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Immunological Responses Triggered by Photothermal
Therapy with Carbon Nanotubes in Combination with
Anti-CTLA-4 Therapy to Inhibit Cancer Metastasis
Chao Wang, Ligeng Xu, Chao Liang, Jian Xiang, Rui Peng, and Zhuang Liu*
Despite tremendous effects devoted into cancer research,
cancer remains one of the major causes of death. The main
reason for such a high mortality from cancer is due to the
highly invasive behavior of cancer cells that cause metas-
tases.[1] Despite improvements in diagnosis and surgical tech-
niques, most deaths from cancer are due to metastasis that is
resistant to conventional therapies. Therefore, the next gener-
ation of cancer therapy should not only be able to remove or
destroy the primary tumor, but also to recognize, track down,
and destroy any remaining tumor cells at site of distant metas-
tases. Particularly, immunotherapy, in which the patient’s own
immune system is trained to recognize and destroy tumor cells,
has shown great promise in treating patients with metastatic
tumors.[2]
Photothermal therapy (PTT) to treat tumors by light-
induced-heating with the assistant of light-absorbing photo-
thermal agents has been wildly studied in recent years.[3] In
the past decade, a large variety of nano-agents including many
inorganic and organic nanomaterials, all with strong absorb-
ance in the near-infrared (NIR) tissue transparency window,
have been extensively studied as photothermal agents by our
and other groups.[4] Among them, carbon nanotubes (CNTs),
including both single-walled nanotubes (SWNTs) and multi-
walled nanotubes (MWNTs), have been intensively explored for
various biomedical applications including photothermal cancer
treatment.[5] While being rather effective in killing tumors by
photothermal ablation with lasers,[6] CNTs with appropriate
surface coatings have been found to be not obviously toxic to
animals and could be gradually excreted from mice over time.[7]
The interactions of CNTs with the immune system, on the
other hand, have also been studied in recent years.[8] Although
the results may vary under different experimental conditions, [9]
it has been reported that CNTs were able to activate both innate
and adaptive immune responses as potential adjuvants.[10] Par-
ticularly, Xu and co-workers reported that oxidized MWNTs by
themselves could induce anti-tumor effect on Babl/c mice upon
C. Wang, Dr. L. Xu, C. Liang, J. Xiang,
Prof. R. Peng, Prof. Z. Liu
Institute of Functional Nano &
Soft Materials (FUNSOM)
Collaborative Innovation Center of Suzhou
Nano Science and Technology
Soochow University
Suzhou, Jiangsu 215123, China
E-mail: zliu@suda.edu.cn
DOI: 10.1002/adma.201402996
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subcutaneous injection, promising the use of CNTs for cancer
immunotherapy.[11] Despite the above encouraging results,
whether and how CNT-based photothermal therapy would
trigger any immulogical response and play any effect in inhib-
iting tumor metastasis, remain largely unknown.
Herein, we report that photothermal ablation of primary
tumors with single-walled carbon nanotubes (SWNTs) in com-
bination with anti-CTLA-4 antibody therapy is able to prevent
the development of tumor metastasis in mice. It is found
that polymer-coated SWNTs could not only be used for photo-
thermal tumor destruction, which releases tumor-associated
antigens, but also act as an immunological adjuvant to greatly
promote maturation of dendritic cells (DCs) and production of
anti-tumor cytokines, a unique behavior among several types of
PTT agents. Furthermore, CTLA-4 blockade applied in SWNT-
based photothermal ablation of primary tumors would induce
infiltration of effective T cells while greatly abrogate regula-
tory T cells at distant tumors. As the results of such combined
immunological effects, SWNT-based PTT in combination with
anti–CTLA-4 therapy is able to inhibit the growth of remaining
cancer cells in both a subcutaneous tumor model and a lung
metastasis model. Our results promise the use of adjuvant
nanomaterial-based PTT in combination with immunotherapy
to treat cancer metastasis, a major cause of cancer death.
In our work, SWNTs were modified by a non-covalent
approach with a polyethylene glycol (PEG)-grafted amphiphilic
polymer following our well established protocol (Figure 1A).[12]
The obtained PEGylated SWNTs were highly stable in var-
ious physiological solutions including PBS, cell medium and
serum. Atomic force microscopy (AFM) images revealed
that those PEGylated SWNTs showed lengths in the range
of 100–200 nm (Figure 1B). With a strong absorbance in the
NIR region (Figure S1A, Supporting Information), PEGylated
SWNTs could be rapidly heated up under exposure to an 808-nm
NIR laser (Figure S1C–D, Supporting Information), enabling
their use as an effective photothermal agent. PEGylated SWNTs
showed strong resonance Raman scattering (Figure S1B, Sup-
porting Information), indicating that the pristine structure of
SWNTs was largely retained after polymer coating.
As professional antigen presenting cells (APCs), dendritic
cells play pivotal roles in the initiation and regulation of innate
and adaptive immunities. The expression of co-stimulatory
molecules (CD80, CD86), which are the hallmarks of DCs
maturation, is a critical factor that determines the quality
of the adaptive immunities.[13] Therefore, to understand
how our PEGylated SWNTs interact with the immunological
system, we firstly studied the effects of PEGylated SWNTs on
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Figure 1. PEGylated SWNTs interact with the immunological system. A) A schematic drawing of our PEGylated SWNTs. B) AFM image of PEGylated
SWNTs. Inset: photograph of PEGylated SWNTs in saline. C) Quantification of CD80+ and CD86+ expression, an indicator of DC maturation, on
bone marrow-derived DCs after in vitro incubation with various concentrations of SWNTs for 12 h. Incubation with PEGylated SWNTs could obviously
enhance the maturation of DCs in vitro by a concentration-dependent manner. Lipopolysaccharides (LPS) was the positive control. D,E) Secretion of
IL-1β (D) and IL-12P70 (E) by DCs after incubation with SWNTs as determined by ELISA. F,G) In vivo DC maturation induced by SWNTs. BALB/c mice
were s.c. injected with SWNTs. Cells in the nearest lymph nodes were examined for the expression of CD11c, CD80, and CD86 by flow-cytometry. H-K)
Cytokine levels in serum from healthy BALB/c mice isolated at 12 h, 72 h and 168 h post SWNT-injection. SWNTs injection enhanced the secretion of
IL6 (H), IL 1β (I), TNF-α (J) and IL 12p70 (K). Data are presented as the mean ± standard errors of the mean (SEM).

DC maturation. For in vitro experiments, bone marrow-derived
DCs separated from BALB/c mice[14] were incubated with var-
ious concentrations of SWNTs for 12 h. Confocal fluorescence
imaging was conducted for DCs after incubation with fluores-
cently labeled SWNTs, and revealed effective internalization
of SWNTs by DCs (Supporting Information, Figure S2). Flow
cytometry was then used to determine the expression of DC
maturation markers, CD80 and CD86 (Supporting Informa-
tion, Figure S3). Remarkably, we found that PEGylated SWNTs
could significantly up-regulate the expression of CD86 & CD80
(Supporting Information, Figure S3B–C) and enhance the per-
centage of matured DCs (CD80+CD86+ DCs) (Figure 1C). In
addition, we studied the DC stimulation effects of several other
commonly used PTT agents including PEGlyated graphene

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oxide (GO), PEGlyated gold nanorods (AuNRs), as well as indo-
cyanine green (ICG), which, however, all showed much weaker
effect to induce DC maturation compared to PEGylated SWNTs
(Figure S4). Our data indicated that SWNTs appeared to be a
unique nano-agent to activate DCs, acting as a potential immu-
nological adjuvant.
Next the DC supernatants were harvested to measure inter-
leukin 12 (IL-12) and interleukin 1β (IL-1β) by enzyme-linked
immune sorbent assay (ELISA). IL-12 and IL-1β are interleukins
naturally produced by DCs in response to antigenic stimula-
tion. Particularly, IL-12 plays an important role in the activation
of cytotoxic activity of NK cells and CD8+ T lymphocytes. It was
found that the secretion levels of IL-1β and IL-12 by DCs were
both obviously enhanced after SWNTs incubation by a concentra-
tion-dependent manner. All these data suggested that DCs could
be activated by PEGylated SWNTs in vitro. The exact activation
pathway, however, remains to be understood in future studies.
To further determine if SWNTs treatment could result in the
activation of DCs in vivo, healthy mice were subcutaneously
(s.c.) injected with SWNTs (dose = 0.33 mg/kg). Mice were
sacrificed at 96 h post injection of SWNTs and the maturation
status of DCs from the nearest lymph nodes was assessed using
flow cytometry by co-staining for CD11c, the specific marker of
DCs, together with their maturation markers CD80 and CD86.
It was found that SWNTs could induce the migration of a larger
number of DCs into lymph nodes and significantly promote the
maturation of DCs compared to untreated group (Figure 1F–G
& Supporting Information, Figure S5). In addition, we studied
the in vivo DC stimulation by several other commonly used
PTT agents (GO-PEG, AuNR-PEG, and ICG at the same dose),
which again showed much weaker effects to induce DC matura-
tion compared to PEGylated SWNTs (Supporting Information,
Figure S6), consistent to our in vitro results. Our data suggest
that SWNTs may as a potential adjuvant which could be recog-
nized by DCs and lead to DC activation both in vitro and in vivo
to amplify the subsequent immune responses.
In the following study, we examined multiple cytokines
including IL-1β, IL-12, interleukin 6 (IL-6) (the typical marker
of humoral immunity), and tumor necrosis factor α (TNF-α)
(typical markers of cellular immunity) in the mouse serum after
s.c. injection of SWNTs. It was found that the secretion levels of
IL-1β, IL-12, IL-6, and TNF-α all exhibited apparent elevation
from the untreated control (P value <0.05) within 7 days after
SWNTs injection (Figure 1H–K). While IL-12 could positively
regulate the activity of natural killer cells, which are the impor-
tant composite of innate immunity, TNF-α is a critical mediator
for the cellular immunity and plays an important role in tumor
immunotherapy. Therefore, we wondered whether SWNTs
alone would show any tumor inhibition effect on BALB/c mice.
In our experiment, mice were firstly s.c. injected with SWNTs
on their left flanks, and then inoculated with 4T1 murine breast
cancer cells on their right flanks. Although the tumor growth
in the SWNT-treated group was slightly delayed, such thera-
peutic effect was not significant enough particularly at later
time points (Figure S7). Therefore, although PEGylated SWNTs
upon s.c. injection at the current dose could induce a substan-
tial level of immunological responses including DC activation
and cytokine secretion, such responses are not strong enough
to inhibit tumor growth by themselves.
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Recent studies have shown that ablative treatments such as
photodynamic therapy, radiofrequency ablation, hyperthermia,
high-intensity focused ultrasound (HIFU), as well as cryoabla-
tion (e.g. ultra-low temperature treatment by liquid nitrogen) of
tumors are able to cause tumor-specific immune responses.[15]
We thus wonder whether and how SWNT-based photothermal
ablation of tumors would trigger immunological responses in
animal models. Considering the facts that local administra-
tion (e.g. s.c. injection) instead of systemic intravenous (i.v.)
injection is usually used for immunotherapy, and the dose
with local injection of SWNTs could be much lower than that
required with i.v. injection (∼5 mg/kg),[6] in our experiments,
mice bearing subcutaneous 4T1 murine breast tumors were
intratumorally injected with SWNTs (dose = 0.33 mg/kg). After
irradiation with an 808-nm NIR laser at 0.5 W/cm2 for 10 min,
the tumor temperature jumped to 53 °C, a temperature high
enough to effectively ablate tumor cells (Figure 2A–C). After
SWNT-induced photothermal treatment, all tumors on mice
were completely eliminated, without showing a single case of
tumor relapse at their original sites after treatment.
The effects of SWNT-based PTT towards DC maturation
and cytokine elevation were then studied. In our experiment,
mice were sacrificed at 96 h after SWNT-based PTT to ablate
the 4T1 tumors. The maturation status of DCs within tumor-
draining lymph nodes (TDLN) was assessed using flow cytom-
etry. It was found that SWNT-based PTT treatment could
greatly promote DC maturation in lymph nodes to a much
greater extent compared to that resulted from SWNT injec-
tion alone without PTT treatment (Figure 2D–H). On the other
side, we analyzed various cytokines in the serum of SWNT-
based PTT-treated mice at 12, 72 and 168 hours post-ablation
(Figure 2I–M). Both SWNTs alone and SWNT-based PTT were
able to increase the secretion of pro-inflammatory cytokines
IL-1β, IL-12p70, IL-6 and TNF-α. Particularly, the serum level
of TNF-α, which plays an important role in anti-tumor immune
responses, was dramatically enhanced after SWNT-treated PTT.
Our data suggest that SWNTs-based PTT is able to initiates a
significant pro-inflammatory immune response. In situ tumor
destruction by SWNTs-based PTT may provide the immune
system with an antigen source for the induction of specific
antitumor immunity as more tumor antigen epitopes may be
exposed after tumor ablation. Therefore, upon contact with
SWNTs and tumor cell debris, DCs which are enriched in the
skin dermis would be stimulated and migrate to nearby lymph
nodes including tumor draining lymph nodes, transit to their
mature state up on arrival, activate T lymphocytes and then reg-
ulate subsequent immunities.
Inspired by the immune responses induced by SWNT-based
PTT, we then wondered whether those effects would be helpful
to prevent tumor metastasis. In our experiment, we inoculated
the first tumor by s.c. injection of 4T1 tumor cells on the left
flank of each BALB/c mice. One week after injection, when the
first tumor reached ∼150 mm3, a second tumor was then inocu-
lated on the right flank. At the following day, the first tumor
was intratumorally injected with SWNTs and treated with PTT,
or simply removed by surgery (Figure 3A). The growth of
the secondary tumors, as a mimic of metastatic tumors,
was then monitored afterwards (Figure 3B). Unfortunately,
we found that the immunological responses induced by
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A B
55 oC

25 oC

C 1min 2min 5min 8min 10min

Tempeture (oC)
55

45 Saline PTT
35

25
0 5 10
Time (min)
D F
104 104 104
Untreated SWNTs SWNT based PTT
38.7% 46.3% 67.6%
103 103 103
Counts

CD80 102 102 102

101 101 101

100 100 100

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
CD11c CD86

E G H
40 40 80

% CD80+ CD86+ DC
% CD11c+ DC

30 30 60
MFI CD80

20 20 40

10 10 20

0 0 0
Untreated SWNTs SWNT Untreated SWNTs SWNT Untreated SWNTs SWNT
baesd PTT baesd PTT baesd PTT

I J K M
20 20 20
Untreated Untreated Untreated Untreated
80
SWNTs SWNTs SWNTs SWNTs
IL12P70 (pg/ml)

15 SWNT-based PTT 15 SWNT-based PTT SWNT-based PTT 15 SWNT-based PTT

TNF-α (pg/ml)

IL1β (pg/ml)

60
IL6 (pg/ml)

10 10 10
40

5 5 20 5

0 0 0 0
12 h 72 h 168 h 12 h 72 h 168 h 12 h 72 h 168 h
12 h 72 h 168 h

Figure 2. SWNT-based PTT induces DC maturation and stimulates the expression of pro-inflammatory cytokines. A) Schematic illustration of the
PTT treatment. B) Representative IR thermal images of tumor-bearing mice exposed to the NIR laser (808 nm, 1 cm2, 10 min) after i.t. injection with
SWNTs. C) Tumor temperature changes for the control mice and SWNT-injected mice under NIR laser irradiation. D-H) DC maturation induced by
SWNT-based PTT to ablate 4T1 tumors on mice. Cells in the TDLN were isolated and stained for CD11c, CD80, and CD86 expressions under flow
cytometry. D) Representative histograms of CD11c expression on DCs analyzed by flow cytometry. E) Qualification of CD11c+ cells according to (D).
F) Representative FACS plots of CD80, CD86 expression gated by CD11c+ cells. G) Corresponding mean fluorescent intensity (MFI) quantification of
CD80. H) Qualification of CD80+ CD86+ cells according to (F). SWNT-based PTT treatment could greatly increase in vivo DC maturation. I-M) Cytokine
levels in sera from mice isolated at 12 h, 72 h and 168 h post SWNT-based PTT. Data are presented as the mean ± SEM.

SWNT-based photothermal ablation of the primary tumors CTLA-4-blocking antibody (anti-CTLA-4), a clinically approved
could not completely inhibit the growth of their secondary therapeutic antibody, is able to target CTLA-4, which is consti-
tumors, whose average growth speed, however, was indeed tutively expressed by regulatory T cells (Tregs) and up-regulated
slower than those on mice with their primary tumors removed after T cell activation.[16] Researchers have found that blockade
by surgery. Additional efforts were thus needed to further of CTLA-4 could inhibit tumor development by abrogating the
enhance the anti-tumor immunological effects induced by immuno-suppressive activity of regulatory T cells (Tregs) and
SWNT-based PTT. augmenting activities of effector T-cells.[16] Clinical trials have
Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a key negative validated the efficacy of anti–CTLA-4 antibody therapy for
regulator of T cell activation. Recent studies have found that the treatment of several types of cancers.[17] In a number of

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COMMUNICATION
A B Surgery
Surgery +Anti CTLA4
SWNT-based PTT

Tumor volume (mm3)

500
SWNT-based PTT+Anti CTLA4
400

300

200

100
D-8 D-1 D0 D1,3,5
0
0 5 10 15
First tumor Second tumor Removal of the Anti-CTLA4
2nd Tumor analysis DAY
Inoculation Inoculation primary tumor by injection
and size
SWNT-based PTT
measurement
or surgery

C D
7.39% 0.0% 15.5% 0.0%
25

CD8+ T Cells (%)

20

15

38.0% 54.6% 39.7% 44.8% 10

Surgery Surgery +Anti–CTLA-4 0

Surgery Surgery + PTT PTT +
7.83% 0.0% 22.9% 0.0%
E Anti-CTLA4 Anti-CTLA4
100
CD4+ T Cells (%)

80
60

13.1% 64.0% 40
7.59% 84.2%
CD8

20
0
SWNT-based PTT SWNT-based PTT + ANTI CTLA4 Surgery Surgery + PTT PTT +
Anti-CTLA4 Anti-CTLA4
CD4
F G 10
Effector to FoxP3 ratio
CD4+ FoxP3+ T Cells(%)

80 CD4Eff/FoxP3 CD8/FoxP3
8

60 6

40
4

20
2

0
Surgery Surgery + PTT PTT + 0
Anti-CTLA4 Anti-CTLA4 Surgery Surgery + PTT PTT +
Anti-CTLA4 Anti-CTLA4

Figure 3. Anti-tumor effect of SWNT-based PTT plus anti–CTLA-4 therapy. A) Schematic illustration of SWNT-based PTT and anti–CTLA-4 combination
therapy. B) Tumor growth curves of different groups of mice after various treatments indicated (7–8 mice per group). Error bars are based on SEM.
C) Quantification of T cells in secondary tumors. Tumor cell suspensions were analyzed by flow cytometry for T cell infiltration (gated on CD3+ T cells).
D,E) Proportion of tumor-infiltrating CD8+ T cells (D) and CD4+ T cells (E) according to (C). F) Proportion of tumor-infiltrating CD4+ FoxP3+ T cells.
G) Ratios of tumor-infiltrating CD8+ T cells and effective CD4+ T cells over regulatory T cells in the secondary tumors upon various treatments to remove
the first tumors. The combination treatment not only increased the ratio of CD8+ T cells to Tregs, but also the ratio of effective CD4+ T cells to Tregs.

previous studies, it has been found that CTLA-4 blockade in
combination with cryoablation or hyperthermia could enhance
tumor-specific immune responses and thereby inhibit tumor
growth at distant sites.[15d,18] Therefore, we next explored
whether CTLA-4 blockage therapy would be able to enhance the
immunological effects induced by SWNT-based PTT.
The experimental design is shown in Figure 3A. After
removal of the primary tumors by either photothermal abla-
tion with SWNTs or simply by surgical resection, mice were i.v.
injected with three doses of anti-CTLA4 antibody (clone 9H10)
(60 µg per mouse for each injection) on day 1, 3, 5. The growth
of secondary tumors as well as the survival of those mice after

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various treatments were monitored (Figure 3B). It was found
that anti-CTLA4 treatment could only slightly delay the growth
of secondary tumors on mice when their primary tumors
were removed by surgery. Remarkably, for mice with their pri-
mary tumors ablated by SWNT-based PTT, CTLA4 blockage
could greatly inhibit the development of secondary tumors
(Figure 3B, Figure S8&9). This result indicated that although
SWNT-based PTT alone could only partially delay the growth
of a second tumor challenge, its anti-tumor efficacy would be
greatly promoted when combined with the CTLA-4 blockade
strategy, leading to effective rejection of secondary tumors,
which mimic tumor metastasis.
To investigate the underlying mechanism of tumor inhibi-
tion by SWNT-based PTT and anti–CTLA-4 combination treat-
ment, immune cells in secondary tumors were then analyzed.
T-cell infiltration in the secondary tumor was studied 10 days
after their implantation by flow cytometry. CD8+ killer T cells,
also known as cytotoxic T (Tc) cells, could kills cancer cells
by releasing cytotoxins perforin, granzymes and granulysin
that eventually lead to apoptosis of targeted cancer cells. The
percentage of CD8+ T cells in the secondary tumor after the
combined SWNT-based PTT plus anti-CTLA-4 treatment was
3-fold of that in the surgery treatment or PTT treatment alone
groups, and 1.5-fold compared to that in the surgery plus
anti–CTLA4 treatment group (Figure 3C–D). On the other
hand, the percentage of CD4+ helper T (Th) lymphocytes,
which play critical roles in the regulation of adaptive immuni-
ties, showed a significant increase in the secondary tumors of
mice after their primary tumors were ablated by SWNT-based
PTT (Figure 3E). However, for SWNT-based PTT alone,
most of increased CD4+ T cells were found to be regulatory
T cells, which are able to hamper effective anti-tumor immune
responses (Figure 3F & Figure S10). In marked contrast,
CTLA-4 blockade therapy could greatly abrogate the activity of
Tregs and reduce the percentage of Tregs in secondary tumors
after SWNT-based photothermal ablation of primary tumors.
As the results, the combination treatment not only increased
the ratio of CD8+ T cells to Tregs, but also increased effective
CD4+ T cells to Tregs (Figure 3G). Both effects are of great
importance to enhance anti-tumor immunological responses.
Beside tumor-infiltration T cells, CD20+ tumor-infiltrating
B cells have also been reported to be strongly associated
with favorable outcomes in immunotherapy of breast, lung,
ovarian and cervical cancers.[19] Nelson and colleagues dem-
onstrated that CD20+ tumor-infiltrating B lymphocytes (TILs)
were able to work together with CD8+ T cells to promote the
patient survival through serving as APCs and/or generating
cytokines to modulate helper T cells polarizing towards Th1
or Th2 functional phenotype.[20] Therefore, CD20+ TILs were
also analyzed in secondary tumors after various treatments.
Interestingly, we found that SWNT-based PTT increased the
number of infiltrating B cells (CD20+CD44+ cells) within the
tumor (Figure S11A–B). Meanwhile, it was observed that the
level of IgG in the serum of combination treatment group
was apparently higher than that in the groups after surgical
resection plus anti-CTLA4 treatment, as well as surgical resec-
tion alone (Figure S11C). This may be greatly useful for APCs
including B cells to take up, process and present the tumor-
associated antigen to T cells and then to initiate the adaptive
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immunities since there existing IgG Fc fragment receptors on
APCs.[21]
To further assess the potency of this combination therapy, we
tested it in a lung metastasis tumor model by challenging mice
with i.v. injection of 4T1 cells (1*105 cells per mouse), which
afterwards would spread into various mouse organs particu-
larly into the lung. The lung metastasis was introduced one day
before SWNT-based PTT or surgery treatment to remove the
primary subcutaneous tumors. In the following days, mice
were i.v. injected with anti-CTLA-4 antibody (60 µg per mouse
at day 1, 3, 5) (Figure 4A). On day 10, lungs were harvested
for analysis. Compared to mice with only surgical resection
of the primary tumors, those after SWNT-based PTT treat-
ment together with anti-CTLA4 therapy showed remarkably
reduced lung metastasis, as evidenced by photos of India-ink
stained whole lungs and Hematoxylin and Eosin (H&E) stained
lung slices (Figure 4B&C). With the combination therapy, the
average number of metastasis sites in lungs was dramatically
decreased from ∼30 per mouse (surgery) to ∼1 per mouse
(SWNT-based PTT plus anti-CTLA4 therapy) (Figure 4D). A
marked difference was also seen for the average lung weights
between these two groups (Figure 4E).
Next we studied the survival of mice treated with our com-
bination therapy. 1*105 4T1 cells were i.v. introduced one day
before SWNT-based PTT or surgery treatment to remove the
primary tumor. Every group had 7–8 mice. It was found that
57% of mice survived in 50 days after combination therapy
(SWNT-based PTT plus anti-CTLA4 therapy). In contrast, only
25% mice could survive with CTLA-4 blockade plus surgery
treatment in 50 days, and none of mice could survive with sur-
gery or SWNT-based PTT alone treatment without the injec-
tion of anti-CTLA4 (Figure 4F). Our results thus demonstrate
that SWNT-based photothermal ablation of the primary tumor
in combination with anti-CTLA4 treatment would be able to
induce strong anti-tumor immunological responses to inhibit
lung metastasis, showing remarkably therapeutic advantage in
prolonging the animal survival.
At last, to see if the immunological effect of SWNT-based
PTT is a general behavior towards other types of cancer cell
lines, we have conducted in vitro experiments with another cell
line, murine B16 musculus skin melanoma. It was found that
residues of both 4T1 cells and B16 cells after SWNT-induced
photothermal ablation could strongly activate DC maturation
in an in vitro Transwell system experiment to mimic in vivo
tumor model (Supporting Information, Figure S12). Although
further in vivo validations are still required, it is likely that the
immunological responses triggered by photothermal tumor
ablation with SWNTs may be applied for different tumor
types.
Generally speaking, the underlying mechanisms of the com-
bination therapy with ideal inhibition activities on tumor growth
and metastasis may greatly correlate with several parameters as
follows (Figure 5). (1) SWNTs may act as an ‘adjuvant’ being
recognized by DCs and leading to DC activation/maturation in
vivo to amplify the subsequent immune responses. (2) SWNT-
based PTT provides tumor-associated antigens for APCs such
as DCs to trigger anti-tumor immune responses. (3) Anti-
CTLA4 blockade strategy effectively hampers the immune-
suppression activity of Tregs and significant increases the ratios
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A

D-8 D-1 D0 D1,3,5

Removal of the
First tumor Lung metastasis primary tumor by Anti-CTLA4 Lung metastasis
Inoculation induction SWNT-based PTT injection Quantification
or surgery

B Surgery C

PTT + Anti-CTLA4

D E F
50 500
Percent survival

100
NO. of lung metastases

Lung Mass (mg)

40 400

50
30 300

20 200
0
0 10 20 30 40 50
10 100 DAY
SWNT-based PTT + Anti CTL4
0 0 SWNT-based PTT
Surgery PTT + Surgery PTT + Surgery + Anti CTLA4
Anti-CTLA4 Anti-CTLA4 Surgery

Figure 4. Lung metastasis inhibition by SWNT-based PTT and anti–CTLA-4 combination therapy. A) Schematic illustration of SWNT-based PTT and
anti–CTLA-4 combination therapy for treatment of a lung metastasis tumor model. B,C) Representative lung photographs (B) and H&E-stained lung
slices (C) collected from mice post different treatments indicated. D,E) Quantification of lung metastasis nodules (D) and lung masses (E) for mice
after different treatments. Compared to mice with only surgical resection of the primary tumors, those after SWNT-based PTT together with anti-CTLA4
therapy showed remarkably reduced lung metastasis. F) The survival curves of mice in 50 days after various treatments indicated. SWNT-based pho-
tothermal ablation of the primary tumor in combination with anti-CTLA4 treatment would be able to induce strong anti-tumor immunological effects
to inhibit lung metastasis.

between CD4+ Th cells to Treg as well as CD8+ Tc cells to Tregs
in secondary tumors. This is greatly helpful for the induc-
tion of anti-tumor immunities especially cellular immunity.
(4) The CD20+ tumor-infiltrating B cells may also serve as APCs
to effectively take up, process and present the tumor-associated
antigens for the induction of CTL-mediated cellular immunity
for tumor immunotherapy. Those immune responses taken
together would result in the effective rejection of remaining
tumor cells inside the body, useful for the treatment of meta-
static tumors.
In summary, we have shown here that SWNT-based PTT
together with anti–CTLA-4 therapy could modulate the adaptive
immune responses especially cellular immunity for the treat-
ment of metastatic cancer. While being a powerful photothermal
agent to effectively burn the tumor, PEGylated SWNTs could
also act as a potential adjuvant to stimulate DC maturation

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Tumor antigen Dendritic cells Tumor-draining lymph nodes (TDLN)

maturation Th cells
CD4+ Th cells

SWNTs
Tumor antigens
Danger signals (e.g. ROS, HSP…) Tc cells

Primary tumor Inflammatory response

treated with
SWNT-based PTT IL-1β Antigen presentation to naïve T cell
IL-6
IL-12
B cells TNF-α
……
Anti-CTLA4 mAb

e.g. NK cells

Tumor specific antibody

CTLA4
Treg cells
CD8+ Tc cells CD4+ Th cells
Metastasis tumor Tumor specific T cells CTLA-4 blockade

Figure 5. The hypothesized mechanism of anti-tumor immune responses induced by SWNT-based PTT in combination with the anti-CTLA-4
therapy.

and cytokine secretion. Such behaviors are not found or to a Acknowledgements

much weaker extend for several other types of widely studied
photothermal agents including GO, AuNRs and ICG. After
SWNT-based photothermal ablation of the primary tumor, the
induced immunological responses combined with anti-CTLA4
treatment are able to inhibit the challenge of a secondary tumor
or even lung metastasis. Our work indicates that compared
with surgery resection, the most commonly used cancer treat-
ment strategy, nanomaterials-based photothermal treatment
of cancer may be of advantageous in terms of triggering the
adaptive immunological responses against remaining tumor
cells in the body. This work is the first demonstration that
photothermal therapy induced by an adjuvant-like nano-agent
in combination with immunotherapy is able to inhibit cancer
metastasis. The mechanisms of the adjuvant-like behaviors
of PEGylated SWNTs still need further careful investigations.
Besides SWNTs, which exhibit unique multiple functions in
this work, it also remains to be explored how other types of
photothermal nano-agents would behave in such combina-
tion therapy. Nevertheless, the combination between PTT
nanomedicine-treatment together with antibody-based immu-
notherapy may be a novel cancer therapeutic strategy, which
not only is able to destroy the primary tumor, but also to inhibit
cancer metastasis at distant sites. It is thus likely that photo-
thermal nano-therapy is not just burning of tumors; instead it
may offer clinically valuable therapeutic advantages over sur-
gery in cancer treatment.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
This work was partially supported by the National Natural Science
Foundation of China (51222203, 51132006), the National “973” Program
of China (2011CB911002, 2012CB932601), a Juangsu Natural Science
Fund for Distinguished Young Scholars, the Jiangsu Key Laboratory
for Carbon-Based Functional Materials, and a Project Funded by the
Priority Academic Program Development of Jiangsu Higher Education
Institutions. The authors thank Prof. Hongjie Dai for his helpful
suggestions to this work.
Received: July 7, 2014
Revised: August 29, 2014
Published online:
[1] a) C. L. Chaffer, R. A. Weinberg, Science 2011, 331, 1559;
b) J. A. Joyce, J. W. Pollard, Nat. Rev. Cancer 2009, 9, 239.
[2] a) C. M. Britten, H. Singh-Jasuja, B. Flamion, A. Hoos, C. Huber,
K. J. Kallen, S. N. Khleif, S. Kreiter, M. Nielsen, H. G. Rammensee,
U. Sahin, T. Hinz, U. Kalinke, Nat. Biotechnol. 2013, 31,
880; b) H. Ledford, Nature 2014, 508, 24; c) I. Melero, S. Hervas-
Stubbs, M. Glennie, D. M. Pardoll, L. Chen, Nat. Rev. Cancer 2007,
7, 95.
[3] a) M. P. Melancon, M. Zhou, C. Li, Acc. Chem. Res. 2011, 44,
947; b) U. Lindner, R. A. Weersink, M. A. Haider, M. R. Gertner,
S. R. H. Davidson, M. Atri, B. C. Wilson, A. Fenster, J. Trachtenberg,
J. Urology 2009, 182, 1371; c) E. C. Dreaden, M. A. Mackey,
X. Huang, B. Kang, M. A. El-Sayed, Chem. Soc. Rev. 2011, 40, 3391;
d) S. Lal, S. E. Clare, N. J. Halas, Acc. Chem. Res. 2008, 41, 1842;
e) O. Akhavan, E. Ghaderi, Small 2013, 9, 3593.
[4] a) Q. W. Tian, M. H. Tang, Y. G. Sun, R. J. Zou, Z. G. Chen,
M. F. Zhu, S. P. Yang, J. L. Wang, J. H. Wang, J. Q. Hu, Adv. Mater.
2011, 23, 3542; b) K. Yang, L. Z. Feng, X. Z. Shi, Z. Liu, Chem.
Soc. Rev. 2013, 42, 530; c) Z. J. Zhang, L. M. Wang, J. Wang,
X. M. Jiang, X. H. Li, Z. J. Hu, Y. H. Ji, X. C. Wu, C. Y. Chen,

8 wileyonlinelibrary.com © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Mater. 2014,
DOI: 10.1002/adma.201402996

www.MaterialsViews.com
Adv. Mater. 2012, 24, 1418; d) J. F. Lovell, C. S. Jin, E. Huynh,
H. Jin, C. Kim, J. L. Rubinstein, W. C. Chan, W. Cao, L. V. Wang,
G. Zheng, Nat. Mater. 2011, 10, 324; e) M. S. Yavuz, Y. Cheng,
J. Chen, C. M. Cobley, Q. Zhang, M. Rycenga, J. Xie, C. Kim, K. H. Song,
A. G. Schwartz, Nat. Mater. 2009, 8, 935; f) O. Akhavan,
A. Meidanchi, E. Ghaderi, S. Khoei, J. Mater. Chem. B 2014,
2, 3306.
[5] a) H. Gong, R. Peng, Z. Liu, Adv. Drug Deliver. Rev. 2013, 65,
1951; b) Z. Liu, K. Chen, C. Davis, S. Sherlock, Q. Z. Cao,
X. Y. Chen, H. J. Dai, Cancer Res. 2008, 68, 6652; c) K. Welsher, Z. Liu,
S. P. Sherlock, J. T. Robinson, Z. Chen, D. Daranciang, H. J. Dai,
Nat. Nanotechnol. 2009, 4, 773; d) J. W. Kim, E. I. Galanzha,
E. V. Shashkov, H. M. Moon, V. P. Zharov, Nat. Nanotechnol. 2009,
4, 688; e) Z. Liu, S. Tabakman, K. Welsher, H. J. Dai, Nano Res.
2009, 2, 85; f) T. R. Fadel, E. R. Steenblock, E. Stern, N. Li, X. Wang,
G. L. Haller, L. D. Pfefferle, T. M. Fahmy, Nano Lett. 2008, 8, 2070;
g) K. Kostarelos, A. Bianco, M. Prato, Nat. Nanotechnol. 2009,
4, 627; h) B. R. Smith, E. E. Ghosn, H. Rallapalli, J. A. Prescher,
T. Larson, L. A. Herzenberg, S. S. Gambhir, Nat. Nanotechnol. 2014,
9, 481; i) Z. Liu, W. Cai, L. He, N. Nakayama, K. Chen, X. Sun,
X. Chen, H. Dai, Nat. Nanotechnol. 2007, 2, 47.
[6] a) P. Chakravarty, R. Marches, N. S. Zimmerman, A. D. Swafford,
P. Bajaj, I. H. Musselman, P. Pantano, R. K. Draper, E. S. Vitetta,
Proc. Natl. Acad. Sci. USA 2008, 105, 8697; b) X. Liu, H. Tao,
K. Yang, S. Zhang, S. T. Lee, Z. Liu, Biomaterials 2011, 32, 144;
c) H. K. Moon, S. H. Lee, H. C. Choi, ACS Nano 2009, 3, 3707;
d) J. T. Robinson, K. Welsher, S. M. Tabakman, S. P. Sherlock,
H. Wang, R. Luong, H. Dai, Nano Res. 2010, 3, 779.
[7] a) R. Singh, D. Pantarotto, L. Lacerda, G. Pastorin, C. Klumpp,
M. Prato, A. Bianco, K. Kostarelos, Proc. Natl. Acad. Sci. USA 2006,
103, 3357; b) Z. Liu, C. Davis, W. Cai, L. He, X. Chen, H. Dai, Proc.
Natl. Acad. Sci. USA 2008, 105, 1410; c) A. De La Zerda, C. Zavaleta,
S. Keren, S. Vaithilingam, S. Bodapati, Z. Liu, J. Levi, B. R. Smith,
T. J. Ma, O. Oralkan, Z. Cheng, X. Y. Chen, H. J. Dai,
B. T. Khuri-Yakub, S. S. Gambhir, Nat. Nanotechnol. 2008, 3, 557.
[8] a) H. Dumortier, Adv. Drug Deliver. Rev. 2013, 65, 2120;
b) T. R. Fadel, T. M. Fahmy, Trends Biotechnol. 2014, 32, 198.
[9] A. J. Andersen, P. P. Wibroe, S. M. Moghimi, Adv. Drug Deliver. Rev.
2012, 64, 1700.
Adv. Mater. 2014, © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/adma.201402996
www.advmat.de
COMMUNICATION
[10] a) J. Palomaki, E. Valimaki, J. Sund, M. Vippola, P. A. Clausen,
|K. A. Jensen, K. Savolainen, S. Matikainen, H. Alenius, ACS Nano
2011, 5, 6861; b) E. Meunier, A. Coste, D. Olagnier, H. Authier,
L. Lefèvre, C. Dardenne, J. Bernad, M. Béraud, E. Flahaut, B. Pipy,
Nanomed-Nanotechnol. 2012, 8, 987.
[11] M. Yang, J. Meng, X. Cheng, J. Lei, H. Guo, W. Zhang, H. Kong,
H. Xu, Theranostics 2012, 2, 258.
[12] C. Wang, X. X. Ma, S. Q. Ye, L. Cheng, K. Yang, L. Guo, C. H. Li,
Y. G. Li, Z. Liu, Adv. Funct. Mater. 2012, 22, 2363.
[13] C. A. Janeway, K. Bottomly, Cell 1994, 76, 275.
[14] M. B. Lutz, N. Kukutsch, A. L. J. Ogilvie, S. Rossner, F. Koch,
N. Romani, G. Schuler, J. Immunol. Methods 1999, 223, 77.
[15] a) J. P. M. Almeida, R. A. Drezek, A. E. Foster, Immunotherapy 2014,
6, 109; b) S. R. Gameiro, J. P. Higgins, M. R. Dreher, D. L. Woods,
G. Reddy, B. J. Wood, C. Guha, J. W. Hodge, Plos One 2013, 8,
e70417; c) Q. Liu, B. Zhai, W. Yang, L.-X. Yu, W. Dong, Y.-Q. He,
L. Chen, L. Tang, Y. Lin, D.-D. Huang, Mol. Ther. 2009, 17, 2049;
d) R. Waitz, S. B. Solomon, E. N. Petre, A. E. Trumble, M. Fassò,
L. Norton, J. P. Allison, Cancer Res. 2012, 72, 430; e) J. E. Kennedy,
Nat. Rev. Cancer 2005, 5, 321; f) K. F. Chu, D. E. Dupuy, Nat. Rev.
Cancer 2014, 14, 199.
[16] a) F. S. Hodi, Clin. Cancer Res. 2007, 13, 5238; b) T. Pentcheva-Hoang,
E. Corse, J. P. Allison, Immunol. Rev. 2009, 229, 67;
c) J. F. Grosso, M. N. Jure-Kunkel, Cancer Immun. 2013, 13, 5.
[17] T. Yervoy, Princeton, NJ: Bristol-Myers Squibb Company 2011.
[18] M. H. den Brok, R. P. Sutmuller, R. van der Voort, E. J. Bennink,
C. G. Figdor, T. J. Ruers, G. J. Adema, Cancer Res. 2004, 64, 4024.
[19] T. W. Kuijpers, R. J. Bende, P. A. Baars, A. Grummels, I. A. M. Derks,
K. M. Dolman, T. Beaumont, T. F. Tedder, C. J. M. van Noesel,
E. Eldering, R. A. W. van Lier, J. Clin. Invest. 2010, 120, 214.
[20] a) J. S. Nielsen, B. H. Nelson, Oncoimmunology 2012, 1, 1623;
b) J. S. Nielsen, R. A. Sahota, K. Milne, S. E. Kost, N. J. Nesslinger,
P. H. Watson, B. H. Nelson, Clin. Cancer Res. 2012, 18, 3281.
[21] a) S. Amigorena, C. Bonnerot, Semin Immunol. 1999, 11, 385;
b) C. Scuoppo, C. Miething, L. Lindqvist, J. Reyes, C. Ruse,
I. Appelmann, S. Yoon, A. Krasnitz, J. Teruya-Feldstein, D. Pappin,
J. Pelletier, S. W. Lowe, Nature 2012, 487, 244; c) K. Serre, P. Machy,
J. C. Grivel, G. Jolly, N. Brun, J. Barbet, L. Leserman, J. Immunology
1998, 161, 6059.
wileyonlinelibrary.com 9