Journal of Controlled Release 254 (2017) 92–106

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Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Targeting tumor associated macrophages (TAMs) via nanocarriers MARK

a,1 a,1 a a a
Yuvraj Singh , Vivek Kumar Pawar , Jaya Gopal Meher , Kavit Raval , Animesh Kumar ,
Richa Shrivastavab, Smrati Bhadauriab, Manish K. Chourasiaa,⁎
a
Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
b
Toxicology Division, CSIR-Central Drug Research institute, Lucknow 226031, India

A R T I C L E I N F O A B S T R A C T

Keywords: Recruitment of inflammatory cells to tumor has been well documented, with the most frequent inhabitants being
TAMs macrophages termed as tumor associated macrophages, (TAMs). Their presence was thought to be an evidence of
Nanocarriers immune system initiating a fight response towards the tumor, i.e. immune surveillance. This is the case too
Active targeting initially, when TAMs majorly exhibit an M1 phenotype, but their continued presence in tumor microenviron-
Tumor environment
ment brings about their polarization to M2 phenotype, which not only participate in continued sustenance of
Immunosuppression
Recruitment
existing tumor but also open up deleterious avenues for further progression and metastasis of cancer. Current
Polarization perspective is built around this very premise and focuses specifically on TAMs and how they are being targeted
by researchers working in annals of nanomedicine. To do so, we dwell into tumor microenvironment and focus
on nanotechnology based drug delivery aspects which have either been already or can be potentially employed
in the future to target tumor associated macrophages for improved immunoadjuvant therapy of cancer.

1. Introduction of macrophages present in tumor stroma [conveniently christened as

Balance of homoeostatic machinery is an important regulator of
human well-being. Even under exposition of harshest insults, home-
ostasis attempts to preserve body's integrity, i.e. it tries to restore basal
structural and functional levels. An immune response can be purported
as the perfect example, wherein, homeostatic machinery actively tries
to cure the human body when faced with stress. Immune response,
regardless of stimulant, scope, or context, is incessantly ubiquitous.
However, there are circumstances when an intended immune response
jeopardizes entire pretext for which that response was generated in the
first place; case in point being etiologic role of immune system in
progression of cancer. Immunoreactivity towards carcinoma cells is
expressed by lymphoid infiltration into tumor microenvironment and
regional lymphatic alterations, specifically phenotypic differentiation
tumor associated macrophages (TAMs)]. TAMs present two subtypes i.e.
classically activated macrophages (M1) and alternatively activated
macrophages (M2). The process of phenotypic conversion of one sub
type into another is termed as polarization of TAMs. M1 cells have
strong antigen presenting capability and facilitate immunological
response against tumor. They raise proinflammatory cytokines such
as TNFα, IL-12, and IL-23 and enhance release of reactive oxygen
species (ROS) and nitric oxide (NO) which provides an antitumor
response. Moreover, they also express high levels of major histocom-
patibility complex: class I and class II molecules, raising probability of
recognizing tumor specific antigens. Contrarily M2 subset has strong
anti-inflammatory activity. It enhances release of IL-4, IL-10, and IL-13
and has poor antigen presenting characteristics which suppresses T cells
and generation of ROS and/or NO. IL-4 and IL-10 have negative

Abbreviations: TAMs, Tumor associated macrophages; Akt, Protein kinase B; CAT, Catalase; CCL, Chemokine (C-C motif) ligand; CD, Cluster of differentiation; COX, Cycloxygenase; CpG,
Unmethylated sequences of DNA that have immunostimulatory properties; CSF, Colony-stimulating factor; CXCL, Chemokine (C-X-C motif) ligand; CXCL10, C-X-C motif chemokine 10
also known as IP10; CXCL9, Chemokine (C-X-C motif) ligand 9; CXCR1, Interleukin 8 receptor α; CXCR2, Interleukin 8 receptor β; EGF, Epidermal growth factor; EPR, Enhanced
permeability and retention; ERK, Extracellular signal–regulated kinases; GPx1, Glutathione peroxidase 1; HIF, Hypoxia-inducible factors; IKK-β, Inhibitor of nuclear factor kappa-B kinase
subunit beta; IL, Interleukin; IP10, Interferon gamma-induced protein 10; JNK, c-Jun N-terminal kinases; M1, M1 subtype of tumor associated macrophage; M2, M2 subtype of tumor
associated macrophage; MAPK, Mitogen-activated protein kinase; M-CSF, Macrophage colony-stimulating factor also known as CSF1; MDC, Macrophage-derived chemokine also known
as CCL22; MIG, Monokine induced by gamma interferon; MIP-1 β, A protein which in humans is encoded by the CCL4 gene; MMP, Matrix metalloproteinase; MRI, Magnetic resonance
imaging; MYO3A, A gene which encodes for the protein Myosin IIIA; NFκB, Nuclear factor kappa-light-chain-enhancer of activated B cells; NO, nitric oxide; p38, Class of mitogen-
activated protein kinases; PD-L1, Programmed cell death 1; PEG, Polyethyleneglycol; PET, Positron emission tomography; PI3K, Phosphoinositide 3-kinase; ROS, Reactive oxygen species;
SESN 3, A protein; SOD, Superoxide dismutase; STAT, Signal transducer and activator of transcription; TGF, Transforming growth factor; TRAIL, TNF-related apoptosis-inducing ligand;
TNFα, Tumor necrosis factor alpha; VEGF, Vascular endothelial growth factor; VEGFR, Vascular endothelial growth factor receptor
⁎
Corresponding author at: Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031, India.
E-mail address: manish_chourasia@cdri.res.in (M.K. Chourasia).
1
Contributed equally.

http://dx.doi.org/10.1016/j.jconrel.2017.03.395
Received 21 February 2017; Received in revised form 29 March 2017; Accepted 30 March 2017
Available online 01 April 2017
0168-3659/ © 2017 Elsevier B.V. All rights reserved.
Y. Singh et al. Journal of Controlled Release 254 (2017) 92–106

Table 1
Effect of M1 and M2 polarization of TAMs on expression of various receptors and production of cytokines and chemokines.

Cellular component M1 phenotypic TAMs M2 phenotypic TAMs Effector Cytokines References

Receptors Membrane scavenger receptor A and receptor B ↓↓ ↑↑ IL-10 [5,6]

Membrane CD14 ↓ ↑↑ IL-10 [7]
Membrane scavenger receptor CD163 and Fcε receptors (CD23) ↓↓ ↑↑ IL-4 and IL-13 [8,9]
Membrane mannose receptor CD206 ↓↓ ↑↑ IL-4, IL-10 and IL-13 [10,11]
Membrane Toll-like receptor 2 (CD282) and 4 (CD284) ↑↑ ↓ IFNγ [12]
Membrane Fcγ receptors (CD16, CD32, CD64) ↑↑ ↓ IFNγ [13]
Membrane CD80 ↑↑ ↓ IFNγ [14]
Cytokine decoy Interleukin-1 R type II (CD121b) ↓↓ ↑↑ IL-4, and IL-13 [15,16]
C-C chemokine receptor type 7 (CCR7) ↑↑ ↓↓ IFNγ [17]
C-C chemokine receptor type 2 (CCR2) ↓ ↑↑ IL-10 [18]
Interleukin 8 receptor α and β (CXCR1 and CXCR2) ↓ ↑↑ IL-4 and IL-13 [19]
Cytokines TNF-α, IL-1, IL-12 and Type I IFN ↑↑ ↓↓ IFNγ [20,21]
IL-1ra ↓ ↑↑ IL-4, IL-10 and IL13 [22]
IL-10 ↓ ↑↑ IL-4, and IL-13 [23]
Chemokines Chemokine (C-X-C motif) ligand 8 9, 10 and 11 (CXCL8, CXCL9, CXCL10 and ↑↑ ↓↓ IFNγ [24–27]
CXCL11)

Chemokine (C-C motif) ligand 2, 3, 4, and 5 (CCL2, CCL3, CCL4 and CCL5)
Chemokine (C-C motif) ligand 17, 22 and 24 (CCL17, CCL22 and CCL24) ↓↓ ↑↑ IL-4 and IL-13 [23,28]
Chemokine (C-C motif) ligand 18 (CCL18) ↓↓ ↑↑ IL-4, IL-10 and IL-13 [29]

downstream effect on secretion of regulatory cytokines like IP-10
(CXCL10), and MIG (CXCL9) which stall Th1 immune response;
whereas IL-4 and IL-10 released by them upregulates secretion of Th2
immune response mediators like eotaxin-2 (CCL24), CCL18 and MDC
(CCL22) [1,2]. Known list of effectors, chemokines and receptors
modulated by TAMs is very large, however, some selected ones which
sub serve role in cancer dynamics are listed in Table 1.
Accumulation of TAMs in tumor stroma has significant physiologi-
cal implications [3,4]. They initially inhibit, but later due to ‘self and/or
cross’ talk promote tumor progression in various ways including
retardation of antitumor immunologic response, stimulating angiogen-
esis, etc. It is therefore no wonder that scientists have tried to target this
complex interplay, “of originally intended to be good immune cells,
gone awry” to provide immunotherapy of cancer. Current perspective is
built around this very premise and focuses specifically on TAMs and
how they are being targeted by researchers working in annals of
nanomedicine. To do so, we dwell deep into tumor microenvironment
and try to locate chinks in tumor armory, which might be used to
modulate activity of TAMs. We then focus on nanotechnology based
drug delivery aspects which have either been already or can be
potentially employed in the future to target tumor associated macro-
phages for improved immunoadjuvant therapy of cancer.
2. Recruitment of TAMs
Peyton Rous first coined the term “subthreshold neoplastic states”
for chemical and viral carcinogenesis induced somatic changes because
they involve irreversible DNA alterations [30]. But it was Rudolf
Virchow who visualized recruitment of leukocytes in tumorous tissues
and proposed a link between tumor and inflammation [31]. Later Karin
substantiated Virchow's notion by elucidating the role of inflammation
in tumorigenesis, particularly involvement of NFκB. This study even-
tually linked immunity and inflammation to cancer development and
progression; as all known targets of NFκB inhibitor, IKK-β, such as TNF-
α, IL-1, IL-6 were associated with carcinogenesis [32]. These observa-
tions strengthened the link between cancer related inflammation, the
seventh hallmark of cancer, to genetic instability [33]. Thus recruit-
ment of inflammatory cells to tumor became well documented with the
most frequent inhabitants being tumor associated macrophages. Their
presence was thought to be an evidence of body initiating a fight
response towards tumor and referred to as immune surveillance [34]. In
terms of lineage, TAMs are derived either directly from circulatory
monocytes or pre-differentiated macrophages invading the periphery.
Tymoszuk et al. have demonstrated that both monocyte recruitment
and local macrophage proliferation determines TAMs pool size in
HER2/Neu-positive mammary carcinomas [35]. Recruitment in tumor
stroma is instigated by various factors like tumor induced local hypoxia,
prevalent acidosis, cytokines, etc. (Fig. 1). For instance, the soluble
chemokine CCL2, whose expression itself is regulated by activin A, is a
known facilitator of monocyte or macrophage accumulation in inflam-
matory sites in vicinity of prostate [36] and breast cancer [37,38].
Apart from its chemotactic action, CCL2 also partakes in signaling
which leads to polarization of monocytes to M2 subtype instigating Th2
immune response [39]. Mizutani K et al. have demonstrated CCL2
overexpressive luciferase-tagged PC3 cells attract monocytes in both in
vitro and in vivo conditions using a xenograft model. They were even
able to significantly reduce tumor burden by administering CCL2
neutralizing antibodies, which prevented recruitment of macrophages
in tumor microenvironment [40]. Other chemokines such as CXCL1,
CXCL8, CXCL12, CXCL15, CCL5, CCL17 and CCL22 are also observed in
tumor environment, playing variety of overt, permissive or occult roles
in initial inflammation, tumor progression, recruitment and polariza-
tion of TAMs [41–43].
Almost all oxygen-breathing species express a highly conserved
transcriptional complex, hypoxia-inducible factor (HIF-1) that responds
to decrease in available intracellular oxygen. In general, HIFs are vital
to embryonic development and tissue repair. However, several reports
suggest HIF-1 is deregulated in cancers due to distorted tumor milieu
which makes chronic inflammation self-perpetuating and provides axis
for further sustenance and metastasis of cancer [44–46]. HIF activity is
required in angiogenesis which is mandatory to nurture rapidly
dividing cancer cells, and consequently HIF inhibitors like acriflavine
and phenethyl isothiocyanate are under the scanner of clinical inves-
tigation as probable anticancer drug candidates. HIF enhances expres-
sion of cytokines CXCR4 and CXCL12 which not only attract circulatory
monocytes but also induce their polarization to M2 subtype by ERK
signaling [47–50]. Tripathi et al. have elucidated the mechanism for
recruitment of TAMs to hypoxic regions. They identified and described
role of hypoxic cancer cell derived cytokines Oncostatin M and Eotaxin-
1 in promoting recruitment of macrophages and their phenotypic
switching to M2 subtype in solid tumors [51]. In a different study
Wen Z et al. have shown that even transient elevation of 5-lipoxygenase
(a metabolite produced by hypoxic cancer cells) enhances infiltration of
TAMs. This hastening in migratory tendency of TAMs in response to
local 5-lipoxygenase levels is mediated by matrix metalloproteinase
(MMP)-7 via p38 pathway. The mechanism is so prudent that in in vivo

93
Y. Singh et al. Journal of Controlled Release 254 (2017) 92–106

Fig. 1. Concept of tumor associated macrophage and its physiologic implications. Any macrophage found inside tumor stroma is nominated as a tumor associated macrophage (TAMs).
These macrophages undergo phenotypic differentiation into either M1 or M2 subtype to produce opposing effects on their milieu. M1 cells produce antitumor response; whereas M2 cells
are strongly pro-tumor and tend to produce angiogenesis, immunosuppression and metastasis of cancer cells. Hypoxic tumor cells, certain growth factors, cytokines and chemokines are
strong stimuli for recruitment and activation of TAMs.

studies, 5-lipoxygenase inhibitor (Zileuton) effectively wards off infil- discussed next. To aptly signify observations made in the following
tration of TAMs [52]. Hypoxic regions in tumor microenvironment are sections, a list of ongoing clinical trials trying to establish or mitigate
therefore an established stimuli for recruitment of TAMs [53] and role of TAMs in varying cancers has also been compiled in Table 2.
appear to be a loci of substantial significance in immunotherapy of
cancer for the foreseeable future. Semaphorin 3A released from tumor
3. TAMs promote angiogenesis
hypoxic regions is yet another chemoattractant for TAMs. It works by
phosphorylating vascular endothelial growth factor receptor-1
Angiogenesis is a physiological process which generates new blood
(VEGFR1), Neuropilin-1 (Nrp1) and PlexinA1/PlexinA4 holoreceptors
vessels form pre-existing ones and is vital in growth, development, and
[54].
wound healing; however it is also fundamental in transforming a benign
In animals, intermittent hypoxia activates cyclooxygenase-2 (COX-
tumor to a malignant one. M2 phenotype of TAMs are steeply pro-
2) pathway. The consequent increase in prostaglandin E2 (PGE2)
angiogenic [58]. In a study conducted by Lie et al., TAMs were found to
accelerates tumor progression by inducing phenotypic switching of
be major agents of angiogenesis in middle T oncoprotein mice [59].
TAMs to M2 subtype. Campillo N et al. have used celecoxib, a COX-2
They enhance secretion of vascular endothelial growth factor (VEGF),
inhibitor, to prevent intermittent hypoxia induced lung tumor malig-
which is a major contributor towards angiogenesis in various cancers
nancy in a mouse model. They found that TAMs isolated from celecoxib
[60,61]. Among VEGF, the most potent and widely reported factor is
treated mouse themselves had innate ability to reduce proliferation of
VEGF-A [62]. VEGF-A and colony-stimulating factor-1 (CSF-1) have
naïve Lewis lung cancer cells while the opposite occurred when TAMs
been attributed as possible perpetrators of TAMs migration. Linde N
isolated from untreated or placbo treated mice were used. In a different
et al. found that implantation of VEFG-A in non-cancerous skin cells
study Dubey P et al. have also demonstrated role of COX-2 in
induced tumor formation during in vivo experiments [63]. Secretion of
accelerating polarization of TAMs using an antagonist flunixin meglu-
VEGF-A is regulated by hypoxia inducible transcription factors (HIF-1α
mine [55].
and HIF-2α) in tumor hypoxic regions [64,65]. However HIF-1 is not
Grimshaw MJ et al. have investigated inhibition of monocyte and
the only regulator and several other cytokines like IL1β [66], TGFβ1
macrophage chemotaxis under influence of hypoxia and inflammation
[67] and CSF-1 (M-CSF) [68] also have a say. Effect of cytokine
accompanying tumor by mitogen-activated protein kinase (MAPK)
Oncostatin M on angiogenesis has been studied by Vasse et al., on
inhibitors. It was observed that under normal circumstances without
endothelial cells in a rabbit corneal model. It was found that in vivo
inhibitor pretreatment, level of MAPK mRNA was drastically enhanced
blockade of Oncostatin M synergistically potentiated anti-angiogenic
in migratiory monocytes/macrophages whereas level of phosphorylated
effect of bevacizumab, an existing angiogenesis inhibitor [69]. Another
MAPK was much less [56] which sets up MAPK inhibitors as probable
major contributor to angiogenesis is a class of enzyme termed as matrix
immunoadjuvants capable of iterating macrophage infiltration in tumor
metalloproteinase (MMP). MMPs degrade proteins responsible for
stroma. For a more detailed overview on the bridge between TAMs and
turgidity of extracellular matrix of existing endothelial vessels. This
hypoxia, and the resulting therapeutic approaches, readers are encour-
proteolysis allows individual endothelial cells to infiltrate tumor stroma
aged to go through a perspective by Silva et al. [57]. Once recruited to
and combine with preexisting VEGF-A to form new capillaries as seen in
tumor environment, TAMs, especially the M2 subtype produce myriad
sprouting angiogenesis. Since TAMs facilitate expression of MMPs,
of responses, which both in the short as well as long run promote tumor
especially MMP-1 [70], MMP-7 [71] and MMP-9 [72], their overriding
growth and metastasis. Some of the prominent responses of macro-
role in promoting angiogenesis and tumor growth becomes extremely
phage recruitment and polarization in tumor microenvironment are
apparent. Once formed, tumor endothelium releases angiopoietin-2

94
 Table 2
On-going clinical trials investigating role of tumor associated macrophages in varying cancers.
Drug Disease
– Lung Cancer
– Advanced non-small cell
lung cancer
– Wilms Tumor and Other
Childhood Kidney Tumors
Ferumoxytol followed by MM- Solid Tumors
398 administered i.v. ER/PR Positive Breast
Cancer
Triple Negative Breast
Cancer
Metastatic Breast Cancer
With Active Brain
Metastasis
Multitargeted Tyrosine Kinase Stage I, Stage II, Stage III
Inhibitor PLX3397 Prostate Adenocarcinoma
95
– Neuroblastoma
GM-CSF (sargramostim) Stage II or Stage III Colon
Cancer
cSLO imaging Ocular Melanoma
– In vitro model of
pancreatic ductal
adenocarcinoma
carcinoma
– Malignant Mesothelioma
AMG 820 Advanced Solid Tumors
(human IgG2 c-fms
Target Method Study phase Clinical trials. Status Sponsor
Y. Singh et al.
gov identifier
Impact of M1/M2 tumor associated Treatment response and mortality, clinical Observational NCT00690261 Recruiting National Taiwan
macrophage polarization on cancer presentation participants University Hospital
progression and prognosis prediction
Correlate response and outcomes in Response to treatment of advanced NSCLC Retrospective NCT01551251 Completed Chang Gung Memorial
advanced non-small cell lung cancer after with high or low TAMs, CD68 antibody Hospital
first line treatment staining
Relationships between tumor-associated Tumor stage, presence of vascular invasion, Observational NCT01493817 Completed Children's Oncology
macrophages and clinicopathological tumor progression, and survival. Paraffin- Group
factors in Wilms Tumor embedded specimens are analyzed for
macrophage markers
Determine biodistribution of MM-398 and Measure tumor levels of irinotecan and SN- Interventional NCT01770353 Recruiting Merrimack
feasibility of Ferumoxytol as magnetic 38, safety profile of MM-398 in the presence phase I pilot study participants Pharmaceuticals
resonance imaging to measure tumor of ferumoxytol, Tumor response rate
associated macrophages and to predict measured by RECIST, Cmax, AUC
patient response to treatment
Targeting the prostatic tumor Grade of specific types of toxicity, MTD of Interventional NCT02472275 Recruiting Barbara Ann Karmanos
microenvironment with PLX3397, a multitargeted tyrosine kinase inhibitor Phase I participants Cancer Institute
tumor-associated macrophage inhibitor in PLX3397, Change in TAMs levels as measured
men with unfavourable risk prostate by immunohistochemistry, Change in PSA
cancer undergoing radiation therapy and levels
androgen deprivation therapy
Identify subgroups of rNB patients who Gene panel sequencing of tumor specimens, Observational NCT02868268 Recruiting New Approaches to
have potentially targetable genetic (ALK, Identify genomic alterations, presence/ participants Neuroblastoma Therapy
MAPK pathway, Metabolic-related genes) absence of immunologic biomarkers Consortium
and/or immunologic (tumor-associated
macrophage infiltration and/or
programmed death ligand [PD-L1]
expression) biomarkers in rNB
Efficiency of GM-CSF and combination Safety of neoadjuvant and adjuvant Interventional NCT00262808 Completed University of Rochester
chemotherapy work in treating patients sargramostim (GM-CSF), proportion of Phase II
who are undergoing surgery patients with a change in tumor-associated
macrophage, VEGF expression, overall
survival
Determine whether a novel imaging Identification of macrophages Observational NCT02909517 Recruiting St. Michael's Hospital,
technique can identify presumptive tumor participants Toronto
associated macrophages (TAMs) in
patients with ocular tumors to establish
role of inflammation
Impact of macrophages on de-novo Proliferation and migration of PANC1 cell Intervention NCT01921699 Unknown Rambam Health Care
chemoresistance of adenocarcinoma cells line in the tested groups, direct cell-to-cell Campus
interaction or soluble factors participate in
MIC
Diagnostic and prognostic role of new Purification of tumor associated macrophages Observational NCT02300883 Ongoing Armando Santoro, MD
inflammation biomarkers (TAMs) and tumor cells from pleural
effusions,
mass spectrometry analysis of TAMs and
tumor cells. Confocal microscopy analysis of
macrophages, tumor cells
Sequential dose escalation and expansion Evaluating the safety, tolerability and Interventional NCT01444404 Completed Amgen
study of AMG 820 in patients pharmacokinetics of AMG 820, change in Phase I
(continued on next page)
Journal of Controlled Release 254 (2017) 92–106
 Table 2 (continued)
Drug
antagonistic antibody)
– Colorectal Cancer,
GM-CSF
Emactuzumab and RO7009789
– Paediatric Solid Tumors
MCS110, Carboplatin,
96
gemcitabine
Metformide Hydrochloride/
Pioglitazone Hydrochloride
Extended-Release Tablet
Paclitaxel
RO5509554
Anti-Macrophage Migration
Inhibitory Factor (Anti-MIF)
Antibody
Vaccine MAGE-3.A1 peptide, or
the NA17.A2 peptide + IL-2,
IFN-α and GMCSF,
Imiquimod
PD 0360324 Cyclophosphamide
Ferumoxytol-Enhanced MRI
Disease Target Method Study phase Clinical trials. Status Sponsor
gov identifier
Y. Singh et al.
tumor associated macrophages (TAMs) as
assessed by tumor biopsy
Defining the scopes of specific Transcriptomal and molecular Observational NCT00979277 Unknown National University
Nasopharyngeal, immunotherapy strategies to overcome characterization of tumor associated Hospital, Singapore
Hepatocellular and Renal tumor-induced immunosuppression and monocytes/macrophages
cell carcinoma induce monocyte/macrophage-mediated
antitumor response
Hematologic Malignancies Study of low-dose cyclophosphamide, Immunological efficacy as indicated by T-cell Interventional NCT02709993 Yet to open Kiromic, LLC
tumor associated peptide antigen-pulsed cytokine levels Phase II
dendritic cell therapy and low dose
granulocyte- macrophage colony
stimulating factor, as consolidation
treatment
Solid Tumors To assess safety, pharmacokinetics, Total tumor-associated macrophages (TAMs) Interventional NCT02760797 Recruiting Hoffmann-La Roche
pharmacodynamics and therapeutic in paired-tumor biopsies Phase I participants
activity of emactuzumab and RO7009789
administered in combination
Characterize the molecular, cellular and Evaluate tumor-associated macrophages Observational NCT01050296 Recruiting St. Jude Children's
genetic properties of primary and (TAMs) post-therapy to locate gene participants Research Hospital
metastatic neuroblastoma, osteosarcoma, expression patterns, markers and functional
retinoblastoma, Ewing sarcoma family of correlation of the tissue repair properties of
tumors, soft tissue sarcomas, macrophages
adrenocortical tumors and liver
malignancies
Advanced Triple Negative Determine whether MCS110 antibody Tumor associated macrophages (TAMs) and Interventional NCT02435680 Recruiting Novartis
Breast Cancer (TNBC) therapy improves the efficacy of Tumor infiltrating lymphocyte (TIL) content Phase II participants Pharmaceuticals
With High TAMs carboplatin and gemcitabine (carbo/gem) in pre- and post-dose tumor biopsies
Oral Cavity Neoplasm How well ACTOplus met XR works in Change in treg, T4, T8, tumor-associated Interventional NCT02917629 Yet to open National Cancer
Oropharyngeal Neoplasm treating patients with stage I-IV oral macrophage-CD68 positive, PD1, PD-L1 Phase II Institute
cavity or oropharynx cancer that are expression, assessed in tumor tissue by IHC
undergoing definitive treatment
Advanced Solid Tumors Safety, tolerability, pharmacokinetics and Pharmacodynamic marker: tumor associated Interventional NCT01494688 Ongoing Hoffmann-La Roche
pharmacodynamics of RO5509554 in macrophages (TAMs) expressing cells in Phase I
participants surrogate/tumor tissue
Breast Cancer Trans-tissular migration of macrophages Surgical samples of human primitive breast in Interventional NCT02876497 Yet to open Institut Claudius Regaud
associated to human breast cancer terms of histology. Isolation, differentiation
of human monocyte-derived Macrophages
(Mphs) and determination of the Macrophage
(Mph) migration mode,
Malignant Solid Tumors Safety, tolerability, pharmacokinetics, Number of participants who develop binding Interventional NCT01765790 Completed Baxalta US Inc.
and pharmacodynamics of anti-MIF and/or neutralizing anti-anti-macrophage Phase I
antibody migration inhibitory factor (anti-MIF)
antibodies following treatment with anti-MIF
Metastatic Melanoma Study of peptide vaccination associated Tumor response will be assessed in Phase I NCT01191034 Unknown Cliniques universitaires
with tumoral immunomodulation with accordance with the modified RECIST version Phase II Saint-Luc- Université
proinflammatory cytokines Catholique de Louvain
Malignant Neoplasm of Pre-conditioning effects of anti- Change in density of cluster of differentiation Interventional NCT02948101 Yet to open M.D. Anderson Cancer
Female Genital Organs macrophage therapy using PD 0360324 in 8 (CD8+) T cells in tumor, safety of PD Phase II Center Pfizer
recurrent platinum-resistant epithelial 0360324 followed by cyclophosphamide
ovarian cancer
Brain Tumors or Other Monitoring extent of inflammation Obtain measurements of iron concentration Interventional NCT02452216 Recruiting Michael Iv
Conditions of the CNS associated with pathologies in the central (indicative of ferumoxytol uptake), participants
nervous system may be helpful for Determine number of macrophages in
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Journal of Controlled Release 254 (2017) 92–106
Y. Singh et al. Journal of Controlled Release 254 (2017) 92–106

(Tie2) overexpressive monocytes (TEMs), which despite counter evi-

dence, seem to further endorse tumor angiogenesis [73–75] by enhan-

Centre Leon Berard

cing secretion of IL-10 [76], stimulating overexpression of mannose

Duke University
receptors [77] and down regulating TNF-α and IL-12. TEMs also
produce high levels of MMP9 and VEGF-A which continue the vicious
Sponsor

cycle of angiogenesis, recruitment, and polarization [78].

4. TAMs exhibit immunosuppressive activities

participants

participants
Recruiting

Recruiting
Retardation of inherent antitumor immune response due to pheno-
Status

typic polarization of TAMs is a major concern during chronic stages of

cancer. For example, suppression of immune response by TAMs is one
of the major reasons responsible for current incurable status of Non-
NCT02777710

NCT02132182
Clinical trials.
gov identifier

Hodgkin lymphoma. TAMs produce IL-6 and IL-10 leading to over

expression of B7-H4 (a recently discovered immunosuppressive mole-
cule), which inhibits host anti-tumor immunity [79]. Additionally, M2
subtype of TAMs have capability to alter antigen presentation and T-cell
mediated immune response by enhancing secretion of IL-10 and TGF-β
[80]. Whilst attempting to delineate implications of macrophage
Interventional

Observational

polarization in squamous cell carcinoma of oral cavity, Costa NL

Study phase

et al. observed significantly higher percentage of M2 subtype of TAMs

Phase I

(accompanied with higher levels of IL-10 and TGF-β) in cancer inflicted

patients than in healthy individuals [81]. IL-10 decreases synthesis of
granulocyte-macrophage colony-stimulating factor (GM-CSF) which in
resected/biopsied samples at histopathology

turns reduces T-cells production and the subsequently expected anti-

Variations in the intensity of cell surface
receptor expression, Disease progression

tumor immune response [82]. An eicosanoid, PGE2 and chemotactic

factors CCL17, CCL18, and CCL22, derived from TAMs and IL-10 play
important role in induction of T regulatory cells [83]. IL-10 in
pharmacokinetic evaluation,

conjunction with TNF-α also participates in production of a chemokine

named programmed cell death 1 (PD-L1) which has deleterious effects
Dose-limiting toxicities
objective response rate

on cytotoxic T-cell production and functionality [84]. In murine tumor

models, suppression of CD8+ T cell proliferation by TAMs is dependent
on production of ROS. This suppression has been offered as a possible
explanation for failure of cytotoxic T lymphocytes in eliminating
Method

tumors [85].

5. TAMs promote tumor invasion and metastasis

without sarcoma to exploit potential anti-
well as monitoring treatment response of

anti-tumor T-cell response following PD-

between monocytes in patients with and

microenvironment of M2 macrophages,
diagnostic and prognostic purposes as

Phenotypic and functional differences

thus favoring induction of a cytotoxic
CSF1R blockade to deplete the tumor

tumor capabilities of monocytes and

current and future immunotherapies

TAMs play major role in metastasis of cancer by infiltrating lymph

L1 blockade with an anti-PD-L1

nodes draining tumor. In a study undertaken specifically to investigate

role of TAMs in malignant breast cancer metastasis, it was found that
TAMs infiltration density had a negative correlation with 5-year
monoclonal antibody

survival rates of breast cancer patients [86,87]. Fellani et al. have

looked into clinicopathological significance of M1 and M2 macrophages
macrophages

in cutaneous melanoma, by monitoring accumulation of M1 and M2

TAMs in tumor stroma, intratumoral nests, and distant invasive sites.
Target

They observed that population of M2 biased TAMs was overwhelmingly

greater than M1 TAMs in all stages of melanoma progression, thereby
favoring neoplastic growth and rapid dissemination of metastatic cells
[88]. Tumor cells secrete CSF-1 and TAMs release epidermal growth
factor (EGF) and TNF-α to simultaneously control migration of cells to
Pancreatic Cancer
Colorectal Cancer

and from tumor microenvironment [89]. Activation of EGF receptors

Osteosarcoma

increase invasion, invadopodia formation and matrix degradation [90].

Elaine Y. Lin et al. have provided evidences which frame CSF-1 to be
Disease

responsible for metastatic growth of tumor by promoting infiltration of

TAMs and regulating their functions [91]. Involvement of TNF-α/NF-κB
dependent pathway in cancer cell migration and invasion is well
established [92]. TNF-α induces expression of macrophage migration
inhibitory factor to render cancer cells invasive [93]. TNF-α is also
involved in MMPs signaling which further facilitates invasion of tumor
Table 2 (continued)

cells. Lee SJ et al. explored MMP9 activation by TNF-α in human

Durvalumab

urinary bladder cancer (HT1376 cells) and deduced involvement of

Pexidartinib

extracellular signal-regulated kinase (ERK1/2), p38 MAP kinase and

JNK pathways [94]. They were able to block TNF-α dependent
Drug

activation of MMP-9 by administering an antitumor nucleoside cordy-

cepin, which is a known inhibitor of kinase pathways and thereby have

97
Y. Singh et al.
come up with a probable strategy to stall invasive tendency of at least
the investigated cancer. VEGF also activates MMP to induce enhanced
vascularization and opens up new pathways for migration of invasive
cancer cells [95]. Belotti D et al. found that VEGF specifically activates
MMP-9 to cause invasion of ovarian tumor cells in mice bearing 1A9-
VS-1 tumor and treatment of animals with an anti-VEGF antibody
(bevacizumab) inhibits VEGF dependent activation of MMP-9 and
down-stream processes [96].
Tjiu et al. have investigated TAMs induced invasion of human basal
cell carcinoma. To do so, M2-polarized TAMs derived from peripheral
blood monocytes were cocultured with basal cell carcinoma cells in
coculture inserts so as to prevent direct cell to cell contact. It was found
that TAMs induced cyclooxygenase-2 dependent release of MMP-9,
VEGF-A, basic fibroblast growth factor which increased angiogenesis
and subsequent invasion of basal cell carcinoma cells [97].TAMs also
promote metastasis via paracrine signaling. Baghel et al. have reported
the role of MIP-1 β in potentiating invasiveness of both metastatic and
poorly metastatic cells which is dependent on MYO3A. Hence MIP1β-
MYO3A axis could be targeted for devising anti-metastatic therapeutics.
Cytokines found in mileu of TAMs also prompt invasion of cancer cells,
with CCL18 being an example whose expression in breast cancer cells
can be positively correlated to tumor invasion [98]. Other TAMs
derived cytokines like, IL-6 and CCL4, have been shown to induce
STAT3 activation in small cell lung cancer cells which leads to invasion,
spheroid formation, acceleration of tumorogenesis in distant locations
and chemoresistance. Downregulation of IL-6 receptor by anti-IL-6
receptor antibody supresses STAT activation and consequently the cell-
cell interaction between TAMs and cancer cells via intermediary
cytokines can be a target for anti-tumor immunotherapy [99].
6. Targeting tumor microenvironment by phamacotherapy
From the previous sections it is clear as to how accumulation of
TAMs in tumor milieu influences its sustenance, progression and
invasiveness. This has prompted researchers to try and plug relevant
gaps via immunotherapy. Unfortunately though, clinical implications
are still tricky, as stand-alone immune modulation has been found to be
capable of only priming the body to fight tumor, and not actually
eradicate it. The next plausible maneuver in clinically tackling cancer
therefore seems adaptation of a combinatorial strategy amalgamating
circumstantially effective chemotherapy with a facilitatory immuno-
modulative regimen to obtain a focused counter measure free from
incidences of adverse effects or subpar efficacy. Nanomedicine and
colloidal carrier based drug delivery has recently come to the fore with
respect to anticancer therapy [100,101]. Nanocarriers owing to their
size inadvertently accumulate in irregularly fenestrated tumor micro-
environment (EPR effect) paving way for preferential tumoral localiza-
tion of chemotherapeutic agents and utilization of ligand based active
targeting with several products gaining clinical approval [102,103].
This opens doors for selectively killing [104] or reversing polarization
of macrophages infiltrating tumor environment, leaving peripheral or
circulatory monocytes/macrophages unharmed [105,106]. Due to their
miniscule size, nanocarriers furnish novel patterns of drug delivery
inside cancer cells, by presenting maximal payload directly at intracel-
lular locations like nucleus or microtubule [107–110]. They do so by
modulating binding capacity and penetrability of cell membrane and
different cellular organelles. Nanocarriers also mold inherent pharma-
cokinetic properties of drugs they carry by controlling their presenta-
tion to systemic dispositional forces, thereby catalyzing drug's efficacy
and overall safety [111–114] [115–117]. For instance, HMG-CoA
reductase inhibitors (statins), at certain higher doses modulate various
intracellular pathways to exhibit quantifiable anticancer activity
[118,119]. Simvastatin indirectly increases intracellular oxidative
stress and induces apoptosis by quenching free radical scavengers
(Mn-SOD, CAT, GPx1, and SESN 3) [120]. Targeted delivery of statins
to tumor microenvironment can therefore theoretically improve their
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Journal of Controlled Release 254 (2017) 92–106
anticancer efficacy by reducing dose dependent side effects. In a recent
study, simvastatin was encapsulated in a long circulating nanometric
liposomal formulation and administered intravenously in B16F10
tumor bearing animals. Liposomal simvastatin significantly reduced
tumor growth compared to free simvastatin with the purported activity
directly dependent on local infiltrative TAMs population. Liposomal
simvastatin furnished anticancer activity by modulating malondialde-
hyde levels, nitrites levels, catalase activity, and HIF-1α production, all
of which have established roles in the dynamics of TAMs and tumor
[121]. Until now iron supplement ferumoxytol and other iron oxide
nanoparticles have been used for treating iron deficiency or as contrast
agents for magnetic resonance imaging and as drug carriers. However,
recent studies have shown their role in inducing polarization of
macrophages towards pro-inflammatory M1 subtype in tumor tissues.
Macrophages exposed to ferumoxytol displayed increased mRNA
associated with pro-inflammatory Th1-type responses. Intravenous
treatment of ferumoxytol in adenocarcinoma afflicted mice enhanced
the presence of pro-inflammatory M1 macrophages in the tumor tissues
and thus could potentiate macrophage-modulating cancer immu-
notherapies. Inhibiting M2 polarization of TAMs is also an approach
for cancer therapy. Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a
natural plant phenol produced in response to attack by pathogens has
been found to inhibit activation of M2 macrophages. Whilst trying to
decode the underlying mechanisms in a lung cancer model, Liwei Sun
et al. discovered that resveratrol significantly reduced STAT3 activation
and F4/80 positive cells in and around tumor. They exploited this
response to inhibit lung cancer growth, in vitro and in vivo [122].
7. Modification of TAM functionality using nanocarriers
Reversal of M2 subtype to M1 is a viable strategy for chemoimmu-
notherapy of cancer. Polarization of TAMs can be reversed by modulat-
ing Th1 cytokine profile and balancing expression of proangiogenic and
antiangiogenic factors. Working in this direction Huang et al. encapsu-
lated CpG, anti-IL-10 and anti-IL-10 receptor oligonucleotides in a
galactosylated cationic dextran nano-complex which had specific action
directed against TAMs. The nano-complex was fabricated to sense
subtle variation in pH associated with tumor microenvironment
triggering release of its encapsulated oligonucleotides. When tested in
an allograft hepatoma murine model, the delivery system directly
accumulated in TAMs and caused its phenotypic reversion by inducing
secretion of Th1 cytokine IL-12 and limiting Th2 cytokine IL-10.
Expression of M2 related genes (Arg1, Ym1, Msr2, FIZZ1, Mgl1 and
Mgl2) was reduced. Further, expression of proangiogeneic factors
including VEGF and MMP9 was also suppressed which cumulatively
resulted in significant tumor regression [123]. Wang et al. have
developed tumor environment sensitive poly-β-amino ester nanoparti-
cles and used them to deliver IL-12 for reeducating TAMs without
causing any undue cytotoxicity. In in vivo studies, reversing of TAM
polarization was confirmed by identifying relative count of CD206
(marker for M2) and CCR7 (marker for M1) positive cells using confocal
microscopy and flow cytometry. Biodistribution studies employing
surrogates suggested that enhanced therapeutic efficacy of IL-12 loaded
nanoparticles was due to their selective tumoral accumulation and
capability to extend circulation time of IL-12 [124]. Banciu et al. whilst
investigating probable role of TAMs in anticancer activity of Doxil® (an
approved pegylated liposomal formulation of doxorubicin hydrochlor-
ide) found that it had little or no effect on level of proangiogeneic and
antiangiogenic factors [125]. Taking note, they prepared polyethylene
glycol functionalized dipalmitoylphosphatidylcholine liposomes loaded
with prednisolone phosphate, which were capable of preferentially
accumulating in tumor microenvironment and remodel TAMs polarity.
Administration of prednisolone phosphate loaded liposomes in B16F10
induced tumor bearing mice significantly reduced expression of proan-
giogeneic factors (GM-CSF, M-CSF, IL-1α, IL-1β, IL-6, IL-9 and TNF-α)
[126].
Y. Singh et al.
Bisphosphonates have been reported to act and modify phenotypic
status of TAMs to render them anti-tumor [127–129]. Zoledronic acid is
the most potent bisphosphonate; but its anticancer activity is limited by
short plasma half-life and rapid accumulation in bones. This is where
nanotechnology comes into fray. Zoledronic acid was encapsulated in
pegylated nanoparticles to improve its accumulation in tumor micro-
environment. In, in vivo studies, normal drug had no anticancer activity
whereas drug carrying pegylated nanoparticles possessed substantial
anticancer activity and prolonged animal survival. TAMs population
and neoangiogenesis was also reduced [130]. Another phosphonate
analogue, clodronate, enhances secretion of Th1 cytokine and inhibits
secretion of Th2 cytokines, angiogenic factors, but its use is again
limited by inherent pharmacokinetic constraints [131,132] necessitat-
ing development of liposomal clodronate [133,134]. Zeisberger SM
et al. have successfully employed liposomal clodronate formulation to
reeducate TAMs for beneficial activity in cancer. Immunohistochemical
evaluation showed liposomal clodronate drastically reduced F4/80
positive cells in comparison to free clodronate [135]. In a different
study Fritz et al. showed that liposomal clodronate specifically hampers
TAMs population in mouse lung adenocarcinomas by reducing insulin-
like growth factor-I, CXCL1, IL-6, and CCL2 levels in pulmonary fluids
[136]. A recent study by Pal et al. has confirmed functionality of noble
metal (gold and silver) nanoparticles in modulation of TAMs [137]. The
study addresses functional characterization of TAMs isolated from
murine fibrosarcoma induced by a chemical carcinogen, 3-methylcho-
lanthrene. The fabricated gold and silver nanoparticles were able to
modulate reactive oxygen species and reactive nitrogen species produc-
tion in tumor cells and also activate proinflammatory signaling
cascades resulting in a polarization of TAMs from M2 to M1.
8. Direct killing of TAMs
There is an alternative school of thought which prefers complete
eradication of susceptible M2 subtype from tumor stroma, instead of
reaping advantages from its reversion to M1 cells, as theoretically, there
is always a chance of a phenotypic re-reversal after discontinuation of
therapy. Wu et al. conducted a cytotoxicity experiment on a co-culture
of Hut78tumor cells and M2 macrophages and found that liposomal
clodronate selectively kills TAMs and does not affect viability of Hut78
tumor cells. Implications of selective TAMs eradication were investi-
gated in animals having cutaneous T-cell lymphoma. Intravenous
administration of liposomal clodronate reduced TAMs (measured by
number of F4/80 positive cells) and inhibited angiogenesis (quantified
by CD31 and podoplanin antibodies in tissue sections) which ultimately
led to substantial tumor reduction [138]. Other examples include
packaging of doxorubicin in a TAMs targeted delivery system to
produce a gross cytotoxic response. Biodistribution studies revealed
that doxorubicin loaded system selectively accumulated in tumor and
caused massive reduction in TAMs population and overall B16F10
tumor burden [139]. F. Piaggio et al. and co-workers have developed
clodronate-containing liposomes for intravenous administration. These
novel systems were investigated in vitro to inhibit proliferation and
induce apoptosis of a macrophage-like cell line in a dose and time-
dependent manner. Upon administration in B16/F10 subcutaneous
melanoma-bearing mice model, it significantly lowered the plasma
levels of IL-10, Mo KC, TNF-α and VEGF [140]. Folate receptors are
over expressed on numerous cancer cells as well as on adjoining TAMs,
which renders them as a convenient target for cancer treatment. Luo T
et al. have successfully exploited this receptor-cancer axis to deliver a
combinatorial pay load of oxygen and paclitaxel in xenograft mouse
ovarian cancer model using ultrasound guided microbubbles. It was
found that, microbubbles could penetrate deep into tumor ascites fluid
and tumor nodules, and induce apoptosis and reduce expression of
VEGF. Since this system was cytotoxic to both cancer cells and TAMs, it
can provide an approach of dual intervention at both cytotoxic and
immunologic level [141].
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Journal of Controlled Release 254 (2017) 92–106
9. Magic bullets: Actively targeting TAMs
M1 and M2 macrophages have differing phenotypes, which not only
alters their functionality but also induces in them several structural
differences. The resultant of such variations is especially felt in relative
presence/absence of surface receptors, which participate in uptake
processes like phagocytosis, endocytosis, pinocytosis etc. Since, nanoar-
riers also exploit similar pathways for uptake; a cursory inspection into
intracellular pharmacokinetics of administered nanoparticles might
provide clues towards selecting appropriate construct material, dosing
frequency, etc. For instance, Jones et al. devised an intravital micro-
scopy based assay to determine effect of immune system on nanopar-
ticle clearance. But their results were contradictory; in vivo studies
suggested that nanoparticles were cleared more slowly in Th1 biased
mice in comparison to Th2 biased mice, whereas the trend was opposite
in vitro, where macrophage polarization from M1 to M2 increased
nanoparticle uptake and retention [142]. In a different study Hopp-
stadter et al. obtained conformity in in vitro and in vivo results. Uptake
of investigated fluorescent silica nanoparticles was greater in M2
polarized macrophages in comparison to M1 polarized macrophages
[143].
Wang et al. attempted to detail effect of nanoparticle surface
modification and macrophage polarization on cellular uptake. They
constructed different batches of particles using p(N-isopropylacryla-
mide-co-acrylic acid) (p-(NIPAm-co-AAc)) copolymer and functiona-
lized them with thirteen functional groups. Quantitative structure
activity relationship software deduced that surface charge, number of
hydrogen atoms and number of 2° carbon atoms in nanoparticle
construct had a telling say in decreasing complement activation and
overall uptake in M2 polarized macrophages [144]. Fuchs AK et al. also
investigated the role of surface decoration of delivery vectors on fate of
macrophage subset. They functionalized polystyrene nanoparticles with
carboxyl and amine groups. The decorated nanoparticles did not affect
expression of the M1 markers CD86, NOS2, TNF-α, and IL-1β. In
contrast, both carboxyl and amine functional groups impaired expres-
sion of scavenger receptor CD163 and CD200R, and release of IL-10
thus suggesting a probable role for functionalized nanoparticles as a
tool in selective reprogramming of M1/M2 polarization [145]. It
therefore seems rational that drug loaded nanoparticles should be
decorated with phenotype receptive ligands to vindicate notion of
Ehlrich's magic bullet, i.e. specific activity with little/no off target
effects. Following section details some bio-inspired ligands which have
been employed to target TAMs.
9.1. Carbohydrates
Scavenger receptors like lectin (CD206/mannose receptors) are
overexpressed selectively on M2 subtype of TAMs. CD206 receptors
respond to mannose and fucose residues on any trailing entity,
initiating its endocytosis via clathrin mediated pathway. This has
prompted extensive utilization of carbohydrate moieties (Mannose,
dextrans and galactose) for decoration of drug and nucleotide loaded
nanoparticles to target TAMs via CD206 receptors [139,146]. In a
recent study by Yu et al., a triblock copolymer was functionalized with
mannose using click chemistry. The synthesized mannose copolymer
was used to develop a micellar system for delivery of siRNA to TAMs.
Mannose functionalization significantly enhanced macrophage uptake
of micelles and produced knockdown of target gene [147]. The same
group also mannosylated nanoparticles for delivering siRNA to TAMs.
Depending upon the experimental model, the said nanoparticles
accumulated selectively in ovarian tumor and lung tumor, undergoing
rapid internalization by targeted tumor associated macrophages to stop
cancer metastasis without producing any side effect on liver and kidney
functions [146]. Proficiency of mannose ligated delivery system has
been validated by PET imaging. A dual imaging mannosylated liposo-
mal system was prepared by tagging 64Cu to a fluorescent dye
Y. Singh et al.
encapsulated in a lipidic membrane. PET and fluorescent imagining
reveled that liposomes specifically accumulate in TAMs upon adminis-
tration in an animal model with lung adenocarcinoma [148]. Zhu et al.
have amalgamated advantages of mannose receptor based macrophage
recognition with a sheddable PEG cloak to come up with a full proof
method of ensuring targeted delivery of doxorubicin. They coupled PEG
to mannosylated nanoparticle template with a pH sensitive hydrazone
bond. It was assumed that upon intravenous administration, PEG
component would prevent unwanted reticuloendothelial uptake of
nanoparticles until they reach slightly acidic tumor environment. This
pH change would trigger loss of hydrazone bond and shedding of PEG
cloak, exposing mannosylated nanoparticle surface (capable of inter-
acting with scavenger receptors on TAMs). Results suggested that
payload accumulated explicitly in tumor and TAMs, away from
unnecessary sites like heart or liver to significantly reduce tumor
volume in B16F10 tumor bearing mice [139,149]. Zhan et al. have
conjugated glucomannan to a bisphosphonate (alendronate) for specific
targeting and elimination of TAMs in a subcutaneous S180 tumor-
bearing mice model. Glucomannan has very high affinity for mannose
receptors and consequently its conjugation allows alendronate to
overcome its inherent tumor environment susceptibility and instead
be rapidly taken up by TAMs to elicit specific immunoactivity which
leads to suppression of angiogenesis and tumor [150]. Another member
of lectin subfamily of receptors overexpressed on TAMs includes
Sialoadhesin (Siglec-1) receptor, which correspond specifically to
carbohydrates possessing sialic acid residue (O - or - N substituted
derivatives of neuraminic acid). Siglec-1 is an endocytic receptor, and
therefore is ideal for receptor oriented nanocarrier delivery. Zhou et al.
have exploited this receptor using sialic derivative of cholesterol as a
targeting moiety for epirubicin loaded liposomes. In vitro and in vivo cell
uptake studies suggested selective accumulation of these liposomes in
TAMs which in turn resulted in depletion of macrophage population
and several fold enhancement in antitumor activity of epirubicin [151].
Dextran's (complex branched glucan) has also been used to target
TAMs for purpose of imaging and delivery of small molecules.
Weissleder et al. synthesized a library of 146 nanoparticles using
different synthetic small molecules and among them dextran coated
iron oxide nanoparticles (CLIO680 and AMTA680) exhibited highest
affinity towards TAMs [152]. CLIO680 and AMTA680 nanoparticles
were consequently conjugated with a fluorochrome (VT680) to speci-
fically label TAMs for imaging applications [153]. Daldrup-Link et al.
have used carboxydextran coated iron oxide nanoparticles for magnetic
resonance imaging of TAMs. In an experimental model treated with
CSF1 monoclonal antibodies to kill TAMs, there was quantifiable
reduction in accumulation and the consequently generated MRI signal
by carboxydextran coated iron oxide nanoparticles showcasing efficacy
of adapted treatment [154]. Glactose type lectin receptors are also
highly expressed over surface of M2 phenotype of TAMs and are worth
a try as targeting moiety. These receptors recognize terminal GalNAc
residue on glycans and mediate endocytosis [155–157] (Fig. 2).
9.2. Antibodies
Antibody (immunoglobulin; Ig) is a Y shaped protein which can
identify specific regions on a target antigen. Each tip of Y has a
paratope which recognizes epitope available on the antigen. The
hypervariable region, also known as Fab(ab')2 fragment, on the Y tip
has large number of slightly variable biding sites which further enlarges
the catalogue of recognizable antigens. Fc fragment present at the base
of Y however is very specific. Antibody ligation on nanoparticle surface
thus in principle could facilitate site specific internalization if the
tethered antibody recognizes any antigen present on target cell.
However, physiological stability and capability to trigger unintended
immune response makes antibody usage tricky. Sun et al. have
synthesized near-infrared fluorescence responsive probe by conjugating
anti-mouse CD206 antibody with DyLight680 succinimidyl ester. The
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Journal of Controlled Release 254 (2017) 92–106
probe, when tested in a 4T1 tumor bearing animal model specifically
tags onto TAMs, generating pertinent in vivo images [158]. In another
study, Movahedi et al. used mannose receptor targeted nanobodies
(single domain antigen binding fragments prepared from Camelidae
heavy-chain antibodies). 99mTc tagged nanobodies specifically label
TAMs in tumor microenvironment [159] and should be tested in
delivery of cytotoxics for therapeutic benefit. Hemoglobin scavenger
receptor CD163 are also overexpressed on TAMs and are involved in
progression of cancer [160]. Therapeutic significance of this receptor
has been illustrated by Etzerodt et al. They ligated CD163 responsive
monoclonal antibody on pegylated liposomes to enhance its TAMs
uptake and efficacy of loaded drug [161].
9.3. Proteins and peptides
Proteins have specific three dimensional confirmations, which is
responsible for their selective affinity towards a substrate. This
specificity can be exploited for development of targeted delivery
systems. Legumain, an asparaginyl endopeptidase, found in plant,
parasites and mammals is overexpressed in variety of solid tumors
leading to their invasion/metastasis [162–164]. More importantly, it is
overexpressed on TAMs; a fact which has been used in development of
TAMs targeted vaccine for eradication of tumor growth and metastasis
[165–168]. A doxorubicin based prodrug, responsive to legumain
expression has also been developed to suppress TAMs and related
tumor growth [169]. In a recent study, a dual approach based upon
legumain and cell penetrating peptide was employed to develop a
liposomal formulation for improved efficacy of doxorubicin by target-
ing tumor associated macrophages [170]. In a conceptually similar
study, Zhang X and fellow researchers have loaded hydrazinocurcumin,
a synthetic analogue of curcumin in legumain-targeting liposomal
nanoparticles and investigated their effects on suppression of tumor
in vivo. When fabricated nanoparticles were intravenously administered
in Balb/C mice model afflicted with breast cancer, they were able to
considerably “re-educate” M2-like macrophages and caused suppres-
sion of tumor growth, angiogenesis and metastasis [171].
Peptides are small amino acid sequences possessing linear or cyclic
structures. Their small molecular size facilitates easy synthesis and
provides environmental stability. Recently, Cieslewicz et al. created a
peptide sequence M2pep (YEQDPWGVKWWY). M2pep demonstrated
high affinity towards TAMs in mixed cells population and has been used
as targeting vector for intracellular delivery of an apoptotic peptide.
When tested in vivo, the system specifically binds to TAMs and delays
mortality in tumor bearing mice [172]. M2pep has also been used to
decorate a nanoparticle system for delivery of siRNA to TAMs in order
to inhibit expression of VEGF and associated tumor burden [173]. In
another study, Yan el al, synthesized a Y shaped peptide [Ala-Ala-Asn-
Leu-His-Lys-(His-Lys)2] for targeting ligumain overexpressed on TAMs.
The synthesized peptide and paramagnetic Fe3O4 nanoparticles were
conjugated on to a carbon nanotube and used for selective MRI imaging
of TAMs. Interestingly, the histidine residue present in Y peptide
facilitated endosomal escape of nanotubes due to proton sponge effect
[174]. Lyp-1 peptide can recognize lymphatic vessel, tumor cells and
TAMs [175,176]. Lyp-1 has been used to decorate a liposomal
formulation for specific binding with TAMs so as to inhibit growth of
metastatic tumor [177]. RVG peptide derived from rabies virus
glycoprotein has been used for active targeting of TAMs of glioma
region [178]. For instance, RVG peptide has been conjugated on to shell
of biodegradable nanoparticles containing paclitaxel to treat malignant
glioma. Owing to their spherical shape, small size, narrow distribution,
and ligand conjugated surface, the said nanoparticles were able to
penetrate the normally impermeable blood brain barrier and accumu-
late in TAMs populating the glioma, keeping away from regular neurons
to provide safe and effective anti-glioma therapy [179]. Table 3
summarizes various ligands and the capacities in which they have been
used to target TAMs.
Y. Singh et al. Journal of Controlled Release 254 (2017) 92–106

Fig. 2. Utilization of phenotypic differentiators for selective targeting of M2 subtype of tumor associated macrophages. Scavenger receptors such as CD206, CD163 are found only on M2
cells and not on M1 cells. Researchers have therefore come up with full proof surface decorated nanocarriers loaded with cytotoxic or immunomodulatory drugs which recognize these
receptors to either kill or reeducate TAMs, sparing other unwanted organs or cells from cytotoxic action. Nanocarriers often exploit subtle variations such as hypoxia; altered pH,
enhanced permeability and retention effect to selectively accumulate in tumor environment. Thereafter receptor mediated endocytosis is initiated which involves recognition and binding
of specific ligands (carbohydrates, proteins, antibodies) appended on nanocarriers by receptors present on cell surface. The payload is subsequently endocytosed and delivered to
intracellular machinery to produce further effects.

10. Future prospects
With improved knowledge of genesis, sustenance, and propagation
of cancer there has been a spurt of probable targets especially with
respect to signaling involved in TAMs. It has been reported that
recruitment of TAMs in tumor microenvironment is facilitated by
certain chemoattractants like CCL2 [180] and CCL5 [181]. CNTO88
(carlumab), an antibody of CCL2, currently under clinical trial has
demonstrated strong anticancer activity in preliminary testing [182].
CSF-1 can be targeted using antisense oligonucleotides [183,184],
monoclonal antibodies [185] and tyrosine kinase inhibitors [186,187]
to modulate recruitment of TAMs in tumor microenvironment [188].
Th1 cytokine IFNγ [189], toll like receptor agonists [190,191] and
CD40 molecule activated antibodies (anti-CD40 mAb) [192] can all be
used to reeducate TAMs to furnish an antitumor immune response.
WP1066, an inhibitor of STAT3 can induce TH1 biased cytokines like
IL-12 and receptors CD80, CD86 in peripheral as well as tumor
associated macrophages, can reverse immune tolerance in malignant
glioma patients and is currently clinical under clinical investigation
[193]. Immunomodulatory compounds like thymosin and β-glucan
have also been explored to reeducate TAMs and increase immune
response in patients suffering from cancer [194,195]. Direct killing of
TAMs has also been tried using trabectedin, which causes apoptosis of
macrophages through TNF-related apoptosis-inducing ligand (TRAIL)
receptors [196]. PF-04136309, a CD192 antagonist, prevents recruit-
ment of CD192 + monocytes from bone marrow to tumors in a mouse
model bearing pancreatic cancer leading to depletion in TAMs popula-
tion, which in turn reduces tumor growth and metastasis. In hypoxic
regions of hepatocellular carcinoma, LY294002, a protein inhibitor, has
shown credible anti-tumor activity by interfering in HIF-1 expression
and subsequent activation of PI3K/Akt headed epithelial-mesenchymal
transition (EMT metastasis) and chemoresistance [197]. Overall hypox-
ia has been associated with malignant phenotype and poor prognosis of
cancer cells [198]. It is studied as a key target due to the fact that
cancerous cells acquire a partial oxygen pressure which is at least 33%
less than normal live cells [199]. Hypoxia induces abnormal vascular-

Table 3
Examples of ligands utilized for targeting TAMs through nanocarriers.

Ligand Class Target Active moiety and imaging agent Nanocarrier system References

Mannose Carbohydrate CD206 receptors siRNA Micelles [147]

Mannose Carbohydrate CD206 receptors Doxorubicin Nanoparticles [136]
Mannose Carbohydrate CD206 receptors 64
Cu and a fluorescent dye Liposomes [148]
Dextran Carbohydrate CD206 receptors Fluorochrome VT680 Nanoparticles [153]
Carboxydextran Carbohydrate CD206 receptors Iron oxide for MRI Nanoparticles [154]
Galactosylated cationic dextran Carbohydrate Glactose type lectin receptors Oligonucleotide Nanoparticles [123]
Anti-mouse CD206 antibody Antibody CD206 receptors DyLight680 succinimidyl ester – [158]
Nanobodies Antibody fragments CD206 receptors 99m
Tc – [159]
Monoclonal antibody Antibody Hemoglobin scavenger receptor CD163 Liposomes [161]
Legumain Proteins Legumain receptors Hydrazinocurcumin, Doxorubicin Liposomes [170,171]
M2pep Peptide – siRNA Nanoparticles [173]
Y shaped peptide Peptide – Anti-apoptotic peptide and iron oxide Carbon nanotube [174]
Lyp-1 Peptide Lymphatics and tumor cells Fluorescein and doxorubicin Liposomes [177]

101
Y. Singh et al.
ization at tumor site creating a barricade in drug access and results in
variable bioavailability which in turn may cause therapeutic chemo-
resistance of anticancer agents. Certain drugs such as melphalan,
bleomycin, etoposide, etc., and radiation therapy necessarily require
molecular oxygen for maximal cytotoxicity, and therefore produce sub
maximal effects in vivo due to prevalent hypoxia [200]. Therefore
researchers have tried various strategies to directly address tumor
hypoxia and the subsequently expected downstream effectuality, i.e.
‘recruitment of TAMs’ [201]. Examples include development of bio-
reductive pro-drugs that are activated by enzymatic reduction specifi-
cally in hypoxia affected region and inhibition of molecular events that
favor proliferation of hypoxic cells [202]. Chemists have developed
various bio-reductive pro-drugs from five chemical moieties (nitro
groups, quinones, aromatic N-oxides, aliphatic N-oxides and transition
metals) which have potential to undergo enzymatic metabolism under
hypoxic condition although the expression level of the activating
enzyme remains a critical parameter. For instance, aromatic N-oxide
tirapazamine is 200 fold more toxic to hypoxic cells than norm-oxic
cells and the activation is governed by single electron reduction process
that damages the DNA replication fork.
It is our firm belief that, if packaged in a rationally devised
nanotechnology platform, the aforementioned chemo-attractants and
endogenous/exogenous compounds would open up even more antic-
ancer avenues by targeting TAMs more efficiently.
Acknowledgements
We would like to thank CSIR for providing financial assistance with
Network Project titled “Advanced Drug Delivery Systems” [CSC0302].
This is CSIR-CDRI communication 9473.
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