti-CD47 monoclonal IgG4 anti-
body and is the most recent drug to move from the preclinical
phase to Phase 1 trials, where it is currently being evaluated
against advanced malignancies as a monotherapy and in
combination with rituximab [26,27]. The trials dosed their
first patients in late February 2019. Blockade of CD47 :
SIRPa in patients is thus being pursued by a growing list of
companies even though single agent efficacy is not especially
compelling. Safety issues that are most easily measured and
that are widely reported include significant anaemia, throm-
bocytopenia and haemagglutination, which must be balanced
in turn with establishing efficacy—most often by combining
with an IgG that binds to abundant antigens on a tumour
cell and thereby strongly activates adhesion-initiated phago-
cytosis. Consequently, the drug space aimed at CD47 :
SIRPa has expanded dramatically, with significant
investment in new antibodies, small molecules and peptides.
royalsocietypublishing.org/journal/rstbPhil.Trans.R.Soc.B
374:20180217
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4. Macrophage checkpoint blockade plusopsonizing antibodies—initial success withliquid tumours, but are solid tumours next?Targeting the SIRPa side of the CD47 : SIRPa interaction
[54,55] rather than CD47 has not yet been attempted in the
clinic but could prove analogous to blocking PD-1 (on the
T cell), which shows clinical efficacy, and certainly, preclini-
cal anti-SIRPa development efforts are encouraging [50].
What is appearing clinically is the utility of combination
therapies with tumour-specific opsonizing antibodies that
seek to strongly shift from anti-phagocytic signals to pro-
phagocytic activity. This is especially interesting for patients
refractory to tumour-specific monotherapies such as rituxi-
mab. The combination approach is now seen in virtually all
of the trials discussed above: Hu5F9-G4 with rituximab,
cetuximab and avelumab, CC-90002 with rituximab, and
ALX148 with trastuzumab (anti-HER2) and rituximab, to
name a few (complete list in table 1). It is increasingly clear
that elimination of non-‘self’ requires an ‘eat me’ cue, often
by IgG opsonization and subsequent engagement of macro-
phage FcRs, in addition to the ‘don’t eat me’ signal of
CD47 : SIRPa. In further recognition of the merits of this
phenomenon, bispecific antibodies containing CD47-specific
and tumour-specific (e.g. mesothelin, CD20 or CD33)
domains are in preclinical development (reviewed in-depth
in [56]), while anti-SIRPa antibodies may also provide
opsonization on the tumour cell surface.
Effective clearance by innate immune effector cells relies
on much more than one or two cell surface interactions.
Some immunotherapies have shown unprecedented success
in generating durable cures to blood cancers, but have fal-
tered in treatment of solid, stiff tumours. Macrophages
engulf foreign material in all types of native tissues and
thus seem tailor-made to enter and eat solid tumours; it
will therefore be very interesting to see whether the success
with anti-CD47 and rituximab against liquid tumours trans-
lates to solid tumours. CD47 : SIRPa’s regulatory role in
phagocytosis and physical differences in the tumour microen-
vironment between haematological and solid malignancies
motivate a deeper understanding of the mechanobiological
underpinnings of phagocytosis in healthy and pathological
processes. Cells in solid tumours generally adhere strongly
to each other and/or to the extracellular matrix—which is
not true of liquid tumours and which makes it physically
more challenging for a macrophage to completely engulf a
cancer cell integrated into a solid tumour. Knowledge of
mechanosensing by phagocytes is likely to help maximize
the utility of macrophage checkpoint therapies and macro-
phage functions more broadly. The following sections
describe some of the physical factors to consider in macro-
phage biology (figure 2a) and how that information might
be used to inform therapeutic design.
5. Forces in phagocytosis and ‘self ’-signallingPhagocytosis is an ancient process that proceeds by the exten-
sive reorganization of the actin cytoskeleton and (often) via
contractions mediated by myosin motors. Myosins bind,
cross-link and pull on actin filaments, and myosins are well
known to be load-sensitive (i.e. contractile velocity decays
with force), which provides a basis for mechanosensitive
interactions. As with integrin-mediated adhesion, target par-
ticle or cell binding to surface receptors frequently leads to
receptor clustering and activates signalling pathways that
drive maturation of adhesions via actomyosin activity: for
macrophages, this involves the formation of pseudopod pro-
trusions around the target, with actin polymerization and
branching generating forces of protrusion [57]. Although
the pro-phagocytic signals mediating cancer cell engulfment
in the presence of CD47 : SIRPa blocking agents have not
been fully elucidated, it is clear that phagocytosis is improved
with an opsonizing antibody, suggesting an FcR-mediated
mechanism [55,58]. Other pro-phagocytic signals may also
be involved, depending on the target. Phagocytosis of hae-
matopoietic cancer cells by macrophages is reported to
depend on Mac-1 (complement receptor-3, CR3 or aMb2
integrin), which binds to an unknown receptor on its target,
along with a homotypic SLAMF7 interaction and signalling
through ITAM-containing co-receptors [59]. Calreticulin is
also proposed to be a pro-phagocytic signal recognized by
scavenger receptor LRP-1 on macrophages [60]. These pro-
phagocytic cues are an emerging theme in CD47 : SIRPa
blockade trials and may be especially critical for the success
of these agents in shrinking solid tumours.
Several myosin motor proteins localize at phagocytic
synapses (figure 2b) [61–64]. The role of myosin in FcR- and
complement receptor-mediated phagocytosis has been investi-
gated by pharmacological inhibitors as well as overexpression
and knockdown of non-muscle myosin IIa (NMIIA). Target
engulfment is deficient in mouse bone marrow-derived
macrophages and macrophage cell lines treated with inhibi-
tors of myosin light chain kinase (i.e. ML7) or myosin (i.e.
2,3-butanedione monoxime and blebbistatin) [8,62,65,66].
Similarly, the human monocytic cell line THP-1 internalizes
‘non-self’ sheep RBCs less efficiently when transfected with
small interfering (siRNA) against NMIIA and more efficiently
when NMIIA is overexpressed [8]. Scanning electron
microscopy revealed that ML7-treated cells still form phagocy-
tic cups bound to IgG-opsonized sheep RBCs, but the
membrane protrusions are not as closely associated with the
target as in the untreated control [67]. Thus, it appears that
actin assembly and membrane protrusion occur indepen-
dently of myosin IIA in FcR-mediated phagocytosis, but
mature or stable adhesions might require contractility through
NMIIA. By contrast, in complement receptor-mediated phago-
cytosis, NMIIA is implicated even earlier in the process and
mediates actin organization with a requirement for
Rho-ROCK signalling [65]. Other myosins, including class I
myosins and myosin X, have been observed to participate in
various stages of phagocytosis and have potential roles in
phagosome formation and closure [62–64].
Cell spreading on matrix-coated substrates uses many of
the same cellular components as phagocytosis, and cells typi-
cally exhibit greater spreading on stiff substrates than on soft
ones. Similarly, the efficiency of phagocytosis is determined
in part by the stiffness of the target, with softer targets
regarded as more difficult to phagocytose than stiff targets,
a factor to consider in cancer cell engulfment. This has been
demonstrated with engineered hydrogel microbeads [10,68]
and chemically stiffened RBCs [11]. In the light of this
effect, control of particle stiffness has risen as a strategy to
increase the circulation time of drug-loaded nanocarriers
through delaying phagocytic clearance by splenic and liver
macrophages [68,69]. Prolonging clearance has also been
CD47 engages SIRPa:– deactivation of myosin IIa– inhibition of phagocytosis
SIRPa : CD47
anti-CD47 Ab myosin IIa
actin
Fc receptor and opsonizing Ab
Solid tumour mechanics:– cell–ECM adhesion– cell–cell adhesion– target stiffness
Polarizationanti-inflammatory
pro-inflammatoryCD206
MHCII
IL-4, IL-10, etc.
TNFa, etc.
Matrix mechanosensing
lamin A, C
fibrous matrix
podosome adhesion
lamin B1, B2
CD47 antibody blockade:– myosin IIa contractility– efficient phagocytosis
(a)
(b)
P P
P
SHP-1– – –
+
+++
+++
P
PP
PP
PP
PP
PP
Phagocytosis:– ‘eat me’ versus ‘don’t eat me’– cytoskeletal force
Figure 2. Summary of macrophage mechanobiology and forces in phagocytosis. (a) Many factors play roles in macrophage interactions and the immune cells’ abilityto clear ‘foreign’ matter. In solid tumours, macrophages must contend with cell – matrix and cell – cell adhesions that can influence their ability to engage and eatcancer cells. Macrophages sense matrix properties through podosome adhesions and exhibit lamin A levels that scale with matrix stiffness. Such matrix to nucleusmechanosensing might influence macrophage polarization characterized by different cytokine secretions and surface marker expression. (b) Engagement of CD47 bySIRPa recruits the phosphatase SHP-1 to the phagocytic synapse in the macrophage where it inhibits myosin IIa assembly (top). In blockade therapy, activatingsignals from the clustering of Fc receptors drive cytoskeletal reorganization and myosin IIa assembly which promote phagocytosis. (Online version in colour.)
royalsocietypublishing.org/journal/rstbPhil.Trans.R.Soc.B
374:20180217
6
accomplished by decorating particles or viruses with
recombinant CD47 or small ‘self’-peptide mimetics [70,71].
When CD47 on a target cell (or engineered particle or
virus) engages SIRPa on macrophages, the cytoplasmic
immunoreceptor tyrosine inhibitory motif (ITIM) of SIRPa
is phosphorylated and recruits the phosphatase SHP-1
(figure 2b) [72]. NMIIA is a direct target of SHP-1, which inhi-
bits NMIIA accumulation at the phagocytic synapse and
prevents the macrophage from efficiently engulfing the
target [8]. For very stiff opsonized targets, however, such as
dialdehyde-cross-linked RBCs, NMIIA becomes hyperacti-
vated at the phagocytic synapse in macrophages and
inhibitory signalling is unable to prevent target engulfment
[11]. This could have implications for the phagocytosis of
cancer cells and for cancer treatment. Cancer cells can be
softer than normal cells [73], which together with increased
expression of CD47 [12,13] could allow transformed cells to
evade phagocytosis. Alternatively, stiffening of cancer cells
following chemotherapy could make pre-treated tumours
more susceptible to phagocytosis and macrophage check-
point therapies [74]. Future progress on this subject could
identify biophysical signatures of cancer cells that relate to
response to anti-CD47 therapy for predictive or prognostic
purposes.
The interaction of CD47 and SIRPa on juxtaposed
membranes is also governed by physical forces, including
royalsocietypublishing.org/journal/rstbPhil.
7
fluctuations of the flexible plasma membrane. In cell-derivedgiant vesicles displaying human CD47, vesicle spreading and
CD47 accumulation was observed on SIRPa-coated coverslips
[75]. The apparent binding affinity increased with the concen-
tration of CD47 : SIRPa complexes, indicating a cooperative
interaction that arises from a decrease in out-of-plane mem-
brane fluctuations. As a result, even low expression levels of
CD47 or SIRPa alleles with low binding affinity may be
able to achieve sufficient levels of ‘self’-signalling to limit pha-
gocytosis. Decreasing the pH to mimic acidosis that is
frequently observed in tumours rigidifies the vesicle mem-
brane and decreases cooperativity. It is speculated that such
a loss of cooperativity could permit macrophages to phagocy-
tose cells expressing low levels of CD47, and in doing so select
for cancer cells expressing high levels of CD47 [75].
Trans.R.Soc.B374:201802176. Macrophage mechanosensing from matrix tonucleus and SIRPa
In cancer clearance, monocytes and macrophages face a
number of challenges presented by the tumour microenviron-
ment; they must be able to extravasate through small or large
pores, differentiate to a phagocytic phenotype, resist repro-
gramming to an anti-phagocytic state, and ultimately, eat
their targets. This process requires the ability to probe and
respond to the mechanical forces surrounding the cells.
Macrophages lack focal adhesions and stress fibres but form
podosome adhesions with the extracellular matrix that are
capable of sensing the environment [76]. These structures
contain a protruding branched actin core and peripheral acto-
myosin cables attached to integrins through adapter proteins,
and multiple podosomes can be connected into higher-order
structures. Force generation in podosomes involves actin
polymerization in the core and myosin contractility, which
allows cells to probe substrate stiffness [76]. Human mono-
cyte-derived macrophages form more podosomes on stiff
substrates than on soft substrates, and phosphorylation of
myosin light chain and force generation are also correlated
with substrate stiffness [77,78]. How the mechanical stimuli
sensed at podosomes are transduced to alter gene expression
remains unclear but is likely to be critical in understanding
macrophage behaviour and how the tumour microenviron-
ment can directly or indirectly affect the cells’ ability to clear
‘foreign’ targets.
Mechanosensing macrophages exhibit differential spread-
ing, migration and polarization, depending on the physical
properties of the matrix. Alveolar macrophages from rats
are rounded when cultured on soft epithelial monolayers
but flatten and spread on polyacrylamide gels of intermediate
compliance and stiff glass substrates [79]. Similarly, human
monocyte-derived macrophages are more spread on stiff
polyacrylamide gels coated with fibronectin than on soft
ones [80]. A biphasic relationship between cell area and
substrate stiffness has also been reported for human macro-
phages, with a maximal area reached at an intermediate
stiffness [81]. Both studies observed stiffness effects on
macrophage migration in two dimensions. In three
dimensions, human monocyte-derived macrophages exhibit
different migration modes, depending on the structure of
the matrix, but seem to be less sensitive to matrix stiffness
than to matrix organization [82]. In Matrigel or a dense col-
lagen gel, macrophages underwent mesenchymal migration
that was slow and dependent on protease activity. In a fibril-
lar collagen matrix, macrophages underwent amoeboid
migration, which was faster and protease-independent. Podo-
some-like protrusions with collagen-degrading activity were
observed during mesenchymal migration through collagen gel
but not during amoeboid migration through fibrillar collagen.
Macrophages exhibit considerable plasticity with respect
to their polarization or activation states [83]. Very broadly,
macrophages can be polarized to a classical, inflammatory
state or to alternatively activated states with anti-inflamma-
tory or wound healing properties. Biophysical cues,
including matrix stiffness, topography and external forces,
can potentially influence macrophage polarization (reviewed
in [84,85]), although further research into their effects is still
needed. In particular, investigations of the effects of matrix
stiffness on macrophage polarization have reached very
different conclusions as to whether stiffness promotes an
inflammatory or alternatively an activated phenotype [86–91].
This may be due in part to different sources of macrophages
(primary cells versus cell lines), to different substrates (syn-
thetic polymer scaffolds, collagen gels, etc.) and adhesive
ligands (fibronectin, collagen), and to simultaneous acti-
vation with different cytokines (e.g. IFNg, IL-4/IL-13) or
inflammatory signals (e.g. LPS). Alternatively, differences
could be due to the inadequacy of current classification
methods to describe the full range of macrophage polariz-
ation or to the relatively weak effect of substrate stiffness
on this phenotype. Given the altered matrix and different
physical cues potentially present in solid tumours, the effi-
cacy of macrophage checkpoint blockade may be strongly
influenced by the macrophages’ ability to sense and respond
to this matrix while maintaining a pro-phagocytic phenotype.
Several studies have noted a relationship between
macrophage shape and polarization [92,93]. Polarization of
macrophages toward an inflammatory phenotype with lipo-
polysaccharide (LPS) and interferon-g (IFNg) produces
round cells while alternative activation with interleukin-4
and interleukin-13 produces elongated cells [92]. The converse
also appears to be true. When mouse bone marrow-derived
macrophages are cultured on micropatterned lines of fibronec-
tin, they become elongated and upregulate expression of anti-
inflammatory markers Arg-1 (arginase-1) and CD206 while
downregulating inflammatory markers iNOS (inducible
nitric oxide synthase) and IFNg [92]. Pharmacological
inhibition of the actin cytoskeleton with cytochalasin D or cel-
lular contractility with blebbistatin, ML-9 or Y27632 attenuates
this polarization. Perhaps consistent with the effects of shape
on macrophage polarization, the moderate cyclic strain of
macrophages on polymeric scaffolds [94] and interstitial flow
shear forces [95] seem to promote alternative activation.
With some exceptions, tissue-resident macrophages orig-
inate from yolk sac- or fetal liver-derived haematopoietic
cells during embryonic development [96]. In response to
injury or inflammation, monocytes can also be recruited to
tissues from circulation and differentiated into macrophages.
Transcriptomic and epigenetic analyses have revealed that
macrophage phenotype is tailored by the local microenviron-
ment [97,98]. Whether biophysical cues including matrix
stiffness contribute to tissue-specific phenotypes in macrophages
remains unclear but plausible.
Macrophages reside in tissues that span a wide range of
stiffnesses. For example, microglia are the resident macro-
phages in brain tissue, which is very soft with an elastic
royalsocietypublishing.org/journal/rstbPhil.Trans.R.Soc.B
374:20180217
8
modulus of approximately 0.3 kPa. Osteoclasts are the resi-dent macrophages on bone tissue, which is much stiffer
with an elastic modulus of greater than 30 kPa (non-calcified,
and far higher for calcified). Using mass spectrometry-based
proteomics, it was demonstrated that the nuclear intermediate
filament protein lamin A, but not lamin B, is mechanosensi-
tive [99]. Specifically, lamin A protein levels measured in
mouse tissue lysates exhibit a power-law scaling with tissue
elasticity E while lamin B levels remain nearly constant. In a
meta-analysis of transcriptomic data from different mouse
tissue-resident macrophages, the ratio of lamin A to lamin B
mRNA (LMNA : LMNB) also exhibited power-law scaling
with E [100]. Consistent with this finding, immunofluores-
cence staining of lamin A was greater in phorbol myristate
acetate (PMA)-differentiated THP-1 cells when cultured on
stiff polyacrylamide gels than when cultured on soft gels [16].
The increased stiffness of collagenous tumours relative to
normal tissue has implications for lamin A expression in
tumour-associated macrophages (TAMs) and for macro-
phage-adoptive transfer therapies. For example, the LMNA :
LMNB transcript ratio in macrophages isolated from subcu-
taneous A549 lung cancer xenografts conformed to the
power-law scaling relationship with the measured tumour
stiffness [16]. This suggests that TAMs are mechanosensitive
and that increased stiffness in tumours could in principle con-
tribute to the reprogramming of monocytes recruited from
circulation or tissue-resident macrophages to TAMs. In the
macrophage checkpoint context, high-collagen microenviron-
ments in vivo (associated with stiffness) and stiff gels in vitrocause macrophages to upregulate SIRPa expression and also
cause macrophages to switch off a pro-phagocytic phenotype
[16]. For therapy, this likely means that more blockade anti-
body will be required and perhaps more opsonizing
antibody to activate a macrophage.
7. Macrophage infiltration and differentiationWithin dense, fibrotic tissues, including collagenous tumours,
extracellular matrix fibres create constrictions that are generic
barriers to three-dimensional migration. As the largest orga-
nelle in the cell, the nucleus limits migration through
narrow constrictions and must be deformed to pass through
pores smaller than the nuclear diameter. Thus, by controlling
the deformability and mechanical integrity of the nucleus,
lamin expression and scaling with tissue elasticity have impli-
cations for tumour infiltration and macrophage migration as
well as egress from marrow. Lamin levels in haematopoietic
cells are indeed relevant to migration through small pores
such as those encountered while trafficking from the bone
marrow into circulation or from circulation into tissue
[99,101].
Three-dimensional migration through Transwell filters
with 3–8 mm diameter pores has served as an in vitromodel of constricted migration [102,103]. High levels of
lamin A protect the nucleus but can also limit migration
[102]. Nuclear rupture and cell death have been observed in
human monocyte-derived dendritic cells migrating through
2 mm constrictions in microfluidic channels [104]. Whether
nuclear rupture occurs frequently during macrophage
migration in vivo is unknown, but the leakage of DNA into
the cytoplasm or formation of micronuclei could have signifi-
cant inflammatory effects by activating the cGAS/STING
pathway of interferon activation [104,105]. Infiltration is
thus physically modulated but has implications for cell fate
and inflammation—which are likely to impact the success
of checkpoint blockade.
Lamin A has also been described as a differentiation
marker in the monocyte lineage. Early studies showed
lamin A and the alternative splicing product lamin C are
undetectable in rat bone marrow-derived precursor cells but
increase significantly following in vitro differentiation to
monocytes and macrophages [106]. A similar increase was
observed during the in vitro differentiation of human periph-
eral blood monocytes to macrophages. Differentiation of the
HL-60 cell line to monocyte- and macrophage-like cells by
PMA increases levels of both lamin A and B [107]. Lamin A
and B expression and the A : B ratio vary greatly during hae-
matopoietic maturation in vivo as measured by mass
spectrometry-calibrated, intracellular flow cytometry of
freshly isolated human marrow and blood cells [108]. While
the total lamin levels decrease slightly or remain approxi-
mately constant between human marrow progenitor cells
(CD34þ CD38þ, CD34þ CD382) and peripheral blood or
marrow monocytes and granulocytes, the lamin A : B ratio
increases due to downregulation of lamin B and upregulation
of lamin A. Flow cytometry measurements also revealed that
the total amount of lamin A and B is lower in haematopoietic
cells that traffic into circulation (e.g. lymphocytes and periph-
eral blood granulocytes and monocytes) than cells residing
primarily in the bone marrow compartment (e.g. CD34þ pro-
genitor cells, erythroblasts, megakaryocyte lineages and
mesenchymal stem cells). Like cancer cells, the constricted
migration of haematopoietic cells through Transwell pores
approximating marrow sinusoidal capillaries is strongly
influenced by lamins [108]. Through such processes and
others, lamin A levels can also change during macrophage
activation in vivo. Mouse peritoneal macrophages collected
5 days after stimulation with thioglycollate stain strongly
for lamin A/C whereas unstimulated macrophages do not
[106]. Together, these results indicate that the nuclear
lamins, which are key components of the matrix to nuclear
mechanosensing pathway, exhibit different expression
during lineage maturation and across different tissues. It
will be important to determine whether such changes are
accompanied by changes in levels of SIRPa.
8. ConclusionThe CD47 : SIRPa axis is an intriguing, rapidly emerging
therapeutic target involving innate immune macrophages. It
is likely dependent not only on strongly opsonizing anti-
bodies (such as rituximab) but also on other processes and
factors, including mechanical ones. Safety issues include
clearance of RBCs and platelets, and these mechanically
related processes and factors could play a role. Phagocytic
effector cells are well known to be sensitive to their microen-
vironment, which motivates the careful study of the physical
forces involved in phagocytosis and the broader mechano-
biology of macrophages, including precursor monocytes.
Matrix mechanics in solid tumours affect cancer cells and
likely immune cells as well, and physical forces also govern
the level of SIRPa as well as the engagement of CD47 :
SIRPa, which inhibits phagocytosis. Moreover, the inflamma-
tory state of a macrophage depends on biophysical cues
royalsocietypub
9
including local matrix stiffness and external forces such asfluid shear, which also influence the ability of these cells to
infiltrate tumours and phagocytose cancer cells. Therapeutic
designs that use macrophages and other phagocytes as effector
cells are likely to be advanced by recognition and careful
consideration of the relevant forces and related physical factors.
lishing.org/jour
Data accessibility. Clinical trial information is available at https://clinical-trials.gov. Screenshots of cited conference abstracts and press releasesare available in the electronic supplementary material.
Authors’ contributions. All of the authors contributed to drafting andrevising the manuscript and gave their approval for the final versionto be published. J.C.A. and L.J.D. contributed equally to this work.
Competing interests. The authors declare no competing interests.
Funding. This work was supported by the National Cancer Institute ofthe National Institutes of Health under U54CA193417 (to D.E.D.) andF32CA228285 (to L.J.D). J.C.A. was supported by the NationalScience Foundation Graduate Research Fellowship Program underDGE-1845298. The content of this article is solely the responsibilityof the authors and does not necessarily represent the official viewsof the National Institutes of Health nor the National ScienceFoundation.
nal/rstbPh
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The macrophage checkpoint CD47:SIRP for recognition of ‘self’ cells: from
clinical trials of blocking antibodies to mechanobiological fundamentals
Jason C. Andrechak1,2, Lawrence J. Dooling1 and Dennis E. Discher1
1Biophysical Engineering Labs and 2Bioengineering Graduate Group, University of
Pennsylvania, Philadelphia, PA, USA
Author for correspondence:
Dennis E. Discher
e-mail: [email protected]
Supplemental Material
Contains: information about referenced abstracts and press releases
Figure S1: Screenshot of conference abstract for the 2018 American Society of Clinical Oncology
(ASCO) Annual Meeting (reference 39, Advani et al.).
Figure S2: Screenshots of conference abstract for the 23rd Congress of the European Hematology
Association (reference 41, Agoram et al.).
Figure S3: Screenshots of conference abstract for the 23rd Congress of the European Hematology
Association (reference 42, Brierley et al.).
Figure S4: Screenshots of April 2018 press release from Trillium Therapeutics Inc. (reference
46). Red highlighted box indicates downloaded press release.
Figure S5: Screenshots of conference abstract for the 2018 American Society of Hematology
Annual Meeting (reference 47, Querfeld et al.).
Figure S6: Screenshot of conference abstract for the 2016 American Society of Hematology
Annual Meeting (reference 48, Holland et al.).
Figure S7: Screenshot of conference abstract for the 2017 American Society of Hematology
Annual Meeting (reference 51, Kauder et al.).
Figure S8: Screenshot of conference abstract for the 2018 ASCO Annual Meeting (reference 53,
Lakhani et al.).
Forces in cancer: interdisciplinary approaches in tumour mechanobiology
A Discussion Meeting issue organized and edited by Chris Bakal and Julia Sero Published July 2019