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TARGETING MACROPHAGES: THERAPEUTIC
APPROACHES IN CANCER
Luca Cassetta1 and Jeffrey W. Pollard1,2 *
1MRC Centre for Reproductive Health, College of Medicine and
Veterinary Medicine, Queen’s Medical Research Institute, The
University of Edinburgh, Edinburgh, UK
2Department of Developmental and Molecular Biology, Albert
Einstein College of Medicine, New York, USA
Correspondence: [email protected],
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“The concept that has emerged over the past century suggests that macrophages
represent an evolutionarily ancient, dispersed, homeostatic system, on a par with
the nervous and endocrine systems (…) Macrophages are essential for survival and
provide an attractive target to manipulate the host, for both immunologic and
metabolic purposes”
Prof. Siamon Gordon, The macrophage, past, present, future, 2007.
Abstract
Infiltration of macrophages in solid tumours is associated with poor prognosis
and correlates with chemotherapy resistance in most cancers. In mouse models
of cancer, macrophages promote cancer initiation and malignant progression by
stimulating angiogenesis, enhancing tumour cell migration, invasion and
intravasation and by suppressing anti-tumour immunity. At metastatic sites,
macrophages promote tumour cell extravasation, survival and subsequent
growth. Each of these pro-tumoral activities are promoted by a sub-population of
macrophages that express canonical markers but have unique transcriptional
profiles, which makes tumour-associated macrophages (TAMs) good targets for
anti-cancer therapy in humans either through their ablation or re-differentiation
away from pro-tumoral to anti-tumoral states. In this review, we evaluate the
state of the art of TAM-targeting strategies, focusing on the limitations and
potential side effects of the different therapies such as toxicity, rebound effects
and compensatory mechanisms. We provide an extensive overview of the
different types of therapy used in the clinic and their limitations in light of
known macrophage biology, and propose new strategies to targeting TAMs.
Introduction
Resistance to cancer treatment can be intrinsic to the tumour cells but it is often
conferred by non-malignant cells that comprise the tumour microenvironment
(TME). The TME is composed of tissue resident cells and a large proportion of
recruited immune cells that can constitute up to 50% of the tumour mass in
certain solid tumours such as breast cancer. Original theories assumed that these
immune cells were part of the body’s response to reject tumours. Indeed, it is
still proposed that at the earliest stage of tumor onset, the immune system reacts
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to the presence of cancer by activating T cells and macrophages, which clear the
tumour and reduce the incidence of cancer 1. But once tumours progress past
this initial state, the immune tumor microenvironment is modified to support the
tumor and promote its progression while suppressing any immune cell mediated
cytotoxicity 2. In this process, substantive clinical and experimental evidence
indicates that macrophages — present abundantly in most tumour types — have
a major regulatory role in promoting tumour progression to malignancy3.
Tumour associated macrophages (TAM) at least in mouse models largely
originate from bone marrow monocytes that are recruited through inflammatory
signals released by cancer cells in the primary and metastatic tumour, where
they differentiate into TAMs and facilitate tumour progression 4,5. However, in
cancers such as gliomas and pancreas TAMs can also result from an expansion of
a tissue resident yolk sac derived population 6 7,8 (Text Box 1). Nevertheless in
either case the microenvironment differentiates the cells to new tumour
associated phenotypes that vary depending on location in the tumour but are
generally pro-tumoural.
Several strategies in immuno-oncology have been developed in the past few
years to re-activate the adaptive and innate immune system to mount a robust
anti-tumoral immune response as an alternative approach to classic anti-cancer
treatments, to which tumours generally develop resistance. Cinical trials with
immune checkpoint inhibitors (such as anti-CTLA4, anti-PD1, and anti-PDL1 that
potentiate the activity of cytotoxic CD8 T cells) have shown successful treatment
of cancers such as melanoma and lung cancer. However, in most cases only a
small fraction of patients fully respond to immunotherapy for unknown reasons 9.
In mouse models TAMs promote the recovery of tumors from biological, chemo-
and radio- therapies through a mix of activities including promotion of
angiogenesis, maintenance of stem cells and inhibition of immune responses 10,11.
Macrophage infiltration in some tumors has also been shown to interfere with
efficacy of immunotherapy, neutralizing efforts to re-activate CD8 T cells. For
this reason several therapeutic strategies to modulate TAM function, infiltration
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or activation are emerging both to block resistance to conventional therapies but
also to promote T cell based therapies 3,10,12 . This article will discuss the biology
behind these macrophage activities and also the manner in which they may be
targeted therapeutically. It will describe macrophage diversity and how this
promotes tumour progression in primary and metastatic sites. It will also discuss
ways to therapeutically exploit this diversity to create an anti-tumoral
microenvironment or to enhance chemo-, radio- or immuno-therapy.
Macrophages in the tumour microenvironment
Macrophages are innate immune cells that populate all tissues 13 and play
multiple roles in development, homeostasis and tissue repair 14. They have
different embryological origins (Box1) and not only expand in response to
infiltration of monocytes but also through local proliferation of tissue resident
macrophage progenitors. Studies indicate significant transcriptomic diversity
between macrophage populations even within a tissue consistent with their
diverse origins, adaptation to different tissue niches and involvement in different
pathologies — although in mice they express canonical markers such as of
Colony Stimulating factor-1 (CSF1R) and F4/80 15. CSF1R a transmembrane
tyrosine kinase Class III receptor ( the c-fms proto-oncogene) is required for the
presence of the vast majority of macrophages 16. It binds two ligands, CSF1 and
IL34 and regulates macrophage differentiation, proliferation and survival in
humans and mice 17. IL-34 shows overlapping functions but no sequence
similarity to CSF1 and binds to a different binding site on CSF1R, with a
significantly higher affinity than CSF1 18.. It regulates a sub-set of macrophages
particularly microglia and Langerhans cells.
Although in the late 19th century the scientific community accepted that tumors
were a mix of malignant and normal cells it was not until the 1970s that the
modern concept of the TME was introduced and the idea that immune cells can
actually promote cancer growth 19. However, even at this time the dominant view
was that macrophages were tumoricidal, as different studies showed that
activated macrophages could kill tumor cells in vitro 20-22. This view began to
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change in the early 1990s when several key studies identified a potential role of
macrophages in tumor progression by showing correlations of high intra-
tumoral density and increased expression of macrophage growth factors such as
Colony Stimulatory Factor-1 (CSF1) with clinical markers of poor prognosis
(grade, stage etc.) 23. In 2001, the genetic ablation of Csf1 and thereby
macrophages in the Polyoma Middle T model of breast cancer resulted in the
inhibition of tumor progression and metastasis thereby formally proving that
macrophages can be pro-tumoral 24. After this discovery several independent
studies in different cancers confirmed the pro-tumoral role of TAMs and
identified several subtypes of TAMs in mice and their roles in promoting primary
tumor progression and metastasis 3,25-27. Consistent with these experimental
mouse models, a meta-analysis study of 55 studies of different human cancers
indicated that high infiltration of TAMs correlated with poor overall survival in
breast, gastric, oral, ovarian, bladder and thyroid cancers, but not in colorectal
cancer 28. The correlation between TAMs infiltration and progression in cancer
was further confirmed by additional recent meta-analysis in breast cancer,
gastric cancer, Hodgkin Lymphoma and non-small cell lung cancer 29-32. More
specifically overexpression of CSF1 in CSF1R positive ovarian cancer cell lines
enhanced their invasive properties, with a potential involvement of the
urokinase plasminogen activator 33. At the clinical level, high serum levels of
CSF1 are found in several cancers including metastatic breast cancer patients 34
and they correlate with poor prognosis in ovarian and endometrial cancer
patients 35-37. In endometrial cancer epithelial CSF1 synthesis is an independent
predictor of survival 38. In breast cancer a “CSF1 expression signature” was
described that predicted poor survival 39. CSF1R expression combined with CD68
positive macrophage infiltration is considered an independent predictor of short
progression-free survival in Hodgkin’s lymphoma 40. Both normal breast and
ovarian tissue do not express CSF1R and CSF1 34,41 but sometimes breast and
ovarian tumor cells can express this receptor ligand pair 42-44. Nevertheless
expression of CSF1R on cancer cells is rare and is probably due to activation as a
result of de-methylation of a recently acquired retroviral element in humans and
thus is not found in mice 45. The role of IL-34 in cancer progression is still largely
unknown although a recent study by Baghdadi et al, showed that IL34 produced
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by chemo-resistant cancer cells was able to sustain the immunosuppressive
functions of TAMs and promote the survival of cancer cells 46. These data over
the last 18 years, in many different cancer contexts, have supported the
conclusion that in general, TAMs promote tumour progression to malignancy
through their interactions with cancer cells (Fig 1).
TAMs in solid tumors
Cancer initiation
The activation of key transcriptional factors such as NfκB, STAT3 and HIF1α by
chronic inflammation (caused by persistent infection, exposure to irritants or
autoimmune disease) or by oncogene activation results in the production of
cytokines and chemokines that engage the innate immune system and especially
macrophages 47. Macrophages in turn can contribute to cancer initiation by
producing pro-inflammatory mediators such as IL-6, TNFα and IFN , growthϒ
factors including EGF and WNTs, proteases and through the production of
reactive oxygen and nitrogen species that may create a mutagenic
microenvironment 48,49. Thus chronic inflammation infection or irritation is
associated with the initiation of many cancer types such as those in the colon or
stomach 50. Genetic ablation of STAT3 in macrophages causes exuberant
inflammation and the formation of invasive tumors in the colon 51. Similarly, the
deletion of the anti-inflammatory cytokine IL-10 that signals through STAT3 in
macrophages is also associated with tumor initiation and promotion 52,53.
Furthermore, deletion of ROS production in myeloid cells including macrophages
also inhibits carcinogenesis in an intestinal cancer model 49. These chronic
inflammatory responses are involved with complicated interactions with the
microbiome with changes in dominance of bacterial species and with breakdown
of mucosal barriers that allow infiltration of pathogenic types 54 . Thus, in animal
models in some cases the initiation of inflammation-induced cancer can be
suppressed by prophylactic treatment with broad spectrum antibiotics 55.
The role of macrophages in the transition from benign to malignant tumour has
only been studied in a few cancer models (mammary, skin and pancreatic
cancer) 6,24,56,57 and, at least in mammary tumours, premature recruitment of
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macrophages by over-expression of CSF1 promotes the transition to malignancy 24. It is thought that in the first stages of tumor formation macrophages are
mainly tumoricidal as they reflect an activated state. However, the evidence for
this conclusion is limited. What is certain, however, is that as the tumor grows,
the presence of a T-helper 2 (Th2) polarized microenvironment that “educates”
macrophages to become pro-tumoral and biases the immune response from a
cytotoxic to a supportive role begins to dominate. In mouse models of breast
cancer this state is driven by IL4 secreted from CD4+ T cells 58,59 recruited to the
tumour through unknown mechanisms.
The transition to malignancy is also enhanced by macrophages through i) their
production of proteases such as MMP9 or cathepsins that enhance tumour cell
escape from their restraining basement membranes and invasion, ii) angiogenic
factors such as VEGF that enable vascularization and iii) growth factors including
those of the EGF family that promotes tumour cell survival and migration as
discussed below. Critically tumour progression to malignancy also requires the
creation of a vascular network for their oxygenation, nutrition and waste
disposal. This process, known as the “angiogenic switch”, is mainly characterized
by a dramatic increase in new vessel formation that often involves vessel dilation
and recruitment of perivascular cells 60-62. TAMs support these processes by the
production of pro-angiogenic factors such as vascular endothelial growth factor
(VEGFA) 63 and angiogenic CXC chemokines (CXCL8, CXCL12) 64. Additional
factors such as WNT7b, TGFβ, TNFα and thymidine phosphorylase contribute to
the angiogenic process by targeting endothelial cells or other cells such as
fibroblasts or pericytes that further support the generation of vascular networks
in the microenvironment 62,65. The identification of a sub-population of TAMs
characterized by the expression of the angiopoietin-1 receptor, TIE2, that lie in
close association with vessels, confirmed the central role of TAMs in tumor
angiogenesis 66 as their depletion inhibited tumor growth and metastasis 67.
Highly invasive tumor cells are able to move from the primary tumor and
intravasate into the blood or lymphatic vessels where they migrate to distant
sites to establish micro-metastasis 27. TAMs are in part responsible for the
creation of this invasive tumor microenvironment. TAMs directly help tumor
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cells to migrate through a paracrine loop between macrophages and tumour cells
involving macrophage synthesized EGF family ligands and tumour cells produced
CSF1 that enhances tumor cell invasive properties. TAMs also produce
cathepsins and matrix re-modelling enzymes that stimulate this process, and
enhance intravasation by the upregulation of metalloproteases 68-70. These tumor
cells are attracted to vessels where they engage with perivascular Tie2+
macrophages that act as a conduit for the escape of tumour cells in part through
expression of VEGF that allows busts of vessel permeability 71. This tripartite
structure consisting of perivascular TAMs, cancer cells that have heightened
motility due to the expression of the invasive isoform of the actin binding protein
mammalian enabled (MENA) and endothelial cells has been called the Tumor
Microenvironment for Metastasis, (TMEM). Similar structures identified in
clinical samples led to the identification of a prognostic clinical score that
predicts chance of metastasis in breast cancer, regardless of tumor stage or
clinical subtype 72.
Transcriptional profiling of invasive TAMs also revealed upregulation of key
components of the WNT and Hedgehog gene families, mainly involved in tissue
patterning and development in homeostatic conditions 73,74. The importance of
WNT signaling was confirmed by the identification of WNT7b as one of the key
factors secreted by TAMs to promote tumor progression and metastasis in part
through effects on angiogenesis but also in promoting tumour cell invasion 62.
TAM mediated immune suppression
Macrophages can potentially mount a robust anti-tumoral response, as they are
able to directly eliminate cancer cells if properly activated by IFNγ. They also can
support the adaptive immune response through presentation of tumor antigens
and the production of chemokines and cytokines to recruit and activate cytotoxic
CD8 T cells and Natural killer (NK) cells. However, these macrophage activities
are restricted by the Th2 dominance in the TME, which profoundly affects
macrophage functions such that their phenotype resembles those involved in
tissue development and repair, with a consequent suppression of anti-tumoral
response 3,14.
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TAMs immune suppression is mediated by the expression of inhibitory
receptors, such as non-classical MHC-I molecules like HLA-E and HLA-G, which
are able to negatively modulate the activation of NK and T cells by the interaction
with CD94 and LIT-2 respectively 75. TAMs also express T cell checkpoint
inhibitors (PD-1, CTLA-4) ligands PDL-1/2 and B7-1/2 that directly inhibit T cell
functions 76,77. TAMs also secrete several cytokines such as IL-10 and TGF-β that
contribute to the maintenance of a strong immune suppressive
microenvironment by inhibiting CD4 (Th1 and Th2) and CD8 T cells and by
inducing T-regulatory cells expansion; TAMs-mediated release of the
chemokines such as CCL2, CCL3, CCL4, CCL5 and CCL20 further contribute to the
recruitment of regulatory T cells in the TME 3. TAMs also directly inhibit T cell
cytotoxicity by depletion of L-arginine, essential for the re-expression of the TCR
after antigen engagement on T cells, by the release of Arginase I that metabolize
L-arginine to urea and L-ornithine 3. Similarly, depletion of tryptophan or
production of tryptophan metabolites by indoleamine 2,3-Dioxygenase (IDO)
expressed by macrophages can inhibit cytotoxic T cells 78,79.
TAMs in metastasis
To establish metastasis, invasive cancer cells need to avoid eradication by the
immune system, survive in the blood or lymphatic circulation, arrest at distant
site and survive and grow in these often-hostile environments. In mouse models
of breast cancer and colorectal cancer, metastasis-associated macrophages
(MAMs) promote these latter steps by enhancing extravasation, sending survival
and growth signals to tumor cells and inhibiting anti-cytotoxic T cells 5,80,81,82.
Importantly, ablation of these MAMs or blockade of their recruitment results in
an inhibition of metastasis and in some cases prolonged survival of mice 5,80,83 ,
suggesting they represent therapeutic targets.
Mechanistically, metastatic cells attract bone marrow derived classical
monocytes (Ly6C+ in mice and CD14HighCD16high in humans) through a CCL2-CCR2
mechanism 5. Once extravasated, these monocytes differentiate into a distinct
population of MAMs 80 through engagement of a chemokine cascade involving
CCR1-CCL3 autocrine signaling 81 and with an identifiable distinct precursor
stage termed a metastasis associated macrophage precursor cell (MAMPC).
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MAMs in the lungs of mice with metastatic disease are transcriptionally different
from resident macrophages and MAMPCs 84 and are characterized by the
expression of the markers CD11b, VEGFR1, CXCR3, CCR2 80,81. Moreover, lung
intravital and ex vivo experiments showed that macrophages in the lung
physically interact with metastatic cells 85 and support metastatic cell survival
through a VCAM1 and AKT dependent mechanism as well as inducing epithelial
to mesenchymal transition by producing TGF- β 86,87. Moreover MAMs and
metastatic cancer cells engage in a crosstalk that enhances MAMs retention in
the metastatic foci that further supports tumor growth through expression of
unidentified survival and growth factors 81. MAMs and their precursors inhibit
cytotoxic T cells suggesting they are also involved in the maintenance of a
immunosuppressive environment that further promotes metastasis 84. Similar
mechanisms have been observed in bone metastasis where another class of
macrophages, the bone degrading osteoclasts, are activated by metastatic cells to
engage a vicious cycle because the bone resorption liberates entrapped growth
factors that further promote metastatic growth 88. There are currently limited
data available on tumoral infiltration in human metastasis although MAMs that
express high levels of VEGFR1 have been identified in lymph node metastases 83.
TAMs targeting strategies in cancer therapy
The sub-populations of macrophages with identifiable markers, transcriptomes
and phenotypes (Fig 2) described above are therefore attractive therapeutic
targets for combination therapies including standard of care and
immunotherapy. In mouse models and clinical contexts TAMs can synergize with
the anti-cancer therapy or alternatively, induce pro-tumoral functions by the
activation of tissue repair mechanisms. Here we will describe the latest studies
reporting dichotomous behaviors of TAMs after standard therapy and strategies
to enhance anti-macrophage therapeutics (Fig 3-4).
TAMs in Conventional therapies
Radiotherapy
Ionizing radiation is designed to target directly cancer cells by inducing DNA
damage in cancer cells that usually have impaired DNA repair mechanisms.
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However the effect of radiotherapy on the TME, and especially on TAMs, is only
partially understood 89. After ablative radiotherapy (single dose of 10Gy), the
innate immune system is activated by inflammatory cytokines such as IL1 and
TNFα and pro-fibrotic factors such as TGFβ that recruit macrophages with a
tissue reparative phenotype and contribute to tumor recurrence and progression 90-94. A recent study by Pinto et al. showed that fractionated cumulative radiation
doses regimens similar to the ones used during cancer treatment induced a pro-
inflammatory phenotype in macrophages in vitro but did not alter their ability to
promote cancer invasion and cancer angiogenesis 95. It’s becoming a priority to
tailor dose and fractionation of radiotherapy in different cancers as the response
elicited by macrophages and the stroma in general can influence the outcome 96.
Consequently, macrophage targeting in combination with radiotherapy is a
potential therapeutic strategy in order to modulate the stroma and allow better
tumor killing but as yet there are no clinical trials reported.
Chemotherapy
The tumor microenvironment and, in particular, macrophages, play an important
role in chemotherapy response and resistance 97-99. The first observation that
suggested a potential role of TAMs in mediating resistance to chemotherapy was
the demonstration that CSF1 inhibition was able to reverse chemoresistance of
breast cancer cell lines in xenograft mouse models 100. DeNardo et al. confirmed
and extended this initial observation and showed that tumor biopsies from
cancer patients who received neoadjuvant therapy had a much larger infiltrate of
CD45+CD11b+CD14+ macrophages compared with patients who received only
surgery. They also demonstrated that paclitaxel treatment of Polyoma Middle T
mice in combination with anti-CSF1R antibodies significantly reduced tumor
burden, vessel density and increased cytotoxic T cells infiltration compared to
mice treated with paclitaxel alone 101.
TAMs are responsible for the increased production of cathepsins (proteases that
facilitate tumor growth and invasion102) that leads to increased
lymphangiogeneis and metastasis after paclitaxel treatment 103,104. Upon 5-
fluorouracil treatment of colorectal cancer, TAMs release the diamine putrescine,
that confers cancer cells resistance to apoptosis in a JNK-Caspase-3 dependent
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manner 105. Doxyrubicin was also shown to induce the selective accumulation of
perivascular MRC1+ TAMs around the blood vessels and these cells promoted
recurrence after therapy through in part, the expression of VEGF and increased
angiogenesis. Selective targeting of the recruitment of this perivascular TAM
population by CXCR4 blockade showed reduced tumor relapse after
chemotherapy suggesting that this targeting strategy could be of clinical utility 64.
Immunotherapy
Mutated tumor cells can express tumor antigens as a product of oncogenic
viruses, differentiation antigens or as a product of tumour-specific mutations
(tumor neoantigens). The production of neoantigens is not equal across tumors
types with tumors such as melanoma and lung cancers among the top
“neoantigens producers” while hematological malignancies (AML, ALL, CML) are
among the lowest 106. T cells are able to recognize tumor antigens loaded to the
major histocompatibility complex on the cancer cell through binding to the T cell
receptor (TCR); however, to get completely activated they require interaction of
CD28 with co-stimulatory B7 molecules (CD80 and CD86) expressed on the
antigen presenting cell (APC). Cancer cells do not express B7 molecules so,
without a second stimulatory signal provided by other APCs (such as dendritic
cells and macrophages) recruited by inflammatory signals, the anti-tumoral T
cell response will not start.
The identification of tumor antigens led to the development of several tumor
vaccination strategies in the ’80s where tumor-derived antigens (DNA or
peptides) were injected together with cytokines in order to enhance the
immunological response; results from these trials however were not as striking
as expected 107, suggesting that the regulation of T cell activation in the tumor
was complex. Both pre-clinical and clinical studies indicated that tumours are
infiltrated by immune-suppressive cells (T-regs, TAMs, cancer fibroblasts,
myeloid derived suppressor cells) and they are exposed to an
immunosuppressive cytokine milieu (IL-10, TGFβ).
With this increasing understanding the immunotherapy field has had a rapid
expansion particularly with the discovery that immune checkpoint inhibitors,
such as anti-CTLA-4 and anti-PD-1, whose main function is to remove the
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“brakes” on T cells, allowing effective anti-tumoral immune response 108. One
anti-CTLA-4 (ipilimumab) and two anti-PD-1 (pembrolizumab, nivolumab)
neutralizing antibodies are currently FDA approved for the treatment of several
cancers including melanoma, advanced renal carcinoma, gastric cancers, non-
small lung cancer and colorectal cancer; hundreds of clinical trials on multiple
solid cancers are ongoing to evaluate their efficacy. While efficacy in melanoma
and some other cancers is high especially when anti-CTLA4 and -PD1 therapies
are combined, in many other cancers only a small fraction of patients treated
respond. One hypothesis is that the efficacy of checkpoint inhibitors could be
improved by the modulation of the immunosuppressive cells such as TAMs.
Macrophages in the tumor express high levels of PD-L1/2, the ligands of PD-1, as
well as PD-1. TAM PD-1 expression also negatively correlates with the ability of
these cells to phagocytose cancer cells and that TAM specific PD-1 inhibition
reduces tumor growth109 .
Moreover, CSF1 expression was recently shown to correlate with CD8+ T cells
and CD163+ TAMs accumulation in melanoma and anti-PD1 and anti-CSF1R
combination therapy induced regression of melanoma in preclinical studies 110
Clinical trials combining checkpoint inhibitors and anti-TAMs agents (such anti-
CSF1R antibodies, see below) are currently ongoing in different solid tumors
contexts 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121.
Methods of TAM targeting
TAM depletion
The dependence of macrophages on CSF1R signaling makes this an attractive
target to selectively deplete macrophage. Consequently different antibodies and
small molecules mainly targeting CSF1R are being studied in different clinical
trials both as monotherapy or in combination with standard therapy or
immunotherapies.
The small molecules under clinical development and study are PLX3397, JNJ-
40346527, PLX7486, ARRY-382 and BLZ945. Preclinical studies showed that
PLX3397, or pexidartinib, reduced the number of tumor associated microglia and
gioblastoma invasion 122; if used in combination therapy with tyrosine kinase
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inhibitors dovitinib and vatalanib in glioma mouse models PLX3397 made the
tumors sensitive to the anti-tumor agents 123. The Plexicon small molecule
PLX3397 was tested in a phase I dose escalation clinical trial followed by a phase
II extension study on advanced tenosynovial giant cell tumors patients 124, a
tumor characterized by the high expression of CSF1 and CSF1R125,126. Results
from the phase I and II study indicated that PLX3397 was tolerated at the dose of
1000mg and in the extension study that 12 out of 23 patients (52%) showed
anti-tumor responses after treatment 124.
In a phase II study in patients with recurrent glioblastoma, PLX3397 treatment
was tolerated and it was able to pass the blood-tumor barrier, however it did not
show improvement in 6 months progression free survival compared to control 127. JNJ-40346527 another kinase inhibitor, was tested in a phase I/II study on
twenty-one patients with relapsed or refractory Hodgkin Lymphoma; one
patient showed complete remission and 11 patients showed stable disease 128.
Several Phase I clinical trials are ongoing with additional CSF1R inhibitors:
PLX7486 is being tested as single agents on patients with advanced solid tumors 129, ARRY-382 is currently involved in two phase I clinical trials 130,113 on
metastatic patients and advanced solid tumors. BLZ945 was reported to alter
macrophage polarization and to block glioma progression 131 with promising
results if used in combination with IGF1-R and PI3K inhibitors 132; it is currently
evaluated in a trial on advanced solid tumors as single agent or in combination
with the anti-PD1 antibody PDR001 114.
There are three anti-CSF1R monoclonal antibodies under clinical evaluation,
RG7155, IMC-CS4 and FPA008. RG7155 (emactuzumab), is a humanized
monoclonal antibody that binds to CSF1R and blocks its dimerization; preclinical
studies showed that RG7155 depletes CSF1R+CD163+ macrophages in vitro and
in vivo. In colorectal and fibrosarcoma mouse models treatment with a chimeric
version of RG7155 reduced the number of infiltrating TAMs and increased the
CD8:CD4 T cell ratio 126; moreover RG7155 showed significant reduction of
CSF1R+CD163+ macrophages and T cell tumor microenvironment composition in
patients with advanced solid tumors treated with RG7155 as monotherapy or in
combination with paclitaxel 126. These promising results led to a dose escalation
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and dose expansion phase I clinical trial on tenosynovial giant cell tumor
patients to investigate the clinical benefit of RG7155; the dose escalation phase
on 12 patients showed no dose toxicity and common adverse effects reported
were facial oedema, asthenia and pruritus. Out of the 28 patients tested, 24
(86%) showed an objective response and 2 (7%) achieved a complete response 133. Two other antibodies currently under phase I clinical trials; FPA008 is
currently under clinical investigation in 3 ongoing clinical trials134, 118, 135 in
diffuse type tenosynovial giant cell tumor and advanced solid tumors. IMC-CS4 is
currently under clinical investigation in 3 clinical trials136 137,138 on solid tumors
including pancreatic, prostate and breast cancer.
Overall these preliminary results suggest that CSF1/CSF1R targeting could be a
promising targeting strategy for the treatment of cancer. Indeed in this type of
precision therapy against tumours over-expressing CSF1 such as the synovial
giant cell tumours that appear to be successful, it is likely that it will be advanced
as an effective treatment. Nevertheless toxicity has been reported that limited
dose escalation as it is problematic to deplete all macrophages from the body for
a long period of time.
Another therapeutic strategy is to selectively kill TAMs and not the other
components of the stroma. An example of this strategy is the use of
bisphosphonates that are stable inorganic compounds and their structure is
identical to pyrophosphatases of the bone matrix so they can be metabolized
rapidly by osteoclast and inhibits their resorption. Moreover, they are also used
as anti-cancer agents for the treatment of hematological and solid malignancies 139,140. They are mainly subdivided in two classes based on their structure and
mechanism of action: clodronate, etidronate and tiludronate belong to the first
group, while alendronate, ibandronate, pamidronate, risenodrate and
zolenodrate to the second group. At the preclinical level bisphosphonates
exhibited direct and indirect anti-tumor properties. They are reported to inhibit
cancer cell proliferation, to induce tumor cell apoptosis, to block angiogenesis, to
inhibit cell adhesion and invasion and to interfere with immune surveillance
through activation of gamma delta T cells 141. Different studies showed that
bisphosphonates are also able to inhibit proliferation, migration and invasion of
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macrophages causing apoptosis 142,143. They however, mainly affect osteoclasts
that share the same lineage with macrophages and have been used in preclinical
bone metastasis models.
A more directed approach is to encapsulate for example clodronate (clodrolip),
in liposome that are preferentially taken up by macrophages due to their
phagocytic activities. This treatment reduces macrophage tumour infiltration in
a lung cancer experimental bone 144 and lung metastasis models 80 and thereby
limited metastatic outgrowth. Similar results were obtained in mice injected
with human melanoma cancer cells, where clodrolip treatment reduced tumor
mass and angiogenesis 145. Clodrolip in combination therapy with anti VEGF
antibodies showed anti-tumour properties in mice injected with teratocarcinoma
and rhabdomyosarcoma cells with a significant reduction of TAM infiltration 146;
moreover in metastatic liver mouse models the combination treatment with
clodrolip and sorafenib caused decreased tumor burden, angiogenesis and
metastasis 147. Liposomal clodronate was also tested in dogs with spontaneous
soft tissue sarcomas where it showed the ability to deplete CD11b+ macrophages
in tumors and to a decreased IL-8 serum level, though the anti-tumor properties
of the treatment were not significant 148. Zoledronic Acid, another
bisphosphonate was shown to reduced tumor burden in a mouse model of breast
cancer bone metastasis 149 and to modulate the TME by reducing the number of
TAMs and their polarization status 150. Recently Comito et al. demonstrated that
Zoledronic Acid treatment impairs macrophage polarization, reduces
macrophage-induced angiogenesis and tumor invasion in prostate cancer 151.
Current studies using nanotechnology are trying to optimize the delivery of
bisphosphonates by their encapsulation in stealth liposomes or in PEGlyated
nanoparticles; pre-clinical tests were promising, showing better anti tumoral
activity and TAMs reduction compared to treatment with free bisphosphonates 152-154.
At the clinical level Clodronate and Zoledronic Acid treatments were tested in
several clinical trials on a variety of different cancers, with inconsistent results
that suggest the need to optimize better combination treatments and for longer
clinical trials, as discussed in several meta-analysis 155,141,156. There are currently
two ongoing clinical trials evaluating the effect of Zoledronic acid in triple
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negative breast cancer 157 and stage IIIb and IV lung cancer 158; clodronate is
being tested in different clinical trials on breast cancer patients as neoadjuvant
agent 159 and in combination with chemotherapy and hormonal therapy 160 ;
additional trials include a clodronate-chemotherapy combination treatment on
refractory metastatic prostate cancer patients161 .
Trabectedin is a tetrahydroisoquinoline alkaloid that was initially isolated from
the Caribbean tunicate Ecteinascida turbinata 162. It is an anti-neoplastic drug
approved in Europe, in Russia and South Korea for the treatment of advanced
tissue sarcoma and platinum-sensitive relapsed ovarian cancer, in combination
with pegylated liposomal doxorubicin 163-165. Trabectedin in addition to targeting
tumour cells was shown to specifically induce apoptosis of monocytes and
macrophages in the tumour by the activation of Caspase-8 through a TNF-related
apoptosis-inducing ligand (TRAIL)-dependent mechanism 166. These results
suggested that the apoptotic receptor family TRAIL could be a potential
therapeutic target to selectively kill immune cells and especially macrophages. A
recent report by Liguori et al. explored this hypothesis and demonstrated that
monocytes and macrophages express the functional TRAIL receptors R1 and R2,
while neutrophils and lymphocytes express the non functional decoy TRAIL-R3 167. Interestingly, human TAMs in mammary, hepatic and colon carcinoma, but
not resident tissue macrophages, express functional TRAIL-R 167 making these
receptors interesting targets for therapy 168.
Inhibition of TAM recruitment
TAM expansion in the tumor is often mediated by monocytic recruitment
through the CCL2-CCR2 axis. CCL2 is a potent chemoattractant for monocytes, T
cells and Natural Killer cells 169, and several mouse studies have demonstrated a
role for it and other chemokines in macrophage accumulation in the tumors 170-
173. CCL2 synthesised by tumour cells recruits classical monocytes (Mouse
Cd11b+, Ly6cHi; human CD14+ CD16-) that express the receptor CCR2 to the
tumor sites and its inhibition correlate with reduced tumor burden and
metastasis in different experimental models of prostate, breast, lung, liver cancer
and melanoma 174. However, withdrawal of anti-CCL2 treatment accelerated lung
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metastasis in mouse models of breast cancer and resulted in death of mice due to
a rebound in monocyte recruitment raising important concerns on the long term
efficacy of this approach 175,176. However, high serum levels of CCL2 as well in the
tumor are associated with poor prognosis in different cancers such as breast 177,178. For these reasons, different CCL2 neutralizing antibodies are now being
tested in clinical trials. The two main drugs currently tested are carlumab (CNTO
888), an anti-CCL2 monoclonal antibody, and PF-04136309 a small molecule
inhibitor that targets CCR2.
Carlumab is a human immunoglobin G1κ antibody that binds to CCL2. Preclinical
studies using prostate cancer mouse models showed that systemic injection of
the antibody reduced tumor growth, the infiltration of CD68 positive
macrophages and vascular density 179,180. CCL2 inhibition was also able to
enhance the effect of paclitaxel and carboplatin therapies in mouse models of
ovarian cancer 181. A phase I clinical trial was performed in 2013 to assess
tolerance to different doses of carlumab in forty-four patients with different solid
tumors 182. The results indicated that CCL2 levels were only partially suppressed,
with an increase of free CCL2 after treatment over the pre-treatment base-line of
more than 1000 fold, as reported previously in preclinical models 175. A phase II
clinical trial was then performed on forty-six castration-resistant metastatic
prostate cancer patients, but the study was not able to show therapeutic efficacy
of carlumab in the treated patients 183.
The CCR2 small molecule inhibitor PF-04136309 was recently tested in a phase
1b non randomized trial on locally advanced pancreatic cancer patients in
combination with FOLFIRINOX chemotherapy (oxaliplatin and irinotecan plus
leucovorin and fluorouracil); 47 patients were treated, 8 patients received only
FOLFIRINOX, while the remaining ones received FOLFIRINOX plus PF-04136309.
Results showed that PF-04136309 treatment in combination with FOLFIRINOX
was safe and well tolerated compared to chemotherapy alone. Patients treated
only with FOLFIRINOX did not show an objective response to the treatment; on
the contrary, in PF-04136309 plus FOLFIRINOX group, 16 of the 33 patients who
underwent repeated imaging assessments had an objective tumor response and
32 patients had local tumor control achieved 184.
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These trials however, were largely disappointing and indicated that much more
biological knowledge is needed to fashion effective inhibition of monocyte
recruitment. For example, CCL2-CCR2 signaling is required for monocyte egress
from the bone into the blood resulting in a severe depletion of monocytes. This
depletion is recognized and the animal attempts to overcome this deficiency with
a dramatic elevations in CCL2 concentrations thus preventing efficacy of the
reagents. In our own studies using genetic ablation we have demonstrated the
requirement for CCR2 mediated recruitment of monocytes to the primary
Polyoma Middle T antigen (PyMT) tumours but that as these progress this is
completely overcome by unknown redundancy mechanisms 4. In addition, there
may be compensatory proliferation of tissue resident macrophages if
recruitment is blocked6. Thus significantly more biological understanding is
required before this approach to selectively inhibit monocyte recruitment to the
tumour or its metastatic derivative is likely to be effective. Indeed, it may be
better to go downstream to molecules required for retention of monocytes such
as CCL3 or that induce their differentiation 4,81
Reprogramming of TAMs
Despite generally being pro-tumoral TAMs can, depending on context, be
tumoricidal and also suppress tumor growth by activating immune responses 185.
This suggests that macrophage plasticity may be therapeutically exploited to
restore antitumor properties to TAMs 186. Indeed, it might indicate the paucity of
the approach of targeting all macrophages as both pro- and anti-tumoural ones
will be depleted. Instead, macrophage reprogramming is a targeting strategy that
provides an opportunity to therapeutically rebalancing the microenvironment
immune infiltrate from a pro-tumoral one to one that actively rejects the tumor
in synergy with T cell enhancing drugs such as check-point inhibitors. It also
suffers less from the drawbacks and long-term toxicity of ablation of all
macrophages, as is the case for example with anti-CSF1R therapeutics. Different
methods are currently being tested at the pre-clinical and clinical level, as
discussed below.
[H3] Anti CD47 antibodies
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CD47, also known as integrin-associated protein (IAP) is a ubiquitous protein
that regulates cell migration, axon extension, cytokine production and T cell
activation 187-190. CD47 interacts with thrombospondin-1 and signal regulatory
protein alpha (SIRPα), mainly expressed by myeloid cells, including dendritic
cells and macrophages 191. In the latter case, it inhibits phagocytosis by the
prevention of Myosin II-A accumulation at the phagocytic synapse 192. The result
of this interaction is a “do not eat me signal” that prevents phagocytosis of
autologous cells in homeostatic conditions. This mechanism is tightly regulated
and it is mainly activated in pro-inflammatory conditions as CD47 null mutant
mice only show a phenotype against self during inflammatory conditions 193.
CD47 is overexpressed in a variety of tumors 194-197 through the activation of
CD47 specific super-enhancers 198. It is involved in tumor invasion, metastasis
and, more importantly, in the inhibition of phagocytosis by the innate immune
system by interacting with SIRP α 197 expressed on phagocytes.
Several preclinical studies in mouse xenograft models demonstrated that CD47
inhibition is an effective strategy for tumor therapy 199-201 since it enables killing
and phagocytosis of tumour cells by macrophages. Moreover recent studies on
human ovarian and small-cell lung cancer cell lines confirmed the fundamental
role of cancer expressed CD47 in the inhibition of phagocyte-mediated killing 202,
203. A recent pre-clinical study showed that injection of highly phagocytic SIRP -αinhibited marrow-derived macrophages preloaded with anti tumor antibodies
model can effectively reach the tumor and engulf cancer cells causing tumor
regression; the anti tumoral effect is then lost due to their differentiation to
TAMs 204.
At the moment there are 2 anti-CD47 monoclonal antibodies (Hu5F9-G4 and CC-
90002) and one soluble recombinant SIRP -Fc fusion protein (TTI-621)α
currently being tested in phase I clinical trials. Hu5F9-G4 showed promising
results at the preclinical level in human AML 205 and in pediatric brain tumors 206;
there are currently 4 ongoing clinical trials on different solid and hematological
malignancies 207,208,209,210 to study the safety of Hu5F9-G4 in humans. Preliminary
results from the NCT02216409 trial indicated that Hu5F9-G4 was tolerated with
reversible side effects observed such as anemia, headache, nausea and retinal
toxicity 211. CC-90002 is currently being tested in two ongoing clinical trials212,213
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in patients with haematological malignancies but results have not been
published yet.
TTI-621 is a fully human recombinant protein that blocks the CD47–SIRP axisα
and enhances killing of cancer cells. A recent study investigated the efficacy of
TTI-621 in aggressive AML and B lymphoma xenografts; TTI-621 successfully
enhanced macrophage-mediated phagocytosis of cancer cells but not normal
cells. Moreover in vivo data suggested that TTI-621 treatment was able to control
the growth of hematological and solid tumors in mouse xenografts models 214.
TTI-621 is currently being tested at the clinical level in two ongoing clinical trials
on hematological and multiple solid tumors 215,216.
[H3] Toll-like receptors agonist
Toll-Like receptors are innate immunity pattern recognition receptors that play
fundamental roles in the activation of innate immune response 217; activation of
TLR by bacterial particles (such as LPS) and viral nucleic acids (RNA or DNA)
polarize macrophages towards a pro-inflammatory phenotype. For this reason
different TLR synthetic ligands have been tested in cancer models in order to
assess their efficacy in the phenotypic switch of TAMs to tumoricidal
macrophages in the TME 218.
In an orthotropic mammary tumor mouse model intra-tumoural delivery of
TLR7 and TLR9 agonists caused increased monocyte infiltration in the tumor
and macrophage repolarization 219; similar results were obtained with a TLR7/8
agonist (3M-052) that induced macrophage repolarization and enhanced
tumoricidal activity in melanoma 220. In preclinical models the TLR7 ligand
imiquimod, the only TLR agonist approved for clinical use, showed antitumoral
activity in basal cell carcinoma, melanoma and breast cancer skin metastasis 221-
224.
At the clinical level two TLR7 (Imiquimod and 852A) and one TLR9 ligand (IMO-
2055) are being tested for their antitumoral properties in clinical trials
Imiquimod has been tested on several cancers; in a prospective clinical trial
topical treatment of breast cancer skin metastasis with Imiquimod was well
tolerated and responders showed histological tumor regression and enhanced
lymphoid immune infiltration 223. 852A has been tested in five clinical trials in
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melanoma, leukemia and gynecological cancers 225-229. A phase I clinical trial on
advanced cancer patients showed that 852A treatment 3 times weekly for 2
weeks was safely administered with reversible side effects 230. IMO-2055 has
been tested in colorectal, head and neck, lung and renal cancers 231-235. Results
from a clinical trial on advanced metastatic non-small cell lung cancer patients
showed that IMO-2055 demonstrated good tolerability and potential anti-
tumoral activity in combination with erlotinib and bevacizumab 236.
[H3] Anti-CD40 antibodies
CD40 is a receptor that belongs to the TNF receptor superfamily and it’s
expressed by antigen presenting cells (APC) such as monocytes, macrophages,
dendritic cells and B cells 237 but it can be expressed by endothelial and epithelial
cells as well. The natural ligand of CD40 is CD154 or CD40L, mainly expressed by
CD4+ T cells, basophils and mast cells 238. CD40:CD40L interaction upregulates
the expression of MHC molecules and the production of pro-inflammatory
cytokines such as IL-12 that primes naive CD4 and CD8 T cells into T helper and
cytotoxic cells, respectively. Preclinical studies showed that agonistic anti-CD40
antibody treatment exert tumor inhibitory effects on several tumor mouse
models opening the way for the development of clinically relevant anti-CD40
antibodies.
Interestingly, recent studies showed that TAM treatment with CD40 agonists in
combination with anti-CSF1R antibodies results in a profound TAM
reprogramming before their depletion; reprogrammed TAM create a
proinflammatory environment that elicit effective T cell response even in tumors
that were non responsive to immune checkpoints inhibitors 239,240.
Two agonistic anti-CD40 antibodies are being tested in clinical trials, CP-870,893
and RO7009789. In a phase I dose escalation study, 29 patients with solid
tumors were treated with a single dose of CP-870,893 intravenously; common
adverse effects included cytokine release syndrome and alterations of immune
cell number but in general the treatment was well tolerated; CP-870,893
treatment showed an objective response and anti-tumor activity 241. CP-870,893
treatment of 32 patients with advanced solid tumors in combination with
carboplatin and paclitaxel was tested in another phase I trial; 6 out of 30
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evaluable patients showed partial response to treatment and depletion of B cells
together with upregulation of immune co-stimulatory molecules 242. Similar
results were obtained in malignant pleural mesothelioma patients treated with
CP-870,893 in combination with cisplatin and pemetrexed 243 and in patients
with advanced pancreatic ductal adenocarcinoma 244. RO7009789 is being
studied in 4 ongoing clinical trials on advanced solid tumors 116,245-247 .
[H3] HDAC Inhibitors
Histone deacetylases (HDACs) of which there are 18 in mammals are broken
down into four classes 248,249. They are responsible for removing the acetyl groups
on histones, a crucial process in epigenetic regulation of gene expression.
TMP195, a specific inhibitor of class IIA HDAC 250, can modify the epigenomic
profile of monocytes and macrophages resulting in for example, altered CCL1
and CCL2 expression in monocytes and promote a pro-inflammatory phenotype.
In a model of luminal B-type breast cancer, intraperitoneal injection of TMP195
increased infiltration of CD11b+ myeloid cells from blood into the tumor, where
they differentiated into anti-tumoral macrophages 251. This resulted in reduced
vessel permeability as well as reduced vasculature and tumor cell proliferation.
Moreover, the antitumour macrophage phenotype induced by TMP195
treatment enhanced the efficacy and durability of both standard
chemotherapeutic regimens (carboplatin and paclitaxel) and immunotherapy
(anti-PD1 antibodies). The findings suggest that Class IIA HDAC inhibitors can
selectively reprogram monocytes and macrophages in the tumor and opens
interesting therapeutic opportunities but it remains to be seen if targeting the
subclass HDACs will be sufficiently specific if given systemically to humans.
[H3] Anti-MARCO antibody therapy
The macrophage receptor with collagenous structure (MARCO) is a pattern
recognition receptor belonging to the class A scavenger receptor family; it is
mainly expressed by macrophages 252 and its expression was linked to poor
prognosis in breast cancer 253. A recent report by Georgoudaki et al. showed that
MARCO is expressed in TAMs of breast cancer and metastatic melanoma patients 254. MARCO neutralization with antibodies inhibits tumor growth and metastasis
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in the 4T1 mammary carcinoma model. Similarly using a B16 melanoma mouse
model anti-MARCO antibody treatment inhibited tumor growth and enhanced
anti-CTLA4 immunotherapy 254. The anti-tumour activity of anti-MARCO therapy
was dependent on the ability of the Fc of the anti-MARCO antibody to engage
with the inhibitory Fc-receptor Fc -RIIB, as previously demonstrated with otherγ
Ab-mediated reprogramming strategies 255. This study highlighted the feasibility
of antibody-mediated reprogramming of macrophages by using TAM-derived
targets and stresses the importance of the correct design of the antibodies,
especially the Fc region, for future clinical interventions.
[H3] PI3K γ inhibitors.
Phosphoinositide 3-kinases (PI3Ks) are involved in almost all types of signalling
in cells 256. There are several sub-classes of which class 1B PI3Kγ is mainly
expressed by hematopoietic cells. Mice lacking PI3Kγ expression show impaired
recruitment of inflammatory cells, mainly macrophages and neutrophils 257.
Recently Kaneda et al. showed that PI3K is a key regulator of tumor immuneγ
suppression exerted by TAMs; genetic and pharmacological inhibition of this
target induced the expression of MHCII together with upregulation of IL12 and
decreases secretion of IL10. As a result, the inhibition of PI3K in TAMs causedγ
recruitment of anti-tumor adaptive immunity and tumor growth inhibition 258. At
the clinical level, head and neck and lung cancer patients with low PI3K activityγ
had a better prognosis and longer overall survival, suggesting that PI3K couldγ
be a potential future therapeutic target.
[H3] Inhibition of miRNA activity .
MicroRNAs (miRNA) are small non-coding RNAs that regulate transcription and
translation in a sequence specific manner and their maturation is regulated by
the RNAse-III enzyme DICER 259. A recent study showed that macrophage
inhibition of DICER affects TAM programming and it’s associated with tumor
regression and altered infiltration of immune cells 260. DICER inhibition
reprogrammed TAMs to express an IFN/STAT1 signature and to become anti-
tumoral. DICER inhibition in TAMs was also associated with a better response to
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immunostimulatory antibodies. These data raise the possibilities of identification
and targeting miRNAs to re-polarise TAMs.
DISCUSSION AND PERSPECTIVES
TAMs represent heterogeneous populations with defined functions that differ
between primary and metastatic tumours. Immune cell infiltrates also vary
according to tumour type and thus dynamic interactions between TAMs and
other immune cells are likely to vary according to cancer type and stage of
progression. In addition to pro-tumoral effects, TAMs can also have anti-tumor
effects that in some cases might be dominant. Consequently it is necessary to
understand tumour heterogeneity and how this evolves during the progression
to malignancy and also following therapy. It is also judicious to realize that much
of these data derives from mouse models and there is scant knowledge except at
the most descriptive level, about TAMs in human cancers.
Despite these caveats, given the consensus that TAMs are overall pro-tumoral
several anti-TAM strategies aimed at depletion (i.e. CSF1R inhibitors),
reprogramming (i.e. PI3K and HDAC inhibitors) and targeting of functionalγ
molecules such as Arginase 1 and Fc receptors are in preclinical and clinical
trials (Fig 3-4). Despite these advances each strategy needs further investigation
as they all have limitations. For example, the general depletion of monocytes and
macrophages exerted by CSF1R inhibitors is not TAM specific and thus has
significant toxicity over time 261. Furthermore, given the complexity of TAM
populations there is growing awareness that the functioning of
macrophages/dendritic cells is required for anti-PD1 therapy 262 and anti-CTLA4 263 , respectively. Thus, potential alternative strategies would be to ablate TAMs
transiently followed by recovery periods during which time monocytes can be
recruited into the tissue and promote anti-tumor immune reactions before
differentiating into pro-tumoral TAMs. This strategy is attractive but it will
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require a careful timing and a better knowledge of the immune interactions
ongoing in all phases of tumor formation 264.
Going forward therefore a better strategy would be to specifically target pro-
tumoral macrophages and enhance the activity of anti-tumoral ones or by
repolarize existing ones to have anti-tumoral activities. In this context TAM
therapy aimed at the functional modulation of TAM subpopulations through the
use of monoclonal antibodies showed promising preclinical results. This
strategy, combined with the use of Fc receptors inhibitors, seems to be the most
promising one for the modulation of the TME as macrophages are able to rapidly
take up anti-PD1 antibodies through their Fc receptors, limiting the efficacy of
the checkpoint inhibitors. Thus, it will be fundamental to identify TAM specific
targets (such as MARCO) to improve therapy specificity.
An interesting approach in this context of re-polarization might also be to
understand the interactions between the microbiome and macrophages. Such a
concept is based upon recent studies suggesting that manipulation of the
microbiome alters cancer incidence as well as responses to therapy. Since
macrophages are sentinel cells for changes in the microbiota usually through
TLR receptor engagement, understanding the exact nature of these interactions
and their consequences could potentially help in tailoring an anti-cancer
microbial cocktail 265.
Another strategy is to target cancer-associated myeloid immature progenitors 266, cells that are mainly found in the blood of cancer patients and they are
thought to be the progenitors of tumor-infiltrating myeloid cells. Several studies
indicated that these immature cells have intrinsic immunosuppressive function
in vitro and therefore could represent a very promising target in combination
with immunotherapy 267. However, again the challenge is to identify specific
markers which will allow the selective targeting of these abnormally expanded
immature populations as there are currently not enough markers available to
distinguish them from mature myeloid cells.
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To enhance all therapeutic modalities a thorough understanding of the TME is
required in humans and in different cancer types with identification of specific
population of cells, compensatory mechanisms between cells of the innate
system and their mechanisms of immunosuppression and tissue repair. The
extensive use of single cell RNA sequencing, multiplex immunohistochemistry
and mass cytometry will in this context considerably enhance our knowledge
and it will allow the selection of novel TAM targets for the modulation of the
TME to enhance therapy.
TEXT BOX1.
The understanding of the origins of macrophages has recently undergone a
profound shift due to the use of modern lineage tracing techniques. These
methods use inducible cre recombinases from cell specific promoters such as the
Csf1r crossed with floxed coloured reporter mice that permanently tag progeny
cells at specific times in development. These methods together with single cell
and bulk RNA sequencing have allowed precise developmental origins of
macrophages to be ascertained. A revelation in these studies is that most tissue
resident macrophages are not as previous thought derived from bone marrow
progenitors (BM) but instead from the yolk sac (YS) or fetal liver (FL) 268,269.
Detailed lineage tracing has indicated some tissues such as the brain where
essentially all macrophages called microglia are directly from erytho-myeloid
progenitors (EMPs) in the YS while others such as heart are hybrid with
contributions from the YS and BM while in others the YS-derived macrophages
are replaced from fetal liver monocytes 270,269,271. Furthermore, there are a few
tissues such as the intestine in the adult where the major populations of
macrophages are all derived from BM monocytes that replace the embryonic
populations (BOX 1) 272,273. The presence of persistent embryonic populations
throughout life in most tissues indicates that these tissues harbour progenitors
that can proliferate to give rise to mature macrophages 271. These data have also
revealed different regulatory mechanisms for example the requirement of BM
derived cells on the transcription factor c-MYB that is not required for YS derived
ones 268. Nevertheless the dominant transmembrane receptor controlling the
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differentiation/survival of almost all macrophages regardless of their origins is
the CSF1R. In its absence, mice are depleted dramatically in almost all
macrophages with some notable exceptions such as the lung where they are
regulated by granulocyte-macrophage CSF (CSF2) 274. However, it should be
recognized that overlayed upon this requirement for CSF1R there are tissue
specific growth factors and chemokines as well as micro-environmental cues that
specify their local identity 275 .
These observations open a number of intriguing questions for example about the
importance of the phenotype of macrophages according to their lineage
compared to their tissue environment, whether the replacement of YS or FL
derived macrophages with BM ones results in identical phenotypes and whether
macrophages from different origins can be individually targeted. In the context
of tumours these questions are important since although in many mouse models
most TAMs appear to be BM in origin there are several exceptions such as in
models of pancreatic cancer and glioma where the populations are a mix of fetal-
and BM-derived ones 57, 7,8. For example in pancreatic cancer models it is the YS-
derived macrophages but not BM-derived ones that are pro-tumoural suggesting
that origin is important 57. This different origins might be of clinical relevance as
it could allow independent targeting of sub-populations 6. It also raises
questions whether inhibition of BM-derived macrophage recruitment might
result in compensation by YS/FL-derived tissue progenitors or vice versa. Such
observations again emphasize the importance of understanding macrophage
origin, heterogeneity and their dynamics in the TME.
ACKNOWLEDGEMENTS
We apologise to the many authors whose work we could not cite due to space
constraints. Research in authors’ laboratories is supported by the Wellcome Trust
(101067/Z/13/Z) and MRC Centre grant MR/N022556/1 to JWP.
COMPETING INTERESTS STATEMENT
The authors declare no competing interests.
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Figure 1. Macrophage infiltration and survival in cancerA. Five year survival rates are high if cancer is localized even if it is invasive. However, survival is dramatically reduced if the cancer has metastasized. Data are expressed as five years survival rate (expressed in percentage) in tumors that are localized (blue dots), invasive (yellow dots) and metastasized (red dots). Image adapted from https://www.economist.com/technology-quarterly/2017-09-16/treating-cancer. B. Immune infiltrates vary according to cancer type, however, monocytes and macrophages represent the major population infiltrating human cancer. CIBERSORT analysis of tissue microarray datasets of solid human tumors revealed the average immune cells composition of bladder, breast, bowel, stomach and lung cancers. Data are expressed as estimated fraction of leukocyte RNA. Image adapted from Gentles et al. 2015 Nature Medicine.C. Human macrophage density (violet triangle) correlates with markers of poor survival (red triangle) in most cancer types.
Figure 2. Macrophage diversity drives tumor progression to metastasis
Tumour-Associated Macrophages (TAM) expressing canonical macrophage markers in the mouse (CD 11b, CSF1R, F4/80) can be polarized to adopt different pro-tumoural functions dependent on the environment as indicated. These activities promote tumour initiation through inflammation, tumor progression to malignancy via enhancing angiogenesis, immunosupression, invasion, intravasation and at distant sites promoting tumour cell extravasation and persistent growth. Each of these functions is performed by a sub-population of macrophages with a different transcriptome and cell surface markers.
Figure 3. TAM protumoral activities can be targeted to reduce tumor progression. Left panel. Summary of pro-tumoral TAM activities (angiogenesis, immune escape, dissemination etc) and the molecules/mechanisms involved in these processes.Right panel. Summary of TAM targeting strategies aimed at the reduction of TAM recruitment and survival and their reprogramming into anti-tumor macrophages.
Figure 4. Selective examples of anti-TAM drugs currently under clinical trials investigation. For each of the strategies outlined in the review, (recruitment, survival and re-programming) there are currently drugs being tested in several clinical trials as monotherapy or in combination with chemo – and immuno-therapies. In many cases one anti-TAM drug can affect more than one process, such as CSF1R inhibitors that inhibit recruitment, survival as well as function.
BOX1. Macrophage lineage and TAM origin redefined.
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Macrophages originate from at least three different embryonic sources (i.e. Yolk sac, Fetal liver and Bone marrow) and differentiate into tissue-specific macrophages in the body according to their origin. Some tissues are almost exclusively derived from one source such as the brain from yolk sac and intestine from bone marrow while other tissues are often hybrids with different proportions of macrophages from one source or another (see references 269,271 for different lineage interpretations). In each case the CSF1R signaling whose ligands are IL34 and CSF1, is central to their survival and differentiation with notable exceptions such as the lung that require GM-CSF. In tumours TAM originate mainly from bone marrow derived classical monocytes (Ly6chi) but can also arise from yolk sac derived tissue progenitors.
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