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: discher@seas.upenn.edu

& 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

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mon

othe

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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

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1re

cruiti

ngm

onot

hera

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adva

nced

mali

gnan

cies

NCT0

3717

103

Phas

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recru

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mon

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vsco

mbi

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a NCT

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able

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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-derived

giant 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.B374:20180217

6. 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 as

fluid 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/rstb

Ph

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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: discher@seas.upenn.edu

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