A Fluorescent Activatable AND-Gate Chemokine CCL2 Enables In Vivo Detection of Metastasis-Associated Macrophages

Article abstract of DOI:10.1002/anie.201910955

We report the novel chemical design of fluorescent activatable chemokines as highly specific functional probes for imaging subpopulations of immune cells in live tumours. Activatable chemokines behave as AND-gates since they emit only after receptor bindi

Full text of DOI:10.1002/anie.201910955

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201910955
German Edition: DOI: 10.1002/ange.201910955
Tumour Imaging
A Fluorescent Activatable AND-Gate Chemokine CCL2 Enables In
Vivo Detection of Metastasis-Associated Macrophages
Antonio Fernandez, Emily J. Thompson, Jeffrey W. Pollard, Takanori Kitamura,* and
Marc Vendrell*
Abstract: We report the novel chemical design of fluorescent
activatable chemokines as highly specific functional probes for
imaging subpopulations of immune cells in live tumours.
Activatable chemokines behave as AND-gates since they emit
only after receptor binding and intracellular activation, show-
ing enhanced selectivity over existing agents. We have applied
this strategy to produce mCCL2-MAF as the first probe for in
vivo detection of metastasis-associated macrophages in a pre-
clinical model of lung metastasis. This strategy will accelerate
the preparation of new chemokine-based probes for imaging
immune cell function in tumours.
species,[8] MMPs,[9] cathepsins,[10] and SLC transporters[11]) can
be used to detect active macrophages under physiological
conditions. These probes provide generic readouts of macro-
phage activity but are not specific for the metastasis-
associated macrophages linked to cancer progression.
CCL2 is a potent chemokine that regulates the migration
of immune cells to tumours through the recognition of CCR2
receptors. Recent studies have identified that metastasis-
associated macrophages express high levels of CCR2 on the
cell surface.[2,12] Binding of CCL2 to CCR2 promotes the
recruitment of macrophages into metastatic sites, which
accelerates the seeding and expansion of cancer cells. Given
that chemokines can enter cells via their functionally-active
receptors, we hypothesized that fluorescently-labelled CCL2
analogues would allow us to detect CCR2 + metastasis-
associated macrophages in tumours. Previously, Nibbs et al.
described CCL2 chemokines to broadly identify subpopula-
tions of mouse leukocytes.[13] The groups of Beck-Sickinger
and Liu and Qi also demonstrated that chemically photo-
caged chemokines can be used to temporally control leuko-
cyte migration.[14] Herein, we have designed a strategy to
prepare AND-gate[15] activatable chemokines that selectively
target CCR2 + populations of metastatic macrophages in
vivo.
We designed fluorescent activatable chemokines to work
in a two-step sequence (Figure 1a). First, they recognise
functional receptors that internalise upon ligand binding (step
1) AND second, they emit fluorescent signals in response to
macrophage-related activity (step 2). Compared to fluores-
cently-labelled antibodies and always-on fluorescent chemo-
kines (Figure 2), this AND-gate strategy makes possible the
recognition of both functional receptors as well as intra-
cellular activity, enabling the detection of highly specific cell
populations. In this work, we have applied this concept to
generate the first fluorescent activatable CCL2 chemokine for
live imaging of active metastasis-associated macrophages.
In order to conjugate the chemokine CCL2 to fluorescent
markers of active macrophages, we first examined macro-
phage-activatable fluorophores (MAFs) responding to intra-
cellular metabolites associated with macrophage activity, such
as phagosomal pH, superoxide and reactive oxygen species.
For this, we incubated inactive and LPS-activated RAW264.7
mouse macrophages with different MAFs and measured their
emission by fluorescence microscopy (Figure S1 in the
Supporting Information). We observed that pH-sensitive
BODIPY dyes triggered by phagosomal acidification
showed bright intracellular fluorescence in active macro-
phages with the largest turn-on emission among the different
T
umour metastasis is the leading cause of cancer-related
death. The metastatic potential of tumours is largely defined
by the surrounding immune environment, where macro-
phages are among the most abundant cells.[1] Different
subpopulations of macrophages are found in tumours, such
as resident macrophages and active metastasis-associated
macrophages, with the latter being recruited by chemokines
(e.g., CCL2 or C-C motif chemokine ligand 2) to accelerate
the progression of metastasis.[2] Whereas novel therapeutic
strategies to stop the recruitment of tumour-associated
macrophages—including chemokine inhibitors—are under
evaluation as anticancer treatments,[3] there are no chemical
probes for detecting these subpopulations of macrophages in
live tumours.
The most widespread technology to track the mobilisation
of macrophages are genetically-encoded fluorescent proteins
(e.g., GFP) expressed under macrophage-selective promot-
ers.[4] For instance, Entenberg et al. recently used transgenic
mice to study the interactions between macrophages and
cancer cells in vivo.[5] However, this technique cannot
distinguish macrophage subtypes or provide cell activity
readouts. Alternatively, small-molecule fluorophores have
been developed to monitor the function of macrophages.[6]
Our group and others have shown that fluorophores respond-
ing to different biomarkers (e.g. acidic pH,[7] reactive oxygen
[*] Dr. A. Fernandez, E. J. Thompson, Dr. M. Vendrell
Centre for Inflammation Research, The University of Edinburgh
47 Little France Crescent, EH16 4TJ Edinburgh (UK)
E-mail: marc.vendrell@ed.ac.uk
Homepage: http://www.dynafluors.co.uk
Prof. J. W. Pollard, Dr. T. Kitamura
MRC Centre for Reproductive Health, The University of Edinburgh
47 Little France Crescent, EH16 4TJ Edinburgh (UK)
E-mail: tkitamur@exseed.ed.ac.uk
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under https://doi.org/10.
1002/anie.201910955.
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diethylamine to yield BODIPY 4 with an overall yield of 87%
including all 3 steps. Finally, compound 4 was hydrolysed and
coupled to 1-(2-aminoethyl)maleimide to generate BODIPY
5 for site-specific protein bioconjugation (see Supporting
Information for synthetic and characterisation details).
We envisaged that the incorporation of fluorophores at
the C-terminus of mCCL2 would not disrupt the recognition
of chemokine receptors or impair its chemotactic activity.
Therefore, we employed an analogue of mCCL2 with an extra
cysteine residue at the C-terminal end that would react site-
specifically with the BODIPY maleimide 5. The fluorophore–
chemokine conjugation was performed with an excess of
compound 5 (4 equiv) in aqueous buffer at pH 6.5 for 1 h, and
the resulting mCCL2-MAF (6, Figure 1b) was purified by
HPLC. We used mass spectrometry to confirm single coupling
of the fluorophore to the C-terminal cysteine of mCCL2 in
a 1:1 ratio (Figure S2).
Next, we evaluated the spectral properties of mCCL2-
MAF (6) to corroborate that the turn-on properties of the
fluorophore remained unaffected after conjugation to
mCCL2. Compound 6 showed similar photophysical proper-
ties to the parent BODIPY fluorophore 4 (lexc.: 496 nm, lem.
:
510 nm; Figure S3) with pH-dependent activation and a pKa
around 4.9, suitable to detect active macrophages specifically
undergoing phagosomal acidification (Figures S4 and S5).
This result suggests that the incorporation of alkyl spacers at
the position 3 of the BODIPY core is an effective strategy to
generate bioconjugatable fluorophores with full retention of
their optical properties. Next, we performed cell migration
transwell assays to examine whether mCCL2-MAF (6)
retained the biological function of the native chemokine
mCCL2. We cultured RAW264.7 mouse macrophages on top
of cell-permeable membranes, incubated them with different
concentrations of mCCL2 or mCCL2-MAF (6), and counted
the migrated cells across the membrane. As shown in
Figure 1. a) Schematic representation of the fluorescence activation
mechanism for mCCL2-MAF (6) in active CCR2+ metastasis-associ-
ated macrophages. b) Synthesis of the BODIPY-based macrophage-
activatable fluorophore 5 and protein conjugation to mouse chemokine
CCL2 (mCCL2).
Figure 2. Imaging capabilities of fluorescently-labelled antibodies,
always-on chemokines and activatable AND-gate chemokines.
MAFs. Therefore, we decided to utilise pH-sensitive BOD-
IPYs for the generation of activatable CCL2 chemokines.
We synthesized the pH-dependent BODIPY (5) contain-
ing a maleimide group for site-specific conjugation to the
mouse chemokine CCL2 (mCCL2, Figure 1). The key inter-
mediate 4 was obtained in 3 steps from pyrrole 1 in good
yields. As reported by our group and others,[16] the assembling
of the BODIPY fluorophore proceeded via condensation of
two functionalized pyrroles (1 and 2, Figure 1) followed by
addition of BF3OEt2 to generate the nitro-derivatised
BODIPY 3. Herein, we optimised the catalytic hydrogenation
and subsequent derivatisation with chloroacetyl chloride and
Figure 3. Chemotactic transwell assays in RAW264.7 macrophages.
Migrated cells were quantified after incubation with culture media
containing increasing levels of mCCL2 or 6 (0, 10, 25, 50, 100 and
200 ngmLÀ1) for 2 h. Cells were stained with DAPI (representative
images on the right) and counted using ImageJ. Scale bar: 10 mm.
Values as means Æ s.d. (n=3). n.s. for p>0.05.
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Figures 3 and S6, mCCL2-MAF (6) promoted macrophage
receptor and subsequent endocytosis (Figure 4c). Altogether
migration to comparable levels of unlabelled mCCL2. These
results confirm that the C-terminal modification of mCCL2
with BODIPY dyes endows the chemokine with fluorescent
reporters of macrophage function without affecting the
chemotactic properties of mCCL2.
these results confirm that the fluorescence emission of
mCCL2-MAF (6) follows an activation mechanism with two
sequential events: 1) CCR2-mediated internalisation, and 2)
fluorescence amplification in active macrophages.
In contrast to mCCL2-MAF (6), AlexaFluor647-mCCL2
(always-on chemokine) emitted strong fluorescence in
CCR2 + mouse macrophages regardless of their activation
state (Figure 4d), which highlights the advantages of activat-
able chemokines as fluorescent reporters of intracellular
activity.
Encouraged by these results, we next examined whether
mCCL2-MAF (6) could selectively stain CCR2 + macro-
phages in the presence of other immune cells. For these
experiments, we used wild-type C57/BL6 as well as Ccr2
knock-out (KO) mice so that we could determine the
selectivity profile of compound 6 in cell populations express-
ing variable levels of CCR2 receptors. We prepared single-cell
suspensions from spleens of wild-type and Ccr2 KO mice,
incubated them with mCCL2-MAF (6) (100 nm) and analysed
them by flow cytometry. We employed mouse antibodies (F4/
80, CD45, CD11b, Ly6G, CD3 and CD19) to identify different
immune cells,[17] which included macrophages, neutrophils, B
cells and T cells (Figure S7 for gating strategy). From this
analysis, we found that a subpopulation of wild-type macro-
phages showed bright fluorescence whereas this cell popula-
tion was not detected in macrophages from Ccr2 KO mice
(Figure 5a), indicating that the signals from mCCL2-MAF (6)
are CCR2-dependent. We also found that mCCL2-MAF (6)
did not stain neutrophils, T cells or B cells in either wild-type
of KO mice (Figure 5b). These results highlight the value of
mCCL2-MAF (6) for the selective detection of CCR2 +
macrophages with minimal off-target fluorescence in other
cells.
Given the functional and fluorescent activatable proper-
ties of mCCL2-MAF (6), we then investigated its applicability
as a marker for active CCR2 + metastasis-associated macro-
phages. First, we compared the emission of mCCL2-MAF (6)
in inactive and LPS-activated CCR2 + mouse macrophages
by confocal microscopy and flow cytometry. We found that
inactive macrophages showed weak fluorescence emission,
which was dramatically increased in activated cells due to
LPS-induced phagosomal acidification (Figures 4a,b). Impor-
tantly, the fluorescence enhancement was reduced to basal
levels when macrophages were pre-treated with bafilomy-
cin A, which inhibits LPS-mediated phagosomal acidification,
or anti-CCR2 antibodies, which block ligand binding to the
Finally, we examined whether mCCL2-MAF (6) could
detect metastasis-associated macrophages in vivo. To this end,
we used a mouse model of lung metastasis where E0771-LG
Figure 5. Ex vivo staining profile of compound 6 (100 nm) in different
immune cells from spleens of wild-type and Ccr2 KO mice. a) Repre-
sentative fluorescence histograms of compound 6-stained and
unstained macrophages from wild-type mice (green) and Ccr2 KO
(blue) mice (n=3 for each group). b) Geometric fluorescence intensity
of immune cells: macrophages (Ly6GÀF4/80+CD11b+), neutrophils
(Ly6G+F4/80À), T cells (CD3+CD19À), and B cells (CD3ÀCD19+).
Values as means Æ s.d. (n=3). * for p<0.05, n.s. for p>0.05.
Figure 4. Fluorescence microscopy (a) and flow cytometric analysis
(b–e) of inactive and active CCR2+ mouse macrophages upon treat-
ment with LPS (100 ngmLÀ1 for 18 h), compound 6 (100 nm, b,c), and
AF647-mCCL2 (100 nm, d,e). Cells were counterstained with Hoechst
33342 for nuclear staining. In panel (a) white arrows point at
fluorescent acidic phagosomes inside macrophages. Scale bar: 10 mm.
Values are presented as means Æ s.d. (n=3).
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mouse mammary tumour cells are injected into the tail vein of
syngeneic C57BL/6 mice to mimic the systemic distribution of
cancer cells. In this model, metastasis-associated macro-
phages (MAMs) and their progenitor cells (MAMPCs)
accumulate in the metastatic lungs via CCL2-CCR2 signal-
ing.[17,18] Since the accumulation of MAMs and MAMPCs
promotes metastasis, these cells are regarded as attractive
targets in anticancer therapy.[17–19] Animals injected with
E0771-LG cancer cells formed metastatic tumours in their
lungs after 2 weeks, as confirmed by whole-body biolumines-
cence imaging (Figure S8). At this point, we injected mCCL2-
MAF (6) via the tail vein (10 ng in 50 mL saline) and harvested
the metastatic lungs after 2 hours. Histological examination of
the lungs corroborated the presence of metastatic nodules
(Figure 6a). Notably, in the metastatic nodules, we found F4/
80 + macrophages showing green fluorescence (Figure 6b),
which indicates that the in vivo injection of mCCL2-MAF (6)
labels a defined subpopulation of macrophages in metastatic
tumours. We also analysed the tissues by flow cytometry and
measured the fluorescence signals in major immune cells
found in metastatic lungs (Figure S9 for gating strategy and
immune cell markers). As shown in Figure 6c, we found high
levels of green fluorescence in active MAMPCs and MAMs
that require CCR2 signaling for their accumulation in
metastatic sites. Remarkably, all other immune cells in the
metastatic lungs [for example, resident macrophages
(RMAC), neutrophils, T cells, B cells, natural killer (NK
cells)] remained unstained by compound 6 (Figure 6c).
These results confirmed that the AND-gate activation
mechanism of mCCL2-MAF (6) was recapitulated in vivo.
Specifically, we did not observe staining in RMAC, which do
not express CCR2, or in NK cells, which express high levels of
CCR2 but cannot trigger the emission of macrophage-
activated fluorophores (Figure 6c). In contrast, the always-
on chemokine AF647-mCCL2 showed bright fluorescence in
NK cells from metastatic tumours under the same experi-
mental conditions (Figure S10). This observation confirms
that the combination of functional chemokines with activat-
able fluorophores can generate highly specific probes for
immune cells that cannot be targeted with conventional
reagents, and validates mCCL2-MAF (6) as the first chemical
probe for selective detection and imaging of active metastasis-
associated macrophages in tumours in vivo.
In summary, we have designed AND-gate activatable
chemokines as fluorescent agents for imaging active subpo-
pulations of immune cells in live tumours. We have synthe-
sized mCCL2-MAF as the first chemical probe for metastasis-
associated macrophages by coupling a Cys-modified chemo-
kine CCL2 with a thiol-reactive macrophage-activatable
fluorophore. mCCL2-MAF fully retains chemotactic activity
and emits bright intracellular fluorescence in a CCR2- AND
macrophage activity-dependent manner. We also demon-
strated the utility of our strategy by showing selective staining
of metastasis-associated macrophages in vivo, outperforming
always-on fluorescent chemokines. This chemical strategy will
accelerate the design of highly specific chemokine-based
imaging agents to study immune cell subpopulations with
enhanced spatial and temporal resolution.
Acknowledgements
We acknowledge funding from MSCA Individual Fellowship
(A.F.: 704912), Wellcome Trust (J.W.P.: 101067/Z/13/Z),
Wellcome-UoE Institutional Strategic Support Fund ISSF2
(T.K.: 615KIT/J22738), MRC Centre Grant (J.W.P. and T.K.:
MR/N022556/1), ERC Consolidator Grant (M.V.: 771443).
We also thank for the support from the Flow Cytometry and
the Confocal Advanced Light Microscopy units at UoE.
Conflict of interest
Figure 6. In vivo characterisation of metastatic lungs after tail vein
injection of mCCL2-MAF (6). a) Histological H&E staining of meta-
static nodules from lungs of tumour-bearing mice. b) Confocal micros-
copy images of metastatic lungs (blue: Hoeschst 33342 for nuclei;
green: compound 6, red: anti-F4/80 for all macrophages). White
arrows point at 6-stained MAMs and yellow arrows point at 6-
unstained RMACs. Scale bar: 100 mm. c) Fluorescence profiling of
immune cells after in vivo staining with compound 6 (Figure S9 for
gating strategy). Values presented as means Æ s.d. (n=3).** for
p<0.01, *** for p<0.001.
The authors declare no conflict of interest.
Keywords: fluorophores · imaging · immunology ·
molecular logic · tumour microenvironment
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[11] S. J. Park, B. Kim, S. Choi, S. Balasubramanian, S. C. Lee, J. Y.
[1] a) B. Z. Qian, J. W. Pollard, Cell 2010, 141, 39 – 51; b) A.
Fernandez, M. Vermeren, D. Humphries, R. Subiros-Funosas,
N. Barth, L. Campana, A. MacKinnon, Y. Feng, M. Vendrell,
ACS Cent. Sci. 2017, 3, 995 – 1005; c) T. Kitamura, J. W. Pollard,
M. Vendrell, Trends Biotechnol. 2017, 35, 5 – 6.
[2] a) B. Z. Qian, J. Li, H. Zhang, T. Kitamura, J. Zhang, L. R.
Campion, E. A. Kaiser, L. A. Snyder, J. W. Pollard, Nature 2011,
475, 222 – 225; b) T. Kitamura, B. Z. Qian, J. W. Pollard, Nat.
Rev. Immunol. 2015, 15, 73 – 86.
[3] a) H. Mathsyaraja, K. Thies, D. A. Taffany, C. Deighan, T. Liu, L.
Yu, S. A. Fernandez, C. Shapiro, J. Otero, C. Timmers, M. B.
Lustberg, J. Chalmers, G. Leone, M. C. Ostrowski, Oncogene
2015, 34, 3651 – 3661; b) T. Kitamura, J. W. Pollard, Pharmacol.
Res. 2015, 100, 266 – 270; c) S. I. van Kasteren, J. Neefjes, H.
Ovaa, Nat. Rev. Chem. 2018, 2, 184 – 193.
[4] For a review, see: D. Hume, J. Leukocyte Biol. 2011, 89, 525 –
538.
[5] D. Entenberg, C. Rodriguez-Tirado, Y. Kato, T. Kitamura, J. W.
Pollard, J. Condeelis, Intravital 2015, 4, 1 – 11.
[6] A. Fernꢀndez, M. Vendrell, Chem. Soc. Rev. 2016, 45, 1182 –
1196.
[7] a) A. Vꢀzquez-Romero, N. Kielland, M. J. Arꢁvalo, S. Preciado,
R. J. Mellanby, Y. Feng, R. Lavilla, M. Vendrell, J. Am. Chem.
Soc. 2013, 135, 16018 – 16021; b) H. Maeda, T. Kowada, J.
Kikuta, M. Furuya, M. Shirazaki, S. Mizukami, M. Ishii, K.
Kikuchi, Nat. Chem. Biol. 2016, 12, 579 – 585.
[8] a) B. C. Dickinson, C. Huynh, C. J. Chang, J. Am. Chem. Soc.
2010, 132, 5906 – 5915; b) K. Xu, B. Tang, H. Huang, G. Yang, Z.
Chen, P. Li, L. An, Chem. Commun. 2005, 5974 – 5976; c) M.
Abo, Y. Urano, K. Hanaoka, T. Terai, T. Komatsu, T. Nagano, J.
Am. Chem. Soc. 2011, 133, 10629 – 10637; d) D. Cheng, Y. Pan, L.
Wang, Z. Zeng, L. Yuan, X. Zhang, Y. T. Chang, J. Am. Chem.
Soc. 2017, 139, 285 – 292.
Lee, H. S. Kim, J. Y. Kim, J. J. Kim, Y. A. Lee, N. Y. Kang, J. S.
Kim, Y. T. Chang, Nat. Commun. 2019, 10, 1111.
[12] a) B. Z. Qian, Y. Deng, J. H. Im, R. J. Muschel, Y. Zou, J. Li,
R. A. Lang, J. W. Pollard, PLOS One 2009, 4, e6562.
[13] L. B. Ford, V. Cerovic, S. W. Milling, G. J. Graham, C. A.
Hansell, R. J. Nibbs, J. Immunol. 2014, 193, 400 – 411.
[14] a) L. Baumann, A. G. Beck-Sickinger, Angew. Chem. Int. Ed.
2013, 52, 9550 – 9553; Angew. Chem. 2013, 125, 9729 – 9732; b) X.
Chen, S. Tang, J. S. Zheng, R. Zhao, Z. P. Wang, W. Shao, H. N.
Chang, J. Y. Cheng, H. Zhao, L. Liu, H. Qi, Nat. Commun. 2015,
7, 7220.
[15] For representative examples on logic gates and activatable
molecular probes, see: a) A. Prasanna de Silva, N. D. McClena-
ghan, J. Am. Chem. Soc. 2000, 122, 3965 – 3966; b) S. Erbas-
Cakmak, S. Kolemen, A. C. Sedwick, T. Gunnlaugsoon, T. D.
James, J. Yoon, E. U. Akkaya, Chem. Soc. Rev. 2018, 47, 2228 –
2248; c) C. Muniesa, V. Vicente, M. Quesada, S. Saez-Atienzar,
J. R. Biesa, I. Abasolo, Y. Fernandez, P. Botella, RSC Adv. 2013,
3, 15121 – 15131; d) S. Sahu, N. Sinha, S. K. Bhutia, M. Maji, S.
Mohapatra, J. Mater. Chem. B 2014, 2, 3799 – 3808; e) Y. Xu, W.
Shi, H. Li, X. Li, H. Ma, ChemMedChem 2019, 14, 1079 – 1085.
[16] a) J. S. Lee, N. Y. Kang, Y. K. Kim, H. K. Kim, A. Samanta, S.
Feng, M. Vendrell, J. H. Park, Y. T. Chang, J. Am. Chem. Soc.
2009, 131, 10077 – 10082; b) J. C. Er, M. Vendrell, M. K. Tang, D.
Zhai, Y. T. Chang, ACS Comb. Sci. 2013, 15, 452 – 457; c) M.
Sintes, F. De Moliner, D. Caballero-Lima, D. W. Denning, N. D.
Read, N. Kielland, M. Vendrell, R. Lavilla, Bioconjugate Chem.
2016, 27, 1430 – 1434; d) L. Mendive-Tapia, R. Subiros-Funosas,
C. Zhao, F. Albericio, N. D. Read, R. Lavilla, M. Vendrell, Nat.
Protoc. 2017, 12, 1588 – 1619.
[17] T. Kitamura, B. Z. Qian, D. Soong, L. Cassetta, R. Noy, G.
Sugano, Y. Kato, J. Li, J. W. Pollard, J. Exp. Med. 2015, 212,
1043 – 1059.
[18] T. Kitamura, D. Doughty-Shenton, L. Cassetta, S. Fragkogianni,
D. Brownlie, Y. Kato, N. Carragher, J. W. Pollard, Front.
Immunol. 2017, 8, 2004.
[9] A. Cobos-Correa, J. B. Trojanek, S. Diemer, M. A. Mall, C.
Schultz, Nat. Chem. Biol. 2009, 5, 628 – 630.
[10] a) K. Oresic Bender, L. Ofori, W. A. van der Linden, E. D.
Mock, G. K. Datta, S. Chowdhury, H. Li, E. Segal, M.
Sanchez Lopez, J. A. Ellman, C. G. Figdor, M. Bogyo, M.
Verdoes, J. Am. Chem. Soc. 2015, 137, 4771 – 4777; b) L. E.
Edgington-Mitchell, M. Bogyo, M. Verdoes, Methods Mol. Biol.
2017, 1491, 145 – 159.
[19] D. Argyle, T. Kitamura, Front. Immunol. 2018, 9, 2629.
Manuscript received: August 27, 2019
Revised manuscript received: September 14, 2019
Accepted manuscript online: September 19, 2019
Version of record online: && &&, &&&&
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Communications
Tumour Imaging
A. Fernandez, E. J. Thompson,
J. W. Pollard, T. Kitamura,*
M. Vendrell*
&&&& — &&&&
A Fluorescent Activatable AND-Gate
Chemokine CCL2 Enables In Vivo
Detection of Metastasis-Associated
Macrophages
Live show: Fluorescent activatable che-
mokines have been designed as the first
chemical probes for in vivo detection of
tumour-associated macrophages in lung
metastasis. Activatable chemokines
behave as AND-gates since they emit only
after receptor binding and intracellular
activation, showing enhanced selectivity
over existing agents.
6
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