Chlorin-Based Nanoscale Metal-Organic Framework Systemically Rejects Colorectal Cancers via Synergistic Photodynamic Therapy and Checkpoint Blockade Immunotherapy

Article abstract of DOI:10.1021/jacs.6b06663

Photodynamic therapy (PDT) can destroy local tumors and minimize normal tissue damage, but is ineffective at eliminating metastases. Checkpoint blockade immunotherapy has enjoyed recent success in the clinic, but only elicits limited rates of systemic ant

Full text of DOI:10.1021/jacs.6b06663

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
A Chlorin-based Nanoscale Metal-Organic Framework
Systemically Rejects Colorectal Cancers via Synergistic
Photodynamic Therapy and Checkpoint Blockade Immunotherapy
Kuangda Lu, Chunbai He, Nining Guo, Christina Chan, Kaiyuan Ni, Ralph R Weichselbaum, and Wenbin Lin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b06663 • Publication Date (Web): 30 Aug 2016
Downloaded from http://pubs.acs.org on August 30, 2016
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 free 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 accessible to all
readers and 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.
Journal of the American Chemical Society 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 10
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
A Chlorin-based Nanoscale Metal-Organic Framework Systemically
Rejects Colorectal Cancers via Synergistic Photodynamic Therapy
and Checkpoint Blockade Immunotherapy
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
Kuangda Lu,a,† Chunbai He,a,† Nining Guo,a,b,† Christina Chan,a Kaiyuan Ni,a Ralph R. Weichsel-
baum,b Wenbin Lina,*
aDepartment of Chemistry, The University of Chicago, Chicago, IL 60637, USA and bDepartment of Radiation and Cel-
lular Oncology and The Ludwig Center for Metastasis Research, The University of Chicago, Chicago, IL 60637, USA
E-mail: wenbinlin@uchicago.edu
KEYWORDS (Word Style “BG_Keywords”). If you are submitting your paper to a journal that requires keywords, pro-
vide significant keywords to aid the reader in literature retrieval.
ABSTRACT: Photodynamic therapy can destroy local tumors and minimize normal tissue damage, but is ineffective at
eliminating metastases. Checkpoint blockade immunotherapy has enjoyed recent success in the clinic, but only elicits
limited rates of systemic antitumor response for most cancers due to insufficient activation of the host immune system.
Here we describe a treatment strategy that combines photodynamic therapy (PDT) by a new chlorin-based nanoscale
metal-organic framework (nMOF), TBC-Hf, and a small molecule immunotherapy agent, that inhibits Indoleamine 2,3-
dioxygenase (IDO), encapsulated in the nMOF channels to induce systemic antitumor immunity. The synergistic combi-
nation therapy achieved effective local and distant tumor rejection in colorectal cancer models. We detected increased T
cell infiltration in the tumor microenvironment after activation of the immune system with the combination of the IDO
inhibition by the small molecule immunotherapy agent and the immunogenic cell death induced by PDT. We also eluci-
dated the underlying immunological mechanisms and revealed compensatory roles of neutrophils and B cells in present-
ing tumor-associated antigens to T cells in this combination therapy. We believe that nMOF-enabled PDT has the poten-
tial to significantly enhance checkpoint blockade cancer immunotherapy, affording clinical benefits for the treatment of
many difficult-to-treat cancers.
INTRODUCTION
(SBUs) facilitate intersystem crossing to enhance 1O2 gen-
eration.4 We further demonstrated in a recent study that
both apoptosis and immunogenic cell death (ICD) con-
tribute to the anticancer efficacy of nMOF-enabled PDT.4b
The acute inflammatory response of PDT can alter the
immunosuppressive microenvironment of the local tumor
to prime the host immune system.5 We hypothesized that
the porous nMOF structure could be leveraged to accen-
tuate immune response by delivering small molecule im-
munostimulatory agents in the open channels, combining
PDT and immunotherapy to eradicate both local, treated
tumors and reject distant, untreated tumors.
Photodynamic therapy (PDT) combines non-toxic pho-
tosensitizers (PSs), light, and tissue oxygen to generate
reactive oxygen species (ROS) such as O2 to achieve local
1
tumor control/rejection.1 PDT is highly effective for the
treatment of esophageal cancer and skin lesions but can-
not completely eradicate deep-seated tumors due to shal-
low penetration of light through tissues.2 The localized
light irradiation also renders PDT ineffective for treating
systemically disseminated disease. Therefore, more effi-
cient PSs and new treatment strategies are needed to en-
hance the efficacy of PDT in both eliminating local tu-
mors and controlling distant metastases.
Immunotherapy has recently emerged as a highly effec-
tive cancer treatment strategy, with high overall response
rates in multiple cancer types and durable tumor control
enjoyed by a small subset of patients.6 Of particular inter-
est is checkpoint blockade immunotherapy which uses
small molecules or antibodies to stimulate the immuno-
suppressive microenvironment of tumors by modulating
protein expressions and/or functions at dysregulated im-
Significant efforts have been devoted to developing na-
noparticle–based PSs to enhance PDT in the past decade.3
We first reported the use of porphyrin- and chlorin-based
nanoscale metal-organic frameworks (nMOFs) as efficient
PSs for PDT of cancer. The stable framework and crystal-
line structure of nMOFs allow for high PS loadings
whereas the heavy metal cluster secondary building units
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 10
mune checkpoints.6b,7 Indoleamine 2,3-dioxygenase (IDO)
RESULTS
1
2
3
4
5
6
7
8
is one such checkpoint, an immunoregulatory enzyme
highly expressed in tumors that catalyzes the oxidative
catabolism of tryptophan (Trp) to kynurenine (Kyn).8 The
results of this catalytic process lead to depletion of Trp
and production of Kyn, thus preventing the clonal expan-
sion of T-cells and promoting T-cell anergy and apopto-
sis.9 Small molecule IDO inhibitors such as 1-methyl-
tryptophan,9b, 10 INCB24360,11 and NLG919,12 can effectively
block Trp catabolism, but show modest effect as mono-
therapies, due in part to ineffective antigen presentation
and insufficient antitumor immunity.
Synthesis and Characterization of nMOFs
The new chlorin derivative 5,10,15,20-tetra(p-benzoato)
chlorin (H4TBC) was synthesized as depicted in scheme 1.
Briefly,
(Me4TBC) was synthesized by a partial reduction of
5,10,15,20-tetra(p-methylbenzoato)porphyrin (Me4TBP)
with toluenesulfonhydrazide in 16% yield. Saponification
of Me4TBC afforded H4TBC in 78% yield. Me4TBC and
H4TBC were characterized by NMR and mass spectrome-
try (Figures S1-S5, supporting information [SI]).
5,10,15,20-tetra(p-methylbenzoato)chlorin
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
Scheme 1. Synthesis of 5,10,15,20-tetra(p-benzoato) chlo-
rin (H4TBC).
We herein report the design of a new nMOF, TBC-Hf,
constructed from a chlorin derivative, 5,10,15,20-tetra(p-
benzoato)chlorin (H4TBC) and Hf6(ꢀ3-O)4(ꢀ3-OH)4 SBUs.
We took advantage of the highly porous structure of the
nMOF to load an IDO inhibitor (IDOi) into TBC-Hf to
afford IDOi@TBC-Hf. The IDOi@TBC-Hf system enables
a novel cancer treatment strategy by combining nMOF-
enabled PDT and IDOi-based immunotherapy (Figure 1).
We demonstrated the eradication of the primary, treated
tumors and the regression of the distant, untreated tu-
mors upon IDOi@TBC-Hf administration and light irradi-
ation using two syngeneic colorectal cancer models, CT26
and MC38, and investigated the underlying immunologi-
cal mechanisms.
MeO2C
CO2Me
CO2Me
H
N
N
H
propionic acid
reflux
+
N
N
H
N
CHO
CO2Me
2. DDQ
MeO2C
1. TsNHNH2, 3 eq,
pyridine, 105
℃
MeO2C
HO2C
CO2H
H
CO2Me
N
H
H
N
H
N
H
H
N
NaOH
H
H
N
N
N
N
H
H
H
THF/MeOH
H
CO2H MeO2C
CO2Me
HO2C
H4TBC
A solvothermal reaction between HfCl4 and H4TBC in
N,N-dimethylformamide (DMF) at 80 °C afforded the
purple powder of TBC-Hf in 87% yield. TBC-Hf was
washed with copious amounts of DMF, 1% triethylamine
(NEt3) in ethanol (v/v), and ethanol successively and
stored in ethanol as a stock suspension. As a control, TBP-
Hf with the corresponding porphyrin ligand 5,10,15,20-
tetra(p-benzoato)porphyrin (H4TBP) was synthesized by a
solvothermal reaction between HfCl4 and H4TBP in N,N-
diethylformamide (DEF) at 120 °C and washed in the same
fashion as for TBC-Hf.
TBC-Hf and TBP-Hf adopt the same structure as the
previously reported TBP-Zr analog (MOF-545), based on
powder X-ray diffraction (PXRD) (Figure 2a).13 The car-
boxylate moieties from the TBC ligands bridge 8 out of 12
edges of octahedral Hf6(µ3-O)4(µ3-OH)4 SBUs. The re-
maining coordination sites on the SBUs are occupied by 4
water and 4 terminal hydroxy groups on the equatorial
plane to afford a hexagonal framework of Hf6(µ3-O)4(µ3-
OH)4(OH)4(H2O)4(TBC)2 (Figure S6, SI). The nMOFs
therefore feature large one-dimensional channels with
diameters of 3.5 nm along the c axis, along with smaller
channels with diameters of 1.0 nm along the c axis and 1.1
nm × 0.9 nm windows perpendicular to the c axis. TBC-Hf
and TBP-Hf exhibit BET surface area of 1077 and 1462
m2/g, respectively (Figure S7, SI). During the course of
this study, a few reports of TBP-based nMOFs with a cu-
Figure 1. Schematic presentation of combined PDT and
immunotherapy by IDOi@TBC-Hf. Local injection of
IDOi@TBC-Hf and light irradiation generate reactive ox-
ygen species, causing immunogenic cell death (ICD), and
releasing tumor-associated antigens which are presented
to T-cells. Meanwhile, the IDO inhibitor released from
IDOi@TBC-Hf modulates tryptophan/kynurenine catabo-
lism to activate the immunosuppressive tumor microenvi-
ronment. The combination of antigen presentation from
PDT and checkpoint blockade by IDO inhibition causes
T-cell proliferation and infiltration, leading to not only
eradication of local, treated tumors but also rejection of
distant, untreated tumors.
ACS Paragon Plus Environment

Page 3 of 10
Journal of the American Chemical Society
gy after incubation in Hank’s balanced salt solution
bic structure for PDT have appeared.14 However, we ex-
1
2
3
4
5
6
7
8
pect that our highly porous TBP-Hf control will outper-
form the cubic TBP-based nMOFs due to the enhanced
1O2 diffusion through the nMOF channels. In TBC-Hf and
TBP-Hf, the porphyrin and chlorin rings are highly rigid
in the nMOF structures to minimize energy transfer and
self-quenching.
(HBSS) for 24 h (Figure 2a,f).
The Hf content was determined by inductively coupled
plasma-mass spectrometry (ICP-MS) to be 30.4% (36.6%
calculated). The TBC weight loss of 61.7% (56.8% calcu-
lated) determined by thermogravimetric analysis further
confirms the framework formula (Figure S11a, SI). We
encapsulated an INCB24360 analogue,11 4-amino-N-(3-
chloro-4-fluoro-phenyl)-N’-hydroxy-1,2,5-oxadiazole-3-
carboximidamide (Figure S11b, SI), into the TBC-Hf chan-
nels to afford IDOi@TBC-Hf for the co-delivery of IDOi
with PSs. The IDOi weight percentage after loading is
determined to be 4.7% by TGA (Figure S11a and S12, SI).
The release of IDOi from IDOi@TBC-Hf was studied in
HBSS (Figure S13, SI). IDOi slowly leached from TBC-Hf,
reaching 83.3% release after incubation in HBSS for 24 h.
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
UV-visible absorption spectroscopy indicates that TBC-
Hf more effectively absorbs red light compared to TBP-Hf
(Figure 3a). H4TBC has a major Soret peak at λmax=420 nm
and four Q-bands at 518, 546, 600, and 652 nm. The Soret
absorption of TBC-Hf almost overlaps with that of the
free ligand (λmax=421 nm) while the Q-bands slightly red-
shift to 520, 548, 600, and 653 nm. In comparison, the
Soret peaks of H4TBP and TBP-Hf are at λmax=420 nm and
418 nm, respectively, and the Q-bands are at 516, 549, 592
and 646 nm for H4TBP and 517, 550, 593 and 647 nm for
TBP-Hf. The lowest energy Q-band of the chlorin there-
fore red-shifts by 6 nm after reduction from the porphyrin
counterpart (652 nm compared to 646 nm). The extinc-
tion coefficient of H4TBC at the lowest energy absorption
(ε=44700 M-1·cm-1) is ~9 fold greater than that of H4TBP
(ε=4800 M-1·cm-1) while the lowest energy absorption of
TBC-Hf (ε=38500 M-1·cm-1) is 6-fold greater than that of
TBP-Hf (ε=6400 M-1·cm-1). Notably, the lowest energy ab-
sorption of TBC-Hf (653 nm) is at a wavelength of 7-nm
longer than that of our previously reported DBC-UiO and
the ε of TBC-Hf is ~1.6 fold higher than DBC-UiO.
H4TBC exhibits two fluorescence emissions at 655 nm
and 715 nm with an intensity ratio of ~6.8 when excited at
420 nm (Figure 3c). H4TBP fluorescence emissions are
close to those of H4TBC, though the 651 nm/713 nm peak
intensity ratio is ~2 (Figure 3b). After forming nMOFs, the
fluorescence intensities drop significantly, consistent with
other nMOF systems.4 For TBC-Hf, the fluorescence
peaks slightly blue-shift to 650 nm and 712 nm and the
intensities decrease by 5.3-fold and 4.7-fold, respectively;
for TBP-Hf, the fluorescence peaks blue-shift to 648 nm
and 704 nm and the fluorescence intensities drop by 9.0-
fold and 6.5-fold, respectively. The decrease in fluores-
cence intensity likely results from the enhanced intersys-
tem crossing after coordination of the chlorin/porphyrin
ligands to Hf on the SBUs. Time-Correlated Single Photon
Counting measurement results also supported this inter-
pretation: the fluorescence lifetime at 650 nm dropped
from 9.56 ns for H4TBC to 8.25 ns for TBC-Hf and from
9.25 ns for H4TBP to 8.38 ns for TBP-Hf (Figure 3d-e and
Table S1, SI).
Figure 2. Morphology and structure of TBP-Hf and TBC-
Hf. (a) PXRD patterns of TBP-Hf, TBC-Hf, and
IDOi@TBC-Hf in comparison to MOF-545. TEM images
of TBP-Hf (b-c), TBC-Hf (d-e) and TBC-Hf after incuba-
tion in Hank’s balanced salt solution for 24 h (f).
Transmission electron microscopy (TEM) revealed na-
norod/nanorice morphologies for TBP-Hf and TBC-Hf
(Figure 2b,d). The rods are typically 50~100 nm long and
30-60 nm wide for TBC-Hf, and 50~100 nm long and 20-30
nm wide for TBP-Hf. High resolution images show the
lattice fringes with the distances matching d200 and d001
spacings of the crystal structure (Figure 2c,e and S8-S9,
SI). The average diameter of TBC-Hf and TBP-Hf are 83.2
nm and 72.7 nm, respectively, by dynamic light scattering
(DLS) measurements (Figure S10, SI). The nMOFs are
stable in biological environments, as evidenced by the
lack of significant changes in PXRD pattern or morpholo-
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 10
Singlet Oxygen Generation
both ligands against CT26 cells was next investigated.
TBC-Hf outperformed TBP-Hf under 90 J/cm2 light irra-
diation. Both nMOFs and ligands outperformed proto-
porphyrin IX (PpIX) upon irradiation, a commonly used
PS, while no cytotoxicity was observed in dark control
groups (Figure S15, SI). The IC50 values of TBC-Hf, TBP-
Hf, H4TBC, H4TBP and PpIX for CT26 cells were
4.82±0.50, 7.09±0.39, 10.04±1.22, 12.83±1.59 and 23.72±0.60
ꢀM, respectively, at an irradiation dose of 90 J/cm2 (Fig-
ure 4a). These results were confirmed by a second colo-
rectal cancer MC38 cell with IC50 values of 2.57±0.85 and
17.9±5.0 ꢀM for TBC-Hf and H4TBC, respectively. The
IC50 values exceeded 50 ꢀM for TBP-Hf, H4TBP and PpIX
for MC38 cells (Figure 4b). To demonstrate the versatility
of our nMOF system, unrelated B16F10 melanoma cells
were employed to confirm the PDT efficacy, yielding IC50
values of 5.48±0.70, 9.72±0.78, 11.03±1.57, 20.10±4.28 and
16.48±0.77 ꢀM for TBC-Hf, TBP-Hf, H4TBC, H4TBP and
PpIX, respectively (Figure S16, SI).
1
2
3
4
5
6
7
8
The 1O2 generation efficiencies of H4TBC, TBC-Hf,
H4TBP, and TBP-Hf were determined by Singlet Oxygen
Sensor Green (SOSG) assay. The nMOF samples and lig-
and controls were irradiated with a LED (λmax=650 nm, 20
mW/cm2) and the fluorescence enhancement upon reac-
tion of SOSG with 1O2 was measured by a fluorimeter (ex-
citation/emission at 504/525 nm). The fluorescence inten-
sity plotted against irradiation time fit exponential decay
equations, as we had previously observed in other nMOF
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
systems,4 indicating a pseudo first-order O2 generation
process (Figure 3f). TBC-Hf outperforms both TBP-Hf and
H4TBC in terms of 1O2 generation efficiency.
1
H
H
TBC
TBP
TBC-Hf
TBP-Hf
a)
b)
6
4
2
0
4
4
H
TBP
4
TBP-Hf
1.0
0.5
0.0
Unlike most conventional cytotoxic agents, PDT is
known to induce ICD via apoptosis and necrosis, thereby
mediating antitumor immune response.5 We confirmed
that both apoptosis and necrosis occur upon treatment
with PDT by flow cytometry of Alexa Fluor 488 Annexin
V-labeled dead cells. MC38 cells were incubated with
TBC-Hf, TBP-Hf, H4TBC and H4TBP at 1 µM (ligand con-
centration of 2 ꢀM) followed by light irradiation at 90
J/cm2 (650 nm, 100 mW/cm2). Significant amounts of cells
underwent apoptosis/necrosis when treated with PDT of
TBC-Hf, TBP-Hf, H4TBC and H4TBP with only 30.0%,
32.7%, 55.5%, and 57.6% healthy cells, respectively, com-
pared to 80-90% healthy cells in dark control or PBS-
treated cells with irradiation (Figure 4c and Figure S17a,
SI). These results were further confirmed in CT26 cells
under the same conditions (Figure S17b, SI). Taken to-
gether, the chlorin-based TBC-Hf is a more efficient PS
than the porphyrin-based TBP-Hf at equivalent nMOF
and light doses.
500
600
400
600
800
600
700
800
Wavelength (nm)
Wavelength (nm)
c)
d)1.0
10
5
TBP-Hf
TBP
H
TBC
4
H
4
TBC-Hf
IRF
0.5
0.0
0
600
700
800
120
140
160
Time (ns)
Wavelength (nm)
e) 1.0
f)
H
H
TBP
TBP-Hf
TBC-Hf
4
4
TBC-Hf
TBC
100
TBC
H
4
IRF
0.5
0.0
50
0
0
5
10
120
140
160
Time (min)
Time (ns)
Figure 3. Photophysics and photochemistry of the ligands
and nMOFs. (a) UV-visible absorption spectra of H4TBP,
H4TBC, TBP-Hf, and TBC-Hf. Inset shows expanded views
of the Q-band regions. The fluorescence spectra of H4TBP
and TBP-Hf (b), H4TBC and TBC-Hf (c) with excitation at
420 nm. Time-resolved fluorescence decay traces of
H4TBP and TBP-Hf (d), H4TBC and TBC-Hf (e) along with
instrument response function (IRF). (f) Singlet oxygen
generation by H4TBP, H4TBC, TBP-Hf, and TBC-Hf with a
650 nm LED irradiation at 20 mW/cm2, detected by Sin-
glet Oxygen Sensor Green.
We also investigated the ability of PDT treatment to in-
duce ICD by determining cell-surface expression of calre-
ticulin (CRT). MC38 cells were incubated with TBC-Hf,
TBP-Hf, H4TBC and H4TBP at 1 µM (ligand concentration
of 2 ꢀM) followed by light irradiation at 90 J/cm2. For flow
cytometry analysis, cells were collected and stained with
Alexa Fluor 488-CRT antibody and propidium iodide (PI,
Figure 4d and Figure S18, SI). The fluorescence intensity
of stained cells was gated on PI-negative cells. For im-
munostaining analysis, the cells were stained with
AlexaFluor 488-CRT and DAPI, and observed under con-
focal laser scanning microscopy (CLSM, Figure 4e and
Figure S19, SI). In both instances, cells treated with nMOF
or ligand showed significant CRT expression on the cell-
surface upon irradiation but none with PBS control or
In vitro PDT efficacy and mechanistic study
We first confirmed efficient cellular uptake of TBC-Hf
and TBP-Hf by cancer cells. TBC-Hf and TBP-Hf were
incubated with colorectal cancer CT26 cells at a ligand
concentration of 25 ꢀM for 24 h. The Hf contents inside
cells were determined to be (2.07±0.26) and (1.79±0.17)
nmol/104 cells, respectively, by ICP-MS analysis (Figure
S14, SI). The in vitro PDT efficacy of TBP-Hf, TBC-Hf, and
1
without irradiation. Due to the higher O2 generation
efficiency and in vitro PDT efficacy of TBC-Hf, we focus
our subsequent in vivo studies on TBC-Hf and use TBP-Hf
as a control wherever appropriate.
ACS Paragon Plus Environment

Page 5 of 10
Journal of the American Chemical Society
using bilateral tumor models of colorectal cancers CT26
1
2
3
4
5
6
7
8
and MC38 in the flank regions of BALB/c mice and
C57BL/6 mice, respectively. When the primary tumors
reached ~100 mm3, mice received a single injection of
H4TBC, H4TBC plus IDOi, TBC-Hf, or IDOi@TBC-Hf into
the primary tumors at a ligand dose of 20 µmol/kg or a
corresponding IDOi dose of 1.5 mg/kg. Twelve hours after
injection, the primary tumors were irradiated at a light
dose of 90 J/cm2 (650 nm, 100 mW/cm2). Mice treated
with IDOi@TBC-Hf without light irradiation served as
dark controls. The primary tumor receiving intratumoral
injection with or without irradiation was designated as
the “treated tumor”, while the contralateral tumor which
received neither direct injection nor irradiation was des-
ignated as the “untreated tumor”. As depicted in Figure 5
and Figure S20-S22 (SI), local nMOF injection with light
irradiation led to near elimination of the treated primary
tumors. At the endpoint, CT26 tumor-bearing mice treat-
ed with IDOi@TBC-Hf or TBC-Hf and PDT therapy had
tumors only (1.1±0.2)% and (0.7±0.4)% the size of PBS
treated tumors, respectively. Similarly, MC38 tumor-
bearing mice had tumors only (0.8±0.3)% and (0.9±0.4)%
of the size of PBS treated tumors, respectively. H4TBC
with light irradiation and IDOi@TBC-Hf dark group failed
to inhibit the tumor growth while H4TBC plus IDOi with
light irradiation slightly inhibits the tumor growth (Table
S2). Moreover, only PDT treatment of IDOi@TBC-Hf suc-
cessfully reduced the sizes of the untreated distant tu-
mors. Tumors began shrinking on Day 6 and 5 after
treatment in the CT26 and MC38 models, respectively,
suggesting the treatment evoked systemic antitumor im-
munity in mice (Table S2-S3). TBC-Hf with light irradia-
tion and IDOi@TBC-Hf dark control slightly inhibited the
distant tumor growth, showing ineffectiveness of mono-
therapies.
9
10
11
12
13
14
15
16
17
18
19
20
Figure 4. In vitro PDT efficacy and mechanistic study.
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
PDT cytotoxicity of H4TBP, TBP-Hf, H4TBC, TBC-Hf, and
PpIX at different PS concentrations in CT26 (a) and MC38
cells (b). (c) Annexin V/PI analysis of MC38 cells incubat-
ed with TBC-Hf or PBS with or without irradiation (90
J/cm2). The quadrants from lower left to upper left (coun-
ter clockwise) represent healthy, early apoptotic, late
apoptotic, and necrotic cells, respectively. The percentage
of cells in each quadrant is shown on each graph. (+) and
(-) refer to with and without irradiation, respectively. (d)
CRT exposure on the cell surface of MC38 was assessed
after incubation with PBS and TBC-Hf with or without
light irradiation (90 J/cm2) by flow cytometry analysis. (e)
The fluorescence intensity was gated on PI-negative cells.
Immunofluorescence microscopy of CRT expression on
the cell surface of MC38 treated with PBS and TBC-Hf
upon irradiation (90 J/cm2).
2
1
0
a)
b)
PBS
PBS
H TBC
4
H TBC+IDOi
4
1.0
0.5
0.0
H
H
TBC
4
TBC+IDOi
4
TBC-Hf
TBC-Hf
IDOi@TBC-Hf
IDOi@TBC-Hf (-)
IDOi@TBC-Hf
IDOi@TBC-Hf (-)
The Abscopal Effect of IDOi@TBC-Hf
Treated
Untreated
The immunoregulatory enzyme IDO is often overex-
pressed in the tumor microenvironment, where it facili-
tates the survival and growth of tumor cells by suppress-
ing antitumor immune responses.15 The small-molecule
IDO inhibitor (IDOi) INCB24360, developed by Incyte,
can reverse immunosuppression and inhibits tumor
growth upon oral administration.16 We propose that by
encapsulation of IDOi into the nMOF channels,
IDOi@TBC-Hf can release IDOi both into the local tumor
environment and enter blood circulation for systemic
IDO inhibition. The effects of IDOi will alter the suppres-
sive tumor microenvironment of both the treated and
untreated tumors. We hypothesize that synergizing local
PDT of IDOi@TBC-Hf with checkpoint blockade therapy
can promote an efficient abscopal effect, regression of an
untreated tumor at a distant site following the local
treatment of another tumor in the same organism.
0
4
8
0
4
8
Day post treatment
Day post treatment
c)
d)
PBS
PBS
TBC-Hf
IDOi@TBC-Hf
IDOi@TBC-Hf (-)
TBC-Hf
IDOi@TBC-Hf
IDOi@TBC-Hf (-)
0.6
0.4
0.2
0.0
2
1
0
Untreated
Treated
0
4
8
0
4
Day post treatment
8
Day post treatment
Figure 5. In vivo anticancer efficacy showing abscopal
effect. Growth curves for treated (a and c) and untreated
(b and d) tumors of CT26 (a and b) or MC38 (c and d)
tumor-bearing mice after PDT treatment. Black and red
arrows refer to the time of injection and irradiation, re-
spectively.
We evaluated the abscopal effect of IDOi@TBC-Hf and
light irradiation in two immunocompetent mouse models
Antitumor Immunity
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 10
We have shown that PDT of IDOi@TBC-Hf caused ef- We further determined the tumor-infiltrating leukocyte
1
2
3
4
5
6
7
8
fective tumor regression of both primary, treated tumors
and distant, untreated tumors in two syngeneic mouse
models of colorectal cancer. We further investigated the
underlying immunological mechanisms in the MC38
model by Enzyme-Linked ImmunoSpot (ELISPOT) and
flow cytometry. We first performed an ELISPOT assay to
detect the presence of tumor antigen-specific T cells 14
days after treatment. As shown in Figure 6a, the number
of antigen-specific IFN-γ producing T cells increased to
799 per million splenocytes in mice treated with
IDOi@TBC-Hf (vs. 128 per million splenocytes in PBS
group, P=0.0879), indicating that PDT of IDOi@TBC-Hf
induced in situ tumor vaccination to effectively generate
tumor-specific T cell response.
profiles 12 days post PDT treatment. As shown in Figure 6
d-f, IDOi@TBC-Hf significantly increased the proportion
of infiltrating CD8+ T cells relative to the total number of
cells in the distant tumor (P=0.0012 vs. PBS), an essential
step to mounting an antitumor immune response to in-
duce the abscopal effect. In both primary and distant tu-
mors, the percentages of infiltrating CD45+ leukocytes
and CD4+ T cells with respect to the total number of cells
in the tumors were significantly increased in mice treated
with PDT of IDOi@TBC-Hf (Treated tumor, CD45+:
P=0.0061 vs. PBS; CD4+ T cells: P=0.0206 vs. PBS. Un-
treated tumor, CD45+: P=0.0001 vs. PBS; CD4+ T cells:
P=0.0388 vs. PBS). Furthermore, the percentage of tumor-
infiltrating NK cells significantly increased in the distant
tumors of mice treated with IDOi@TBP-Hf and PDT
(P=0.0034 vs. PBS, Figure S26, SI). Interestingly,
IDOi@TBP-Hf administration and PDT also led to a sig-
nificant increase in the percentages of tumor-infiltrating
B cells and a decrease in the percentages of tumor infil-
trating dendritic cells with respect to the total number of
cells in the primary tumors 12 days post the treatment (B
cells, P=0.0017 vs. PBS; DCs: P=0.0041 vs. PBS, Figure S27,
SI).
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
Further analysis of the immune response was carried
out by investigating the tumor-infiltrating leukocyte pro-
files in each tumor by flow cytometry. Previous research
has shown that PDT can evoke acute inflammation,5 so
we first evaluated the population of leukocytes responsi-
ble for innate immune response (neutrophils, B cells,
dendritic cells, and macrophages) in both primary and
distant tumors 12 hours after PDT treatment. As shown in
Figure 6 b-c and Figure S23 (SI), IDOi@TBC-Hf admin-
istration and light irradiation led to significant increase in
the percentage of tumor-infiltrating neutrophils and B
cells with respect to the total number of cells in the tumor
compared to PBS (neutrophils: P=0.0369 vs. PBS; B cells:
P=0.0215 vs. PBS). To our surprise, the percentage of infil-
trating dendritic cells appeared to decrease compared to
PBS control in the primary tumor (Figure S23, SI), alt-
hough the difference is not of statistical significance.
We also performed T cell blocking experiments to con-
firm the involvement of T cells in the efficient abscopal
response on bilateral subcutaneous MC38 mouse model.
MC38 tumor-bearing mice were treated with IDOi@TBC-
Hf and light irradiation as described before and received
i.p. injection of Mouse IgG, anti-CD4, or anti-CD8 anti-
body at a dose of 200 µg/mouse/injection on Day 0 and
Day 5 post the PDT treatment. While mice treated with
Mouse IgG showed regression of both primary and distant
tumors, no abscopal effect was observed on mice with
blocking of CD4+ or CD8+ T cells (Figure 6 g-h). Further-
more, blocking of CD4+ and CD8+ T cells also diminished
the effect of IDOi@TBC-Hf in regressing the primary tu-
mors. These results indicated that CD4+ and CD8+ T cells
were essential not only to abscopal effect but also to pri-
mary tumor rejection.
To better understand the roles these types of cells
played in the antigen presentation after PDT, we further
detected the major histocompatibility complex class II
(MHC-II) expression on these cells 12 hours after PDT
treatment. MHC-II mediates establishment of specific
immunity by interacting with CD4 molecules on the sur-
face of helper T cells.17 We found a significant decrease in
MHC-II expression levels on dendritic cells, macrophages,
and neutrophils in the primary treated tumors (DC:
P<0.0001 vs. PBS; macrophages: P=0.0004 vs. PBS; neu-
trophils: P=0.006 vs. PBS, Figure S24, SI). These results
imply that TBC-Hf mediated PDT may have an impact on
the method of antigen presentation, which is typically the
role of dendritic cells.
We profiled the tumor-infiltrating leukocytes on 7 days
post PDT treatment. The mice treated with IDOi@TBC-
Hf plus PDT did not show significant difference in all cell
types except B cells from the control mice (Figure S25, SI).
The percentage of B cells significantly increased with re-
spect to the total number of cells in the primary tumors of
IDOi@TBC-Hf and PDT treated mice (P=0.0018, Figure
S25 c, SI), while the percentage of dendritic cells signifi-
cantly decreased in the distant tumors of treated mice
(P=0.0083, Figure S25 f, SI). We also found that the per-
centage of NK cells was extremely low on day 7 post PDT
treatment both in IDOi@TBC-Hf treated mice and PBS
treated mice (both percentage <0.05%, Figure S25 e, SI).
ACS Paragon Plus Environment

Page 7 of 10
Journal of the American Chemical Society
response rate.6b Here we report a new treatment strategy
a)
b)
1
2
3
4
5
6
7
8
9
that combines local PDT of a new nMOF, TBC-Hf, in
combination with IDO inhibition with nMOF-delivered
small molecules to achieve effective and consistent ab-
scopal responses in mouse models of colorectal cancers.
We have shown that IDOi released from locally injected
IDOi@TBC-Hf reversed the suppressive tumor microenvi-
ronment in both treated and untreated tumors, which
further synergized with TBC-Hf mediated PDT to stimu-
late the immune system for activating both acute innate
and prolonged adaptive immune response to achieve effi-
cient local tumor regression and a consistent abscopal
response. Our treatment method maximizes the benefits
of local treatment with systemic immune response for the
rejection of both primary and distant tumors while mini-
mizing side effects, potentially affording an effective sys-
temic therapy for metastatic colorectal cancers.
c)
e)
g)
d)
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
f)
Owing to the outstanding photophysical properties of
the chlorin-based TBC ligand, TBC-Hf shows potent PDT
efficacy and outperforms its porphyrin counterpart TBP-
Hf. The tetracarboxylate ligand constructs a robust
framework with very large channels for small molecular
inhibitor loading and ROS diffusion, and the coordination
to Hf6 SBUs enhances intersystem crossing of TBC to en-
h)
0.8
0.4
0.0
PBS (-)
Mouse IgG (+)
PBS (-)
Mouse IgG (+)
2
1
0
α
α
-CD4 (+)
-CD8 (+)
α
α
-CD4 (+)
-CD8 (+)
1
Untreated
hance O2 generation. As we19 and others20 have demon-
Treated
strated the synthetic tunability and potential biomedical
applications of nMOFs in the past decade, we believe that
the nMOF compositions and structures can be further
0
4
8
0
4
8
1
optimized in order to enhance light absorption and O2
Day post treatment
Day post treatment
generation/diffusion. We thus believe that nMOF-based
synergistic PDT and immunotherapy have the potential
for future clinical translation for the treatment of meta-
static cancers.
Figure 6. Antitumor immunity of PDT by IDOi@TBC-Hf.
(a) Antigen-specific IFN-γ spot forming cells (SFC) 14 days
after IDOi@TBC-Hf PDT treatment. The percentage of
tumor-infiltrating neutrophils (b) and B cells (c) with
respect to the total number of cells in the tumor com-
pared to PBS, 12h after IDOi@TBC-Hf PDT treatment.
The percentage of tumor-infiltrating CD8+ T cells (d),
CD4+ T cells (e) and CD45+ cells (f) with respect to the
total number of cells in the tumors 12 days after
IDOi@TBC-Hf PDT treatment. Growth curves for prima-
ry, treated (g) and distant, untreated (h) tumors after
PDT treatment (+) with IDOi@TBC-Hf and injections of
Mouse IgG, anti-CD4, or anti-CD8 antibody, compared to
PBS control without PDT treatment (-). Black, red and
blue arrows refer to the time of IDOi@TBC-Hf injection,
light irradiation and antibody injection, respectively.
We propose that IDOi@TBC-Hf administration and
light irradiation causes highly efficient tumor regression
of both primary treated tumors and distant untreated
tumors owing to two factors. First, TBC-Hf based PDT
causes ICD of cancer cells in the primary tumors, which
activates innate immune system and promotes antigen
presentation (Figure 1). The massive stressed and dying
necrotic tumor cells in the PDT-treated primary tumor
sites are engulfed by the innate immune effector cells
followed by presenting tumor-derived antigenic peptides
to T cells, thus stimulating a tumor-specific T cell re-
sponse. Second, the IDOi is released from intratumorally
injected IDOi@TBC-Hf to systemically inhibit IDO activi-
ty to reverse the immunosuppressive tumor environ-
ments. Alternatively, the recruited immune cells likely
undergo IDO inhibition in the treated tumors and mi-
grate to the distant tumors to cause an abscopal effect.
The two treatment modalities, PDT and IDOi checkpoint
blockade therapy, synergize with each other to kill cancer
cells locally and create an immunogenic tumor microen-
vironment systemically, leading to strong and consistent
abscopal effects.
Discussion
Approximately 140,000 patients are diagnosed with col-
orectal cancer in the US annually, with one-third dying
from metastasis.18 Stimulation of the host immune system
has been shown to generate an antitumor immunity ca-
pable of controlling metastatic tumor growth, and thus
represents a promising treatment strategy for metastatic
colon cancer.8 Clinical results suggest that immunothera-
pies have the potential to achieve systemic and adaptable
cancer control. However, combination with other treat-
ment modalities is necessary to maximize the benefits of
immunotherapy and increase the tumor-specific immune
Dendritic cells are one of the most important antigen-
presenting cells and prevalently believed to play a key role
in antitumor immune response.21 However, we observed a
decrease of dendritic cell population percentage at differ-
ACS Paragon Plus Environment

Journal of the American Chemical Society
ent time points along with a significant decrease of MHC-
Page 8 of 10
1
2
3
4
5
6
7
8
II expression level on dendritic cells after PDT treatment
with IDOi@TBC-Hf, indicating an altered role for den-
dritic cells after PDT treatment. Meanwhile, the increase
of both neutrophils and B cell population percentages
implies a compensating effect of other antigen presenting
cells to present tumor-associated antigens and initiate the
antitumor immune response. Previous studies also sug-
gested that neutrophils, rather than dendritic cells, di-
rectly affect T cell proliferation upon PDT treatment.22
We also observed significant increase in the percentage of
tumor infiltrating NK cells from 0.02% on day 7 to 3.83%
on day 12 post PDT treatment in distant tumors, suggest-
ing the important role of tumor-infiltrating NK cells in
tumor rejection in addition to those played by CD8+ T
cells, CD4+ T cells, and B cells after IDOi@TBC-Hf and
PDT treatment. Our future efforts will be directed toward
elucidating the roles of neutrophils and B cells in initiat-
ing antitumor immune response after the PDT of
IDOi@TBC-Hf.
Synthesis of TBC-Hf
To a 2-dram glass vial was added 1 mL of HfCl4 solution
[2 mg/mL in N,N-dimethylformamide (DMF), 6.2 µmol], 1
mL of the H4TBC solution (1.9 mg/mL in DMF, 2.4 µmol),
and 60 µL of 88% formic acid (1.4 mmol). The reaction
mixture was kept in an 80 °C oven for 2 days. The purple
powder was collected by centrifugation and washed with
DMF, 1% triethylamine in ethanol (v/v) and ethanol.
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
Singlet Oxygen Generation
A light-emitting diode (LED) array with peak emission
at 650 nm was used as the light source of singlet oxygen
generation test. The irradiance of LED is 20 mW/cm2.
Singlet oxygen sensor green (SOSG) reagent (Life Tech-
nologies) was employed for the detection of singlet oxy-
gen. H4TBP, H4TBC, TBP-Hf and TBC-Hf samples were
prepared in 1 µM solutions/suspensions in water (for the
nMOFs, the concentration was calculated as ligand equiv-
alents). To 2 mL each of these solutions/suspensions,
SOSG stock solution (5 µL at 5 mM) was added (final con-
centration=12.5 µM) before fluorescence measurement.
For a typical measurement, fluorescence intensity was
acquired on a spectrofluorophotometer (RF-5301PC, Shi-
madzu, Japan) with excitation at 504 nm and emission at
525 nm (slit width 1.5 nm/3 nm for ex/em). Fluorescence
was measured after irradiation by LED for 0 (as back-
ground), 0.5, 1, 1.5, 2, 3, 4, 5, 7 and 10 min.
Conclusion
In this work, we have rationally designed a chlorin-
based nMOF with large channels for highly efficient PDT,
while simultaneously loaded an IDO inhibitor into its
channels to achieve combination therapies of PDT and
checkpoint blockade immunotherapy. We consistently
observed an abscopal effect in mice receiving treatment
with PDT of IDOi@TBC-Hf. The in situ vaccination in-
duced by PDT treatment and IDOi immunotherapy syn-
ergize with each other and effectively generate systemic
antitumor immunity. We believe the present strategy has
the potential to significantly increase the systemic tumor-
specific immune response rates of checkpoint blockade
cancer immunotherapy and lead to clinical benefits for
the treatment of metastatic colorectal cancers and other
difficult-to-treat cancers.
In vitro PDT efficacy
The cytotoxicity of TBC-Hf and TBC was evaluated in
murine colorectal cancer cell CT26 and murine melanoma
cell B16F10, respectively. CT26 cells or B16F10 cells were
seeded on 96-well plates at 1000 cells/well. The cells were
treated with TBC-Hf and H4TBC at various ligand concen-
trations (2, 4, 10, 15, 20, and 50 µM base on ligand concen-
trations). A further incubation of 4 h was allowed fol-
lowed by replacing the culture medium with 100 µL of
fresh medium. The cells were irradiated with LED light
(650 nm) at 100 mW/cm2 for 15 min (total light dose 90
J/cm2) or kept in dark, respectively. The cells were further
incubated to achieve a total incubation time of 72 h. The
cell viability was detected by (3-(4,5-dimethylthiazol-2-
yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium) (MTS) assay (Promega, USA).
Materials and Methods
Materials, cell lines, and animals
All of the starting materials were purchased from Sig-
ma-Aldrich and Fisher (USA), unless otherwise noted,
and used without further purification. INCB24360
analogue,
4-amino-N-(3-chloro-4-fluoro-phenyl)-N’-
hydroxy-1,2,5-oxadiazole-3-carboximidamide
purchased from Medkoo Biosciences, USA.
was
Murine colon adenocarcinoma cells CT26 and MC38
and murine melanoma cells B16F10 were purchased from
the American Type Culture Collection (Rockville, MD,
USA) and cultured in Dulbecco's Modified Eagle's
Medium (DMEM) medium (Gibco, Grand Island, NY,
USA) supplemented with 10% FBS.
Abscopal Effect
The PDT efficacy of IDOi@TBC-Hf was investigated us-
ing a bilateral CT26 mouse colorectal cancer model and a
bilateral MC38 mouse colorectal cancer model. Tumor
bearing mice were established by subcutaneous inocula-
tion of CT26 or MC38 cell suspension (1×106 cells per
mouse on the right flank and 2×105 cells per mouse on the
left flank) into 6-week female BALB/c or 6-week female
C57BL/6 mice, respectively. When the right tumors
reached 100 mm3, the CT26 tumor bearing mice received
intratumoral injection only into the right tumors of PBS,
C57BL/6 female mice (6 weeks, 20-22 g) and BALB/c
female mice (6 weeks, 20-22 g) were provided by Harlan
Laboratories, Inc (USA). The study protocol was reviewed
and approved by the Institutional Animal Care and Use
Committee (IACUC) at the University of Chicago.
ACS Paragon Plus Environment

Page 9 of 10
Journal of the American Chemical Society
48 h at 37 °C and then discarded. The plate was then in-
H4TBC, H4TBC+IDOi, TBC-Hf, or IDOi@TBC-Hf at a lig-
1
2
3
4
5
6
7
8
and dose of 20 µmol/kg and IDOi dose of 1.5 mg/kg.
Twelve hours post injection, CT26 tumor bearing mice
were anesthetized with 2% (v/v) isoflurane and right flank
tumors were irradiated with a 650 nm LED at 0.1 W/cm2
for 15 min (90 J/cm2). IDOi@TBC-Hf without light irradia-
tion served as a dark control. The nMOF injection and
light irradiations were performed every three days for a
total two injections and irradiations. For MC38 tumor
model, mice received intratumoral injection only into the
right tumors of PBS, TBC-Hf, or IDOi@TBC-Hf at a ligand
dose of 20 µmol/kg and IDOi dose of 1.5 mg/kg. Twelve
hours post injection, MC38 tumor bearing mice were
anesthetized with 2% (v/v) isoflurane and right flank tu-
mors were irradiated with a 650 nm LED at 0.1 W/cm2 for
15 min (90 J/cm2). IDOi@TBC-Hf without light irradiation
served as a dark control. The nMOF injection and light
irradiations were performed only once on MC38 tumor
bearing mice. For both CT26 and MC38 models, no nMOF
injection or light irradiation was performed on the left
tumors.
cubated with biotin-conjugated anti-IFN-γ detection an-
tibody at r.t. for 2 h, followed by incubation with Avidin-
HRP for 2 h at r.t. AEC substrate solution (Sigma, Cat.
AEC101) was added for cytokine spot detection.
Flow Cytometry
Tumors were harvested, treated with 1 mg/mL colla-
genase I (Gibco™, USA) for 1 h, and ground by the rubber
end of a syringe. Cells were filtered through nylon mesh
filters and washed with PBS. The single-cell suspension
was incubated with anti-CD16/32 (clone 93; eBiosciences)
to reduce nonspecific binding to FcRs. Cells were further
stained with the following fluorochrome-conjugated anti-
bodies: CD45 (30-F11), CD3e (145-2C11), CD4 (GK1.5), CD8
(53-6.7), Foxp3 (FJK-16s), CD11b (M1/70), Ly6C (HK1.4),
Ly6G (RB6-8C5), F4/80 (BM8), B220 (RA3-6B2), NKp46
(29A1.4) and PI staining solution (all from eBioscience).
LSR FORTESSA (BD Biosciences) was used for cell acqui-
sition, and data analysis was carried out using FlowJo
software (TreeStar, Ashland, OR).
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
To evaluate the therapeutic efficacy, the tumor size was
measured with a digital caliper every day. Tumor volumes
were calculated as follows: (width2 × length)/2. Finally, all
mice were sacrificed when the tumor size of control group
exceeded 2 cm3, and the excised tumors were photo-
graphed and weighed.
MHC-II expression
Tumors were harvested 12 hours after LED irradiation,
treated with 1 mg/mL collagenase I, and ground by the
rubber end of a syringe. Cells were filtered through nylon
mesh filters and washed with PBS. The single cell suspen-
sion was incubated with anti-CD16/32 (clone 93; eBiosci-
ences) for 10 minutes and then stained with CD45, CD3e,
CD11b, Ly6G, B220, F4/80, CD11c and MHC-II. The expres-
sion levels of MHC-II on CD11b+Ly-6G+, CD3e-B220+,
CD3e-CD11c+ and CD3e-CD11c-F4/80 populations were
determined respectively.
T cell blocking
The abscopal effect of IDOi@TBC-Hf was evaluated on
bilateral subcutaneous MC38 model on C57BL/6 mice
with CD4+ T cell or CD8+ T cell depletion. When the right
tumors reached ~100 mm3, IDOi@TBC-Hf was intra-
tumorally injected to the mice at a dose of 20 µmol/kg.
Anti-CD4 (for CD4+ T cell depletion), anti-CD8 (for CD8+
T cell depletion), or mouse IgG (control) were intraperi-
toneally injected to the mice (200 µg/mouse/injection) on
Day 0 and Day 5 post the first treatment. Twelve hours
post injection, mice were anesthetized with 2% (v/v)
isoflurane and the right tumors were irradiated with LED
light irradiation (100 mW/cm2, 90 J/cm2, 650 nm). Single
IDOi@TBC-Hf injections followed by single light irradia-
tions were carried out. To evaluate the therapeutic effica-
cy, the tumor size was measured with a digital caliper
every other day. Tumor volumes were calculated as fol-
lows: (width2 × length)/2.
ASSOCIATED CONTENT
Supporting Information. Experimental details and sup-
porting results for the synthesis and characterization of
H4TBC, TBC-Hf, IDOi@TBC-Hf, cellular uptake, in vitro
PDT efficacy, abscopal effect, and antitumor immunity. This
material is available free of charge via the Internet at
http://pubs.acs.org.”
AUTHOR INFORMATION
Corresponding Author
*wenbinlin@uchicago.edu
ELISPOT
Author Contributions
†These authors contributed equally.
Tumor-specific immune responses to IFN-γ was meas-
ured in vitro by ELISPOT assay (Mouse IFN gamma
ELISPOT Ready-SET-Go!®; Cat. No. 88-7384-88; eBiosci-
ence). A Millipore Multiscreen HTS-IP plate was coated
overnight at 4°C with anti-Mouse IFN-γ capture antibody.
Single-cell suspensions of splenocytes were obtained from
MC38 tumor-carrying mice and seeded onto the anti-
body-coated plate at a concentration of 2×105 cells/well.
Cells were incubated with or without KSPWFTTL (KSP)
stimulation (10 μg/mL; in purity > 95%; PEPTIDE 2.0) for
ACKNOWLEDGMENT
We thank Mr. Christopher Poon and Mr. Zekai Lin for exper-
imental help. We acknowledge the National Cancer Institute
(U01–CA198989), the University of Chicago Medicine Com-
prehensive Cancer Center (NIH CCSG: P30 CA014599), the
Cancer Research Foundation, and the Ludwig Institute for
Metastasis Research for funding support.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 10
6. (a) Couzin-Frankel, J. Science 2013, 342 (6165), 1432-1433; (b)
REFERENCES
Pardoll, D. M. Nature Reviews Cancer 2012, 12 (4), 252-264.
7. Sharma, P.; Allison, J. P. Science 2015, 348 (6230), 56-61.
8. Zou, W. Nature Reviews Cancer 2005, 5 (4), 263-274.
9. (a) Gilboa, E. Nature Reviews Cancer 2004, 4 (5), 401-411; (b) Munn,
D. H.; Shafizadeh, E.; Attwood, J. T.; Bondarev, I.; Pashine, A.; Mellor,
A. L. The Journal of experimental medicine 1999, 189 (9), 1363-1372.
10. Munn, D. H.; Zhou, M.; Attwood, J. T.; Bondarev, I.; Conway, S. J.;
Marshall, B.; Brown, C.; Mellor, A. L. Science 1998, 281 (5380), 1191-
1193.
1
2
3
4
5
6
7
8
1. (a) Hamblin, M. R.; Hasan, T. Photoch Photobio Sci 2004, 3 (5), 436-
450; (b) Celli, J. P.; Spring, B. Q.; Rizvi, I.; Evans, C. L.; Samkoe, K. S.;
Verma, S.; Pogue, B. W.; Hasan, T. Chem. Rev. 2010, 110 (5), 2795-2838;
(c) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Nature Reviews
Cancer 2003, 3 (5), 380-387; (d) Ethirajan, M.; Chen, Y.; Joshi, P.; Pan-
dey, R. K. Chem Soc Rev 2011, 40 (1), 340-362.
2. Stolik, S.; Delgado, J.; Perez, A.; Anasagasti, L. Journal of Photochem-
istry and Photobiology B: Biology 2000, 57 (2), 90-93.
11. Yue, E. W.; Douty, B.; Wayland, B.; Bower, M.; Liu, X.; Leffet, L.;
Wang, Q.; Bowman, K. J.; Hansbury, M. J.; Liu, C. J. Med. Chem. 2009,
52 (23), 7364-7367.
9
3. (a) Ng, K. K.; Zheng, G. Chem. Rev. 2015, 115 (19), 11012-11042; (b)
Lovell, J. F.; Jin, C. S.; Huynh, E.; Jin, H.; Kim, C.; Rubinstein, J. L.;
Chan, W. C.; Cao, W.; Wang, L. V.; Zheng, G. Nature materials 2011, 10
(4), 324-332; (c) Huynh, E.; Leung, B. Y.; Helfield, B. L.; Shakiba, M.;
Gandier, J.-A.; Jin, C. S.; Master, E. R.; Wilson, B. C.; Goertz, D. E.;
Zheng, G. Nat Nanotechnol 2015, 10 (4), 325-332; (d) Carter, K. A.;
Shao, S.; Hoopes, M. I.; Luo, D.; Ahsan, B.; Grigoryants, V. M.; Song,
W.; Huang, H.; Zhang, G.; Pandey, R. K.; Geng, J.; Pfeifer, B. A.;
Scholes, C. P.; Ortega, J.; Karttunen, M.; Lovell, J. F. Nature communica-
tions 2014, 5, 3546; (e) Kim, J.; Piao, Y.; Hyeon, T. Chem Soc Rev 2009,
38 (2), 372-390; (f) Wang, W.; Wang, L.; Li, Z.; Xie, Z. Chem Commun
2016, 52 (31), 5402-5405; (g) Padilla, R.; Maza, W. A.; Dominijanni, A.
J.; Winkel, B. S.; Morris, A. J.; Brewer, K. J. Journal of Photochemistry
and Photobiology A: Chemistry 2016, 322, 67-75; (h) Han, K.; Wang, S.-
B.; Lei, Q.; Zhu, J.-Y.; Zhang, X.-Z. ACS nano 2015, 9 (10), 10268-
10277; (i) Chen, J.-X.; Wang, H.-Y.; Li, C.; Han, K.; Zhang, X.-Z.; Zhuo,
R.-X. Biomaterials 2011, 32 (6), 1678-1684; (j) Wang, C.; Tao, H.;
Cheng, L.; Liu, Z. Biomaterials 2011, 32 (26), 6145-6154; (k) Idris, N.
M.; Gnanasammandhan, M. K.; Zhang, J.; Ho, P. C.; Mahendran, R.;
Zhang, Y. Nat. Med. 2012, 18 (10), 1580-1585; (l) Huang, P.; Lin, J.;
Wang, S.; Zhou, Z.; Li, Z.; Wang, Z.; Zhang, C.; Yue, X.; Niu, G.; Yang,
M. Biomaterials 2013, 34 (19), 4643-4654; (m) Park, Y. I.; Kim, H. M.;
Kim, J. H.; Moon, K. C.; Yoo, B.; Lee, K. T.; Lee, N.; Choi, Y.; Park, W.;
Ling, D. Adv Mater 2012, 24 (42), 5755-5761; (n) Levine, D. H.;
Ghoroghchian, P. P.; Freudenberg, J.; Zhang, G.; Therien, M. J.; Greene,
M. I.; Hammer, D. A.; Murali, R. Methods 2008, 46 (1), 25-32; (o) Cheng,
Y.; C. Samia, A.; Meyers, J. D.; Panagopoulos, I.; Fei, B.; Burda, C. J.
Am. Chem. Soc. 2008, 130 (32), 10643-10647; (p) Rieffel, J.; Chen, F.;
Kim, J.; Chen, G.; Shao, W.; Shao, S.; Chitgupi, U.; Hernandez, R.;
Graves, S. A.; Nickles, R. J. Adv Mater 2015, 27 (10), 1785-1790; (q)
Luo, D.; Carter, K. A.; Miranda, D.; Lovell, J. F. Adv. Sci. 2016, DOI:
10.1002/advs.201600106.
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
12. Khleif, S.; Munn, D.; Nyak-Kapoor, A.; Mautino, M. R.; Kennedy, E.;
Vahanian, N. N.; Link, C. J ASCO Annual Meeting Proceedings, 2014; p
TPS3121.
13. Morris, W.; Volosskiy, B.; Demir, S.; Gándara, F.; McGrier, P. L.;
Furukawa, H.; Cascio, D.; Stoddart, J. F.; Yaghi, O. M. Inorg Chem 2012,
51 (12), 6443-6445.
14. (a) Park, J.; Jiang, Q.; Feng, D.; Mao, L.; Zhou, H.-C., J. Am. Chem.
Soc. 2016, 138 (10), 3518-3525; (b) Liu, J.; Yang, Y.; Zhu, W.; Yi, X.;
Dong, Z.; Xu, X.; Chen, M.; Yang, K.; Lu, G.; Jiang, L., Biomaterials
2016, 97, 1-9.
15. Uyttenhove, C.; Pilotte, L.; Théate, I.; Stroobant, V.; Colau, D.; Par-
mentier, N.; Boon, T.; Van den Eynde, B. J. Nat. Med. 2003, 9 (10), 1269-
1274.
16. Newton, R. C.; Scherle, P. A.; Bowman, K.; Liu, X.; Beatty, G. L.;
O'Dwyer, P. J.; Gajewski, T.; Bowman, J.; Schaub, R.; Leopold, L ASCO
Annual Meeting Proceedings, 2012; p 2500.
17. Janeway, C. A.; Travers, P.; Walport, M.; Capra, J. D., Immunobiolo-
gy: the immune system in health and disease. 2005.
18. Siegel, R.; DeSantis, C.; Jemal, A. CA Cancer J. Clin. 2014, 64 (2),
104-117.
19. (a) Huxford, R. C.; Della Rocca, J.; Lin, W. Curr. Opin. Chem. Biol.
2010, 14 (2), 262-268; (b) Lin, W.; Rieter, W. J.; Taylor, K. M.
Angewandte Chemie International Edition 2009, 48 (4), 650-658; (c)
Della Rocca, J.; Liu, D.; Lin, W. Accounts Chem Res 2011, 44 (10), 957-
968; (d) He, C.; Liu, D.; Lin, W. Chem. Rev. 2015, 115 (19), 11079-
11108.
20. (a) Cui, Y.; Yue, Y.; Qian, G.; Chen, B. Chem. Rev. 2011, 112 (2),
1126-1162; (b) Furukawa, H.; Cordova, K. E.; O’Keeffe, M.; Yaghi, O.
M. Science 2013, 341 (6149), 1230444; (c) Horcajada, P.; Gref, R.; Baati,
T.; Allan, P. K.; Maurin, G.; Couvreur, P.; Ferey, G.; Morris, R. E.; Serre,
C. Chem. Rev. 2011, 112 (2), 1232-1268.
4. (a) Lu, K.; He, C.; Lin, W. J. Am. Chem. Soc. 2014, 136 (48), 16712-
16715; (b) Lu, K.; He, C.; Lin, W. J. Am. Chem. Soc. 2015, 137 (24),
7600-7603.
5. Castano, A. P.; Mroz, P.; Hamblin, M. R. Nature Reviews Cancer 2006,
6 (7), 535-545.
21. Palucka, K.; Banchereau, J. Nature Reviews Cancer 2012, 12 (4), 265-
277.
22. Kousis, P. C.; Henderson, B. W.; Maier, P. G.; Gollnick, S. O. Cancer
Res. 2007, 67 (21), 10501-10510.
ACS Paragon Plus Environment