Imaging of Tie2 with a Fluorescently Labeled Small Molecule Affinity Ligand

Article abstract of DOI:10.1021/acschembio.9b00724

The receptor tyrosine kinase inhibitor, Tie2, has significant roles in endothelial signaling and angiogenesis and is relevant in the pathophysiology of several diseases. However, there are relatively few small molecule probes available to study Tie2, maki

Full text of DOI:10.1021/acschembio.9b00724

Subscriber access provided by BIU Pharmacie | Faculté de Pharmacie, Université Paris V
Article
Imaging of Tie2 with a fluorescently labeled small molecule affinity ligand
Peter David Koch, Maaz S. Ahmed, Rainer H. Kohler, Ran Li, and Ralph Weissleder
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00724 • Publication Date (Web): 06 Dec 2019
Downloaded from pubs.acs.org on December 11, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government
works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 15
ACS Chemical Biology
1
2
3
4
5
6
7
8
Signal
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Rebastinib
BODIPY-TMR
TIE2 +Rebastinib
BODIPY-TMR
TIE2
ACS Paragon Plus Environment

ACS Chemical Biology
Page 2 of 15
1
2
3
4
5
6
7
8
9
Imaging of Tie2 with a fluorescently labeled small molecule affinity ligand
Peter David Koch1,3, Maaz S. Ahmed1,3, Rainer H. Kohler1, Ran Li1, Ralph Weissleder1,2,4
1.Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02115
2.Department of Systems Biology, Harvard Medical School, Boston, MA 02115
3.These authors contributed equally
4.Correspondence: rweissleder@mgh.harvard.edu
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Abstract
The receptor tyrosine kinase inhibitor, Tie2, has significant roles in endothelial signaling and
angiogenesis, and is relevant in the pathophysiology of several diseases. However, there are
relatively few small molecule probes available to study Tie2, making evaluation of its activity in
vivo difficult. Recently, it was discovered that the small molecule, rebastinib (DCC-2036), is a
potent Tie2 inhibitor. We hypothesized that fluorescent derivatives of rebastinib could be used
as imaging agents for Tie2. Based on crystallography structures, we synthesized three
fluorescent derivatives, which we then evaluated in both in vitro and in vivo assays. We found
that the Rebastinib-BODIPY TMR (Reb-TMR) derivative has superior imaging characteristics in
vitro, and successfully labeled endothelial cells in vivo. We propose that this probe could be
further used in in vivo applications for studying the role of Tie2 in disease.
Introduction
The angiopoietin-Tie2 signaling pathway plays an important role in regulating vascularization and
angiogenesis. Angiopoietins comprise a set of 4 growth factors, Ang1 through Ang4, which bind
to Tie2, a receptor tyrosine kinase that activates downstream signaling pathways1,2. Tie2 is
closely related to the receptor Tie1, and sometimes functions as a heterodimer. Both are
predominately expressed in endothelial cells. Binding of Tie2 to its agonist ligand, Ang1, activates
the receptor and promotes angiogenesis via several pathways and downstream nodes, such as
Akt, FOXO, and NFkB1,2. Other angiopoietins, such as Ang2, also bind to Tie2 and can promote
angiogenesis; however, the effects induced by other angiopoietins are context dependent and in
fact can sometimes inhibit angiogenesis.
Dysregulation of the Ang-Tie2 pathway has been linked to several diseases. For example, it has
been observed that Tie2 levels are reduced in model systems of neovascular age-related macular
degeneration (NV-AMD), a condition characterized by irregular, leaky vasculature in the choriod
that leads to progressive vision loss3. It has been shown that activation of the Tie2 pathway with
an agonistic antibody is effective in mouse models of NV-AMD, improving vascularization without
the side effects associated with current standard of care anti-VEGF therapy. Relatedly, activation
of the Tie2 receptor with the same agonistic antibody also has proven beneficial in mouse models
of sepsis, as it normalized the vasculature4
Overactivation of the Ang-Tie2 pathway, on the other hand, is typically pro-tumorigenic5,6,7
.
Hypoxic conditions can trigger production of Tie2, which stimulates tumorigenesis through a
number of pro-wound healing mechanisms8,9,10. Curiously, it has been observed that Tie2 is also
expressed in macrophages, and the presence of Tie2-positive macrophages in biopsies is
associated with higher risks of metastasis5,6. Conversely, there are some studies suggesting that
activation of the Tie2 pathway with a phosphatase inhibitor can enhance immunotherapy11, but to
date, most work suggests that this pathway promotes tumorigenesis. Indeed, it is increasingly
clear that the tumor microenvironment, including both stromal and immune cells, plays a
substantial role in therapy. Further study in the role of Tie2 in cancer tumor microenvironment is
warranted.
ACS Paragon Plus Environment

Page 3 of 15
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
Despite the various roles of Tie2 in disease pathophysiology, it is still comparatively less studied,
especially in vivo. One challenging factor is that there are limited small molecule probes available
to study its activity. Recently however, it was discovered that rebastinib, a kinase inhibitor in
clinical trials for refractory leukemia, exhibits potent activity against Tie2. Rebastinib is a small
molecule drug that functions as a switch control inhibitor; it binds at an allosteric site and induces
the kinase to adopt inactive conformation12. Given the emerging roles of Tie2 in disease, and the
need for more probes to study Tie2, we developed companion imaging agents based off a
rebastinib scaffold to study Tie2 in various model systems. Specifically, we synthesized three
derivatives, evaluated their activities in in vitro cell culture assays, and finally tested the utility of
one lead compound in an in vivo setting.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Methods
Synthesis of fluorescent derivatives
For description of the synthesis of the fluorescent rebastinib analogs, please refer to the
supplementary material.
Cell culture, in vitro assays, and immunofluorescence
HUVEC cells were maintained in Lonza Endothelial Growth Medium-2 (EGMTM-2) with
supplements added as per manufacturer instructions (Lonza, CC-3162). HEK293T and
RAW264.7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented
with 10% fetal bovine serum (Atlanta Biologicals) and 1% penicillin-streptomycin (Thermo Fisher).
Cells were incubated at 37°C, 5% CO2.
TIE2 plasmid was obtained from SinoBiological (HG10700-NY). For transfection of Tie2 plasmid,
the Fugene 6 transfection kit (Promega, E2691) was used, following protocol of manufacturer.
For microscopy assays, Corning 3764 384-well plates were seeded with approximately 1,000
cells/well in 40 µL of media. Cells were treated with fluorescent drug and/or unlabeled drug, for
90 minutes of treatment. Wells were then washed out 6 times with 80 uL of media. Washes were
performed repetitively, with no wait times. Cells were then incubated with 10 µg/mL Hoechst
333412 for 5 minutes, and then washed out, three times with 80 uL of media. Cells were
subsequently imaged on either an Operetta High Content Screening System, or an IXM
MetaExpress. In both cases, cells were imaged in either the 560 nm channel (BODIPY-TMR) or
650 nm channel (SiR and BODIPY-650), as well as a 360 nm channel (Hoechst 333412) to
determine nuclei. Channel information for microscopy is summarized in supplementary table 1.
For co-localization assays, cells were imaged on an upright BX63 microscope (Olympus) or a
Olympus high-throughput imaging system in the same channels as above with either 100x
(HUVEC) or 60x (RAW264.7) magnification. Cells were treated with fluorescent drug as above
and imaged after washing out drug. Cells were then fixed with 4% PFA for 20 min, blocked with
3% BSA in PBS, and then stained with a Tie2 antibody (Abcam, ab24859 for anti-mouse Tie2 and
ab221154 for anti-human Tie2) at 10 µg/mL for 1 hour at room temperature. Cells were then
stained with a secondary anti-mouse or anti-rabbit Alexa 647 antibody (anti-rabbit or anti-mouse
Alexa 647 antibody) at 10 µg/mL and Hoechst 33342 (10 µg/mL) for 30 min.
Image analysis used in figures 3-4 was performed using the Columbus server at Laboratory of
Systems Pharmacology (Harvard Medical School). In brief, nuclei were first identified using the
Hoechst stain, and surrounding cellular regions were identified with the fluorescent drug. Image
intensities were then quantified. Co-localization plot in figure 5 was done in ImageJ FIJI (NIH),
using the Coloc2 plugin. For figure 6, blood half-life measurement was determined by quantifying
fluorescent intensity in a segmented vessel area, using Cell Profiler.
ACS Paragon Plus Environment

ACS Chemical Biology
Page 4 of 15
1
2
3
4
5
6
7
For fluorescent polarization binding assay, recombinant Tie2 (Thermo Fisher, PV3628) was
diluted in PBS to 0.8 uM and mixed with 0.8 uM of Reb-TMR. Tie2 was pre-incubated with
unlabeled rebastinib at indicated concentrations. Assays were performed in Corning 4581 low
volume, 384 well plates.
8
9
All plots presented were made in GraphPad Prism 8.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Intravital imaging
Images were acquired on a modified multiphoton/confocal FV1000 imaging system (Olympus,
America) 13. The 490 nm channel was used to image Tie2-GFP and the 560 nm channel was
used to image Reb-TMR. Examination was performed in dorsal skin-fold window chambers
implanted in STOCK Tg(TIE2GFP)287Sato/J reporter mice (The Jackson Laboratory). Two
million mouse mammary tumor cells were suspended in 20 µl PBS and injected into the dorsal
skin-fold chamber 3 days prior to treatment. Tie2 expression was examined at baseline
immediately before treatment. Images were pseudo-colored and processed in FIJI (ImageJ, NIH)
by adjusting brightness/contrast.
Results
Modeling and synthesis of rebastinib derivatives
We synthesized three derivatives using either BODIPY-TMR, BODIPY-650, or silicon rhodamine
(SiR) as the fluorochrome. Our choice of these fluorochromes was motivated in large part by their
superior performance for in vivo applications14–18. Synthesis of the derivatives are described in
the methods (fig. 1). Ultimately, we synthesized 3 derivatives: Rebastinib-BODIPY TMR (Reb-
TMR), Rebastinib-BODIPY 650 (Reb-650), and Rebastinib-SiR (Reb-SiR). Yields were highest
for the BODIPY analogs, while the SiR analog had a slightly lower yield.
Synthesis was guided by molecular modeling of a previously determined crystal structure of
rebastinib and Tie26. Rebastinib binds to the switch control portion of Tie2. The t-butyl, urea, and
fluorophenyl moieties of rebastinib make interactions with several residues in the switch control
region, which promote the protein to adopt an inactive conformation, thereby inhibiting its activity.
The rebastinib-Tie2 complex is very stable with a half-life of approximately 10 hours. The crystal
structure showed that the terminal amide of rebastinib extends out of the binding pocket. Thus,
we decided to couple fluorophores to this part of the molecule to minimize interference with the
binding motif (fig. 2).
In vitro assays for validation of fluorescent rebastinib
To characterize biochemical activity of the fluorescent derivatives, we first treated HUVEC and
HEK293T cells with the three derivatives at various concentrations and measured cellular uptake.
These two cell lines are Tie2+ and Tie2- respectively19, 20. We found that both Reb-TMR and Reb-
650 stained HUVEC cells brightly at low concentrations (100 nM), and expectedly showed minimal
staining in the Tie2- HEK293T line (fig. 3). The Reb-SiR analog on the other hand, showed weak
staining in HUVEC cells even at doses up to 1µM (fig. 3). This drug also showed high amounts
of background fluorescence, even after thorough washout. These results indicated that the SiR
probe is not suitable for labeling of Tie2. We did not study in depth why the Reb-SiR failed to
stain well. It is has previously been shown that the fluorescence of SiR probes are sensitive to
the cellular environment, which may be one possibility21. Other possibilities include that the Reb-
SiR probe may have a high koff rate and is washed out quickly or that there is plate binding. The
fact that the BODIPY analogs stained HUVEC and HEK293T cells to different degrees suggests
that the drugs bind to protein targets in the cells, not to internal cellular membranes
nonspecifically. Between the Reb-TMR and Reb-650 analogs, the Reb-TMR showed stronger
ACS Paragon Plus Environment

Page 5 of 15
ACS Chemical Biology
1
2
3
4
5
6
differential staining between HUVECs and HEK293T, so we focused on this compound for all
future experiments. Neither drug showed significant cytotoxicity that would prohibit its use in in
vivo applications (fig. S1). Absorption and emission spectra are shown in fig. S2.
7
8
Characterization of Rebastinib-BODIPY TMR (Reb-TMR)
To further characterized Reb-TMR we performed a competition assay with Reb-TMR and
unlabeled rebastinib (fig. 4a, b). In cell culture, we treated cells with a combination of unlabeled
rebastinib and Reb-TMR; we observed that unlabeled rebastinib competes out the fluorescent
signal of Reb-TMR (IC50 = 2.69 µM), indicating that Reb-TMR shares the targets of rebastinib.
Moreover, comparison of Reb-TMR and BODIPY-TMR treatment showed that the free dye was
taken up in negligible amounts compared to Reb-TMR (fig. S3), further confirming specific activity
of the drug. Next, we also performed a complementary experiment in which we transfected
HEK293T cells with a plasmid encoding for Tie2 and treated these cells with Reb-TMR. We show
that fluorescent intensity levels in Tie2-transfected cells, were above that of untransfected cells,
and comparable to the levels of HUVEC cells (fig. S4). In all, these cell-based assays suggest
Reb-TMR binds Tie2.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We also performed a biochemical assay to measure binding to Tie2 directly. Using fluorescent
polarization as an indicator of binding, we showed that Reb-TMR with Tie2 had higher anisotropy
as compared to free Reb-TMR, indicating binding (fig. 4c). Additionally, pre-incubation of
recombinant Tie2 with unlabeled rebastinib (10 µM) reduced the anisotropy to levels comparable
to that of the free drug, suggesting that Reb-TMR can be competed out by unlabeled drug. We
noted that fluorescent intensities of Reb-TMR in vitro changed upon addition of recombinant Tie2;
fluorescent was enhanced about 3-fold when adding Reb-TMR to recombinant Tie2 compared to
Reb-TMR in PBS (fig. S5). However, in the presence of non-fluorescent rebastinib, these levels
returned to baseline.
We next treated cells with Reb-TMR and obtained higher resolution images to investigate the
degree of co-localization of Reb-TMR and Tie2. In HUVEC cells, both Reb-TMR and Tie2 display
a largely cytoplasmic stain, consistent with previous knowledge of biology (fig. 5). Higher signal
was observed in the perinuclear region. Correlation was not perfect (r2 = 0.88), which likely could
arise due to several factors, including non-covalent binding of Reb-TMR, off-target
binding/polypharmacology or delays (Koff) in subsequent immunostaining. Given that Tie2 is also
expressed in macrophages, we performed the same experiment in RAW264.7 murine
macrophages. There, we observed the same cytoplasmic staining of both Reb-TMR and Tie2
(fig. S6).
Use of Reb-TMR in vivo
To determine the suitability of Reb-TMR as an in vivo imaging agent, we treated Tie2-GFP mice
22with Reb-TMR (20 mg/kg, I.V.). This mouse model expresses GFP in endothelial cells, under
the control of the Tie2 promoter; higher levels of GFP correlate with more production of Tie2.
Tie2-GFP reporter mice were implanted with a dorsal skin chamber and injected with mouse
mammary tumor virus cells (MMTV) to both induce a wound and to stimulate tumorigenesis.
Three days after injection, we confirmed development of vasculature in the window chamber, and
proceeded to image various sites. As expected, strong GFP signals could be observed lining the
edges of blood vessels, consistent with the location of endothelial cells. Injection of Reb-TMR
into the tail vein led to rapid, strong increase in signal in the 560 nm fluorescence channel. After
the free drug began to clear out of the vessel, we observed lining of Reb-TMR along the edges of
the vessel, co-localizing with the Tie2+ endothelial cells (fig. 6a). To study pharmacokinetics, we
performed time lapse imaging on a single blood vessel and measured fluorescent intensity
throughout the whole vessel over the course of approximately three hours. Blood PK half-life
ACS Paragon Plus Environment

ACS Chemical Biology
Page 6 of 15
1
2
3
4
5
6
measured in a single vessel was approximately 30 min (fig. S7). The long half-life of Reb-TMR,
combined with the very strong signal of Reb-TMR along the lining of blood vessels, indicates
successful in vivo labeling of Tie2 and is promising for the utility of this probe as an in vivo imaging
agent.
7
8
9
Discussions
Tie2 plays a role in a number of disease, and much study is still warranted to determine its
potential as a biomarker, prognostic, and/or therapeutic target. Tie2 is predominantly expressed
in endothelial cells and to a lesser extent in immune cells. While Tie2 does not necessarily directly
cause pathogenesis of a disease, modulation with a drug could be a useful therapeutic strategy.
Being able to determine levels and activation states of Tie2 with companion imaging agents would
therefore be useful in both experimental animal models and potentially even in clinical setting.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In comparison to other receptor tyrosine kinases, Tie2 is not as thoroughly studied, and there are
limited small molecule probes available. Rebastinib was only discovered activity against Tie2
relatively recently, and as such, we decided to develop small molecule imaging agents using
rebastinib as a backbone. Small molecule imaging agents have proven very successful in many
other example cases, such as BTK, MERTK, and B-Raf14,23,18. Efficacious small molecule imaging
agents can be used in any mouse model, do not exhibit permanent effects, and minimize the need
for costly and timely genetically engineered mouse models.
We found that the BODIPY analogs of rebastinib, in particular Reb-TMR, labeled cells in vitro,
very brightly and potently. Our analog also proved efficacious in a reporter mouse model,
suggesting its utility for use in a broader array of model systems. While we focused on Reb-TMR
in this work, we found that the Reb-650 analog also showed activity (fig. 3, fig. S8), and
optimization of this molecule may be warranted if a imaging agent in the 650 nm channel is
needed. Ultimately, we envision the development of a small molecule agents, including
radioactive probes, that could be translated into clinical use, for measurement of Tie2 levels.
Activation of Tie2 is a potential therapeutic strategy for sepsis and NV-AMD, so an imaging agent
to detect for presence of Tie2 could aid rapid decision making for treatment choice. Alternatively,
another possibility is to encapsulate an imaging agent in a nanoparticle to selectively deliver it to
macrophages. This could allow one to image specifically for the presence of Tie2+ macrophages,
which is an important prognostic marker in cancer. Given that our work so far indicates that Tie2
can be imaged with a small molecule, we hope that further work can optimize companion agents
further for even more efficacious activity.
Supporting Information
Toxicity data, photophysical data, biochemical data, pharmacokinetics data, chemical synthesis
protocols, NMR data, HPLC purity analysis. This material is available free of charge via the
internet at http://pubs.acs.org.
Acknowledgments
We thank the Laboratory of Systems Pharmacology (Harvard Medical School) for the use of the
high content imaging microscopes, C. Yapp for technical assistance with imaging, and O. R.
Weissleder for help with chemical purification and NMR.
ACS Paragon Plus Environment

Page 7 of 15
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
References
1.
2.
3.
Jeltsch, M., Leppänen, V. M., Saharinen, P. & Alitalo, K. (2013) Receptor tyrosine kinase-
mediated angiogenesis. Cold Spring Harb Perspect Biol 5, 1-22
Leligdowicz, A., Richard-Greenblatt, M., Wright, J., Crowley, V. M. & Kain, K. C. (2018)
Endothelial Activation: The Ang/Tie Axis in Sepsis. Front Immunol 9, 838
Kim, J., Park, J. R., Choi, J., Park, I., Hwang, Y., Bae, H., Kim, Y., Choi, W., Yang, J. M.,
Han, S., Chung, T. Y., Kim, P., Kubota, Y., Augustin, H. G., Oh, W. Y. & Koh, G. Y. (2019)
Tie2 activation promotes choriocapillary regeneration for alleviating neovascular age-related
macular degeneration. Sci Adv 5, 6732
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4.
Han, S., Lee, S. J., Kim, K. E., Lee, H. S., Oh, N., Park, I., Ko, E., Oh, S. J., Lee, Y. S., Kim,
D., Lee, S., Lee, D. H., Lee, K. H., Chae, S. Y., Lee, J. H., Kim, S. J., Kim, H. C., Kim, S.,
Kim, S. H., Kim, C., Nakaoka, Y., He, Y., Augustin, H. G., Hu, J., Song, P. H., Kim, Y. I.,
Kim, P., Kim, I. & Koh, G. Y. (2016) Amelioration of sepsis by TIE2 activation-induced
vascular protection. Sci Transl Med 8, 1-11.
5.
6.
Chen, L., Li, J., Wang, F., Dai, C., Wu, F., Liu, X., Li, T., Glauben, R., Zhang, Y., Nie, G.,
He, Y. & Qin, Z. (2016) Tie2 Expression on Macrophages Is Required for Blood Vessel
Reconstruction and Tumor Relapse after Chemotherapy. Cancer Res 76, 6828-6838
Harney, A. S., Karagiannis, G. S., Pignatelli, J., Smith, B. D., Kadioglu, E., Wise, S. C.,
Hood, M. M., Kaufman, M. D., Leary, C. B., Lu, W. P., Al-Ani, G., Chen, X., Entenberg, D.,
Oktay, M. H., Wang, Y., Chun, L., De Palma, M., Jones, J. G., Flynn, D. L. & Condeelis, J.
S. (2017) The Selective Tie2 Inhibitor Rebastinib Blocks Recruitment and Function of Tie2Hi
Macrophages in Breast Cancer and Pancreatic Neuroendocrine Tumors. Mol Cancer Ther
16, 2486-2501
7.
Karagiannis, G. S., Pastoriza, J. M., Borriello, L., Jafari, R., Coste, A., Condeelis, J. S.,
Oktay, M. H. & Entenberg, D. (2019) Assessing Tumor Microenvironment of Metastasis
Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using
Intravital Imaging and Fixed Tissue Analysis. J Vis Exp, 148, 59633
8.
9.
Lee, S. J., Lee, C. K., Kang, S., Park, I., Kim, Y. H., Kim, S. K., Hong, S. P., Bae, H., He, Y.,
Kubota, Y. & Koh, G. Y. (2018) Angiopoietin-2 exacerbates cardiac hypoxia and
inflammation after myocardial infarction. J Clin Invest 128, 5018-5033
Moritz, F., Schniering, J., Distler, J. H. W., Gay, R. E., Gay, S., Distler, O. & Maurer, B.
(2017) Tie2 as a novel key factor of microangiopathy in systemic sclerosis. Arthritis Res
Ther 19, 105
10. Vadivel, A., Alphonse, R. S., Etches, N., van Haaften, T., Collins, J. J., O’Reilly, M., Eaton,
F. & Thébaud, B. (2014) Hypoxia-inducible factors promote alveolar development and
regeneration. Am J Respir Cell Mol Biol 50, 96-105
11. Li, G., Sachdev, U., Peters, K., Liang, X. & Lotze, M. T. (2019) The VE-PTP Inhibitor AKB-
9778 Improves Antitumor Activity and Diminishes the Toxicity of Interleukin 2 (IL-2)
Administration. J Immunother 42, 237-243
12. Chan, W. W., Wise, S. C., Kaufman, M. D., Ahn, Y. M., Ensinger, C. L., Haack, T., Hood, M.
M., Jones, J., Lord, J. W., Lu, W. P., Miller, D., Patt, W. C., Smith, B. D., Petillo, P. A.,
Rutkoski, T. J., Telikepalli, H., Vogeti, L., Yao, T., Chun, L., Clark, R., Evangelista, P.,
Gavrilescu, L. C., Lazarides, K., Zaleskas, V. M., Stewart, L. J., Van Etten, R. A. & Flynn,
D. L. (2011) Conformational control inhibition of the BCR-ABL1 tyrosine kinase, including
the gatekeeper T315I mutant, by the switch-control inhibitor DCC-2036. Cancer Cell 19,
556-568
13. Pittet, M. J., Garris, C. S., Arlauckas, S. P. & Weissleder, R. (2018) Recording the wild lives
of immune cells. Sci Immunol 3, 1-12
14. Kim, E., Yang, K. S., Kohler, R. H., Dubach, J. M., Mikula, H. & Weissleder, R. (2015)
Optimized Near-IR Fluorescent Agents for in Vivo Imaging of Btk Expression. Bioconjug
Chem 26, 1513-1518
ACS Paragon Plus Environment

ACS Chemical Biology
Page 8 of 15
1
2
3
4
5
6
7
8
9
15. Lukinavičius, G., Blaukopf, C., Pershagen, E., Schena, A., Reymond, L., Derivery, E.,
Gonzalez-Gaitan, M., D’Este, E., Hell, S. W., Wolfram Gerlich, D. & Johnsson, K. (2015)
SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy. Nat Commun 6, 8497
16. Lukinavičius, G., Reymond, L., Umezawa, K., Sallin, O., D’Este, E., Göttfert, F., Ta, H., Hell,
S. W., Urano, Y. & Johnsson, K. (2016) Fluorogenic Probes for Multicolor Imaging in Living
Cells. J Am Chem Soc 138, 9365-9368
17. Meimetis, L. G., Giedt, R. J., Mikula, H., Carlson, J. C., Kohler, R. H., Pirovich, D. B. &
Weissleder, R. (2016) Fluorescent vinblastine probes for live cell imaging. Chem Commun
(Camb) 52, 9953-9956
18. Miller, M. A., Kim, E., Cuccarese, M. F., Plotkin, A. L., Prytyskach, M., Kohler, R. H., Pittet,
M. J. & Weissleder, R. (2017) Near infrared imaging of Mer tyrosine kinase (MERTK) using
MERi-SiR reveals tumor associated macrophage uptake in metastatic disease. Chem
Commun (Camb) 54, 42-45
19. Dalton, A. C., Shlamkovitch, T., Papo, N. & Barton, W. A. (2016) Constitutive Association of
Tie1 and Tie2 with Endothelial Integrins is Functionally Modulated by Angiopoietin-1 and
Fibronectin. PLoS One 11, e0163732
20. Sturk, C. & Dumont, D. J. (2010) Tyrosine phosphorylation of Grb14 by Tie2. Cell Commun
Signal 8, 30
21. Lukinavičius, G., Umezawa, K., Olivier, N., Honigmann, A., Yang, G., Plass, T., Mueller, V.,
Reymond, L., Corrêa, I. R., Luo, Z. G., Schultz, C., Lemke, E. A., Heppenstall, P., Eggeling,
C., Manley, S. & Johnsson, K. (2013) A near-infrared fluorophore for live-cell super-
resolution microscopy of cellular proteins. Nat Chem 5, 132-139
22. Motoike, T., Loughna, S., Perens, E., Roman, B. L., Liao, W., Chau, T. C., Richardson, C.
D., Kawate, T., Kuno, J., Weinstein, B. M., Stainier, D. Y. & Sato, T. N. (2000) Universal
GFP reporter for the study of vascular development. Genesis 28, 75-81
23. Mikula, H., Stapleton, S., Kohler, R. H., Vinegoni, C. & Weissleder, R. (2017) Design and
Development of Fluorescent Vemurafenib Analogs for In Vivo Imaging. Theranostics 7,
1257-1265
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 9 of 15
ACS Chemical Biology
1
2
3
4
Figures
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1: Synthesis of fluorescently labeled rebastinib derivatives. The synthesis of labeled
rebastinib was completed using the scheme depicted above.
ACS Paragon Plus Environment

ACS Chemical Biology
Page 10 of 15
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2: Modeling. Addition of a linker to the amide of rebastinib allows for coupling of a
fluorophore to the small molecule. Based on a previously published structure of rebastinib in
complex with Tie2 (PDB: 6MWE), we predicted this addition should not interfere with binding
of rebastinib to Tie2.
ACS Paragon Plus Environment

Page 11 of 15
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3: In vitro testing. Staining of HUVEC and HEK293T cells with the three fluorescent
analogs of rebastinib. (a). Panel of representative images of Reb-TMR (100 nM), Reb-650 (100
nM), and Reb-SiR (1 uM) in HUVEC (Tie2+) and HEK293T (Tie2-) cell lines. Reb-TMR and
Reb-650 stain HUVEC cells strongly, whereas the Reb-SiR analog stains cells weakly. (b)
Quantification of fluorescent intensities in HUVEC and HEK293 cells at single cell resolution.
Reb-TMR shows the strongest differential between HUVEC and HEK293T cells.
ACS Paragon Plus Environment

ACS Chemical Biology
Page 12 of 15
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4: Quantitative binding data and inhibition of Reb-TMR (a) Representative images of
Reb-TMR in HUVEC cells in the presence (below) or absence (top) of unlabeled rebastinib (10
uM). (b) Quantification of Reb-TMR fluorescent intensity as a function of increasing
concentration of unlabeled rebastinib. Reported is the mean single cell fluorescent intensity per
well, N = 2 wells. Error bars correspond to S.D. (c) Biochemical fluorescent polarization assay
of rebastinib-bodipy TMR (0.8 uM) on enzymatic domain of Tie2. Leftmost point corresponds to
polarization of Reb-TMR in PBS, and right points correspond to polarization of Reb-TMR with
Tie2, and in increasing concentrations of unlabeled rebastinib (0,0.1,1,10 uM). Reported is
mean over N = 2 wells. Error bars correspond to S.D.
ACS Paragon Plus Environment

Page 13 of 15
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5: Co-localization (a) High resolution imaging of HUVECs. Treatment of HUVECs with
Reb-TMR (100 nM) followed by a stain for Tie2 shows colocalization of the two markers in the
cytoplasm (b) Correlation of Reb-TMR and Tie2 immunofluorescence signals (Pearson r2 =
0.88)
ACS Paragon Plus Environment

ACS Chemical Biology
Page 14 of 15
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6: Intravital Microscopy. Tie2 reporter mouse, with implanted dorsal skin chambers,
were injected with Reb-TMR (20 mg/kg, tail vein I.V.). About 1 hour after injection, select sites
in the vascularized region were imaged. The two sites above show Reb-TMR lining the
endothelia of the blood vessels, overlaying Tie2.
ACS Paragon Plus Environment

Page 15 of 15
ACS Chemical Biology
1
2
3
4
Table of Contents Graphic
5
Signal
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Rebastinib
BODIPY-TMR
TIE2 +Rebastinib
BODIPY-TMR
TIE2
ACS Paragon Plus Environment