ARTICLE
Received 23 Apr 2016 | Accepted 12 Sep 2016 | Published 21 Oct 2016
A therapeutic strategy that can eliminate primary tumours, inhibit metastases, and prevent tumour relapses is developed herein by combining adjuvant nanoparticle-based photothermal therapy with checkpoint-blockade immunotherapy. Indocyanine green (ICG), a photothermal agent, and imiquimod (R837), a Toll-like-receptor-7 agonist, are co-encapsulated by poly(lactic-co-glycolic) acid (PLGA). The formed PLGA-ICG-R837 nanoparticles composed purely by three clinically approved components can be used for near-infrared laser-triggered photothermal ablation of primary tumours, generating tumour-associated antigens, which in the presence of R837-containing nanoparticles as the adjuvant can show vaccine-like functions. In combination with the checkpoint-blockade using anti-cytotoxic T-lymphocyte antigen-4 (CTLA4), the generated immunological responses will be able to attack remaining tumour cells in mice, useful in metastasis inhibition, and may potentially be applicable for various types of tumour models. Furthermore, such strategy offers a strong immunological memory effect, which can provide protection against tumour rechallenging post elimination of their initial tumours.
DOI: 10.1038/ncomms13193 OPEN
Photothermal therapy with immune-adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapy
Qian Chen1,*, Ligeng Xu1,*, Chao Liang1, Chao Wang1, Rui Peng1 & Zhuang Liu1
1 Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123, China. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Z.L. (email: mailto:[email protected]
Web End [email protected] ).
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13193
Developing effective therapeutic strategies with high specicities and low toxicities to eradicate tumours, particularly post their metastases, and further prevent
their recurrence, is the ultimate goal in the battle against cancer. Currently used gold-standard cancer treatment approaches, surgery chemotherapy and radiotherapy, all fail to achieve this goal. In recent years, along with the growing knowledge on cancers and their interactions with immune systems, cancer immunotherapy by training or stimulating the inherent immunological systems of the body to attack tumour cells, has been progressing rapidly and shown tremendous promises as a next generation of cancer treatment strategy1,2. Several different types of cancer immunotherapies including cytokine therapy3,4, checkpoint-blockade therapy5,6, adoptive T-cell transfer especially the emerging chimeric antigen receptor T (CAR-T) cell therapy711, as well as cancer vaccines1214, have demonstrated some exciting clinical responses. However, until now, most of those immune-therapeutic strategies still have limitations such as extremely high costs15, large individual variations in therapeutic responses, as well as certain immunotoxicity like the cytokine release syndrome14,16,17.
Among above-mentioned cancer immunotherapy strategies, cancer vaccines may own a number of unique advantages18,19. It has been demonstrated that cancer vaccines loaded with tumour-associated antigens are able to induce antigen-specic immunities against tumours, rather than non-specic immunological responses triggered by other methods such as the checkpoint-blockade therapy20. On the other hand, cancer vaccines may offer a long-term immune-memory effect that could be helpful to prevent cancer reccurrence2022. In general, cancer vaccines involve tumour-specic antigens-based vaccines and whole cancer cell vaccines23,24. Although tumour-associated antigens such as specic proteins or peptides with the help of adjuvant agents may induce robust anti-tumour immune responses, the large heterogeneity of patients lead to their limited clinical applications25. Different from the former, whole cancer cell vaccines (for example, using lysates of dissected tumour tissues) can induce immunities against all released potential tumour antigens, and in principle should be applicable to various types of solid tumours18. However, the complicated manufacture process, the uncertainties in characteristics and dosages for whole cancer cell vaccines, as well as their limited efcacies resulted in disappointing clinical results so far26. Therefore, a cancer immunotherapy strategy that is easy to operate and has high specicity and efcacy is urgently needed.
Photothermal therapy (PTT) has been developed as a new cancer treatment strategy that employs the heat generated from the absorbed optical energy by light-absorbing agents accumulated in the tumour to ablate tumour cells27,28. Recently, we and others2933 discovered that photothermal therapy with inorganic nano-agents (for example, carbon nanotubes, graphene oxide or CuS nanoparticles) could generate anti-tumour immunological effects by producing tumour-associate agents from ablated tumour cell residues. Such an effect has also been observed in a preliminary clinical trial study32. Inspired by such interesting ndings, in this work we discover that the tumour-associated antigens generated in situ after photothermal tumour ablation in the presence of immune-adjuvant nanoparticles could show vaccine-like functions, which in combination with checkpoint blockade show strong anti-tumour immune responses for effective cancer immunotherapy (Fig. 1a).
In our formulation, those nanoparticles are composed by three US FDA (Food and Drug Administration) -approved agents, poly(lactic-co-glycolic) acid (PLGA) as the encapsulating polymer, indocyanine green (ICG) as the near-infrared (NIR)
dye to enable photothermal therapy, and imiquimod (R837) which is a potent TLR7 agonist useful in activating immune responses34,35. Upon NIR-induced photothermal ablation of primary tumours injected with PLGA-ICG-R837, the released tumour-associated antigens in combination with R837-loaded nanoparticle adjuvant would show vaccine-like functions, generating strong immunological responses which with the help of anti-CTLA4 checkpoint-blockade therapy to inhibit the activities of immune-suppressive regulatory T cells (Tregs) could attack distant tumour cells remaining in the mouse body. This strategy appears to be particularly effective in inhibiting tumour metastasis post spreading of tumour cells in the mouse body. PLGA-ICG-R837-based photothermal treatment combined with anti-CTLA4 therapy could protect treated mice against tumour cells rechallenging 40 days post ablation of primary tumours, demonstrating the strong immune-memory effect to protect mice from cancer relapse. Such a strategy could also work by systemic injection of reformulated nanoparticles to realize effective cancer treatment. Therefore, the use of immune-adjuvant nanoparticles for photothermal tumour ablation offers vaccine-like functions in situ, which in combination with the clinically adapted checkpoint-blockade method shows efcacy in cancer immunotherapy.
ResultsNanoparticle formulation and immune-stimulation abilities. PLGA, a biodegradable synthetic polymer approved for clinical use by US FDA, was used to encapsulate two types of small molecules, a NIR dye ICG and a TLR7 ligand R837, by oil-in-water (o/w) emulsion method. The obtained PLGA-ICGR837 nanoparticles showed well-dened spherical shape and homogenous sizes as revealed in the transmission electron microscope image (Fig. 1b, inset). The average hydrodynamic size of PLGA-ICG-R837 was B100 nm as measured by the dynamic light scattering (Fig. 1b). The ultravioletvisibleNIR absorption spectrum of PLGA-ICG-R837 showed the characteristic absorption peak of ICG, indicating the successful encapsulation of ICG in the PLGA core (Fig. 1c). The loading of R837 was conrmed by high-performance liquid chromatography (Supplementary Fig. 1).
As one of the most important classes of antigen-presenting cells, DCs play crucial roles in initiating and regulating the innate and adaptive immunities36. Upon exposure to antigens, immature DCs will engulf and then process them into peptides during their migration to nearby lymph nodes. Thereafter, those immature DCs would transform into mature DCs and present the major histocompatibility complex-peptide to T-cell receptor when arriving at lymph nodes37. Therefore, we rstly investigated the immunological effects of PLGA-ICG-R837 nanoparticles towards bone marrow-derived DCs separated from BALB/c mice, by using ow cytometry to analyse the upregulation of co-stimulatory molecules CD80, CD86, which are well-known markers for DC maturation. It was found that PLGA-ICG-R837 nanoparticles could greatly promote in vitro DC maturation similar to free R837 at the same dose, while PLGA-ICG showed no obvious immune-stimulation effect to DCs (Supplementary Fig. 2).
To further investigate if PLGA-ICG-R837 could accelerate DC maturation in vivo, BALB/c mice were subcutaneously (s.c.) injected with PLGA-ICG, free R837, PLGA-ICG-R837 (50 mg kg 1 PLGA, 0.7 mg kg 1 R837, 1.1 mg kg 1 ICG). Three days post injection, mice were killed and the inguinal lymph nodes were collected for assessment by ow cytometry after co-staining with CD11c (the DC marker), CD80 and CD86. The percentage of matured DCs (CD11c CD80 CD86 ) signi
cantly increased from B28 to B45% after treatment with PLGA-
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13193 ARTICLE
a
iDC
Primary tumour Tumour vaccine
Tumour-associated antigen ( )
NIR
Uptake
Migration
Tumour-draining lymph nodes (TDLN)
R837
PLGA
o/w
Memory T cells
mDC
Precaution
Rechallenged tumour cells
ICG
PLGA-R837-ICG
Th
Anti-CTLA4 mAb
......
Metastasis tumour
CTLA4 blockade
b
c d
30
3.0
50
25
PLGA-ICG-R837
2.5
40
20
2.0
Number (%)
Absorption
1.5
DC maturation in
lymph node (%)
30
15
10
20
1.0
5
0.5
10
0
0.0
0
Untreated
PLGA-lCG
100 1,000 10,000
1 10 400 600 800 1,000
200
Diameter (nm)
Wavelength (nm)
Free R837
PLGA-ICG-R837
Figure 1 | Formulation of nanoparticles and their immune-stimulation abilities. (a) The mechanism of anti-tumour immune responses induced by PLGA-ICG-R837-based PTT in combination with checkpoint-blockade. (b) Hydrodynamic diameters of PLGA-ICG-R837 nanoparticles measuredby DLS. Inset: (a) TEM image of PLGA-ICG-R837. (c) UVvisNIR spectra of PLGA-ICG-R837 and free ICG, indicating the successful loading of ICG into PLGA. (d) In vivo DC maturation (CD80 CD86 ) with lymph nodes of BALB/c mice s.c. injected with PLGA-ICG, free R837, or PLGA-ICG-R837
(three mice per group). Data are presented as the means.e.m. Error bars are based on triplicated experiments. DLS, dynamic light scattering; TEM, transmission electron microscopy.
ICG-R837, while the DC maturation percentages from mice treated with PLGA-ICG or free R837 (with the same dose) only increased to B30 or B35%, respectively. Therefore, PLGA-ICG
R837 nanoparticles showed even stronger in vivo immune-stimulation effect compared with the same dose of free R837, although the two induced similar levels of in vitro DC maturation (Fig. 1d and Supplementary Fig. 3).
DCs upon maturation would secrete various types of cytokines to regulate other types of immune cells38. Thus, in the following experiment, various cytokines including interleukin 6 (IL-6) (an important marker in the activation of humoral immunity), tumour necrosis factor a (TNF-a) (an important marker in the activation of cellular immunity), and interleukin 12 (IL-12p70) (an important marker of innate immunity)3942 in the mouse sera after different treatment were analysed by ELISA. It was found that mice treated with PLGA-ICG-R837 showed high serum levels of IL-12p70, IL-6 and TNF-a, which appeared to be higher than those in sera of mice treated with the same dose of free R837 (Supplementary Fig. 4). Such observed stronger in vivo immune-stimulation effect of PLGAICG-R837 than free R837 may be attributed to the sustained release of R837 from nanoparticles.
Photothermal tumour ablation for immune system activation. On the basis of the aforementioned experiment results,
PLGA-ICG-R837 nanoparticles designed in our system is an effective immune-stimulator. It has been reported that many other ablative tumour treatments such as hyperthermia, photodynamic therapy and cryoablation will induce strong tumour-specic immune responses4346. Therefore, we wonder if photothermal therapy with our PLGA-ICG-R837 could trigger further enhanced immunological responses. Firstly, in vitro experiments veried that the residues of 4T1 breast tumour cells after NIR-induced photothermal ablation with PLGA-ICG-R837 could dramatically enhance the DC maturation, to a level much higher than that achieved by simply adding PLGA-ICG-R837 nanoparticles, or cell residues ablated by PLGA-ICG in the absence of R837 (Supplementary Fig. 2). Such results suggest that R837-containing nanoparticles could potentially act as an adjuvant to promote immunological responses of tumour-associate antigens in cell residues.
In our further in vivo experiment, BALB/c mice-bearing subcutaneous 4T1 tumours were intratumourally (i.t.) injected with PLGA-ICG or PLGA-ICG-R837 and then irradiated by an 808 nm laser at the power density of 0.5 W cm 2 for 10 min. As monitored by an infrared thermal camera (Fotric 225), the tumour temperature of mice injected with PLGA-ICG or PLGA-ICG-R837 under laser irradiation quickly rose to B60 C, which was high enough to effectively ablate tumours
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13193
a b
0 min
2 min
4 min
6 min
8 min
10 min
70
PLGA-ICG-R837
PLGA-ICG
Untreated
60
65 C
Temperature (C)
50
40
30
20 0 2 4 6 8
15 C
10
Time (min)
c d e
104
103
102
101
100
104
103
102
101
100
104
103
102
101
100
104
16.89%
14.05%
14.87%
30.82%
32.23%
60.19%
103
102
101
32.95%
19.34%
100
31.78%
21.94%
14.05% 10.89%
100
101
102
103
104
100 104
103
102
101
100
101
102
103
104
Surgery
Surgery + PLGA-ICG
PLGA-ICG + Laser
f g
h
104
103
102
1
12.21%
11.03%
59.09%
72.21%
*
75 PLGA-ICG PLGA-ICG-R837
Surgery PTT
60
***
Percent of mature
DC (%)
45
30
15.40%
8.03%
15
7.54%
CD86
101 104
103
102
101
102
103
104
100
Surgery + PLGA-ICG-R837
PLGA-ICG-R837 + laser
0 Surgery alone
CD80
i k
j
300
60
80
Surgery
Surgery
Surgery
IL-12p70 (pg ml1)
TNF-((pg ml1)
60
IL-6 (pg ml1)
200
40
40
100
20
20
0
0
0
24 h
72 h
168 h
24 h
72 h
168 h
24 h
72 h
168 h
Figure 2 | Immune responses after PLGA-ICG-R837-based PTT. (a) IR thermal images of 4T1-tumour-bearing mice injected with PLGA-ICG-R837, PLGA-ICG or PBS under the 808 nm laser (0.5 Wcm 2) irradiation. (b) The tumour temperature changes based on IR thermal imaging date in a. (ch) DC maturation induced by PLGA-ICG-R837-based PTT on mice-bearing 4T1 tumours (gated on CD11c DC cells). Cells in the tumour-draining lymph nodes
were collected 72 h after various treatments for assessment by ow cytometry after staining with CD11c, CD80 and CD86. (ik) Cytokine levels in sera from mice isolated at 24, 72 and 168 h post different treatments (surgery, surgery and s.c. injection of PLGA-ICG-R837, i.t. injection of PLGA-ICG-R837 and PTT). Three mice were measured in each group in (ak). Data are presented as the means.e.m. P values were calculated by Tukeys post-hoc test (***Po0.001, **Po0.01 or *Po0.05). For (ik), P values were determined between the second group (Surgery PLGA-ICG-R837) and the third group
(PLGA-ICG-R837 laser).
(Fig. 2a,b). To analyse the status of DCs in treated tumours after PTT, tumour cells were collected and co-stained with CD11c/propidium iodide for assessment by ow cytometry 4 h post-PTT treatment with PLGA-ICG-R837. After photothermal
ablation of the tumour with PLGA-ICG-R837 nanoparticles, more DCs would be recruited into the initial tumour site, although DCs pre-existing in the tumour before treatment might have been killed alongside the tumour (Supplementary Fig. 5).
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Three days after photothermal therapy, mice were killed with their tumour-draining lymph nodes collected for assessment using ow cytometry after co-staining with various markers. Interestingly, photothermal tumour ablation with adjuvant nanoparticles (PLGA-ICG-R837 Laser) induced an high level
of DC maturation (B72%), which appeared to be much higher than that observed for adjuvant nanoparticles alone (PLGA-ICGR837 without laser) or PTT with PLGA-ICG in the absence of immune-adjuvant (Fig. 2ch). Therefore, after the tumour is destroyed post PTT, DCs may be recruited to the ablated tumour site as antigen-presenting cells to trigger immune responses. Meanwhile, tumour-associated antigens in tumour debris post PTT may be transported into nearby lymph nodes and then processed by DCs to simulate DC maturation, particularly with the help of adjuvant nanoparticles.
Cytokines secretion is also important in the process of immune responses. In a parallel experiment, sera of mice bearing either 4T1 tumours or CT26 tumours after different treatments were collected at 24, 72 and 168 h to analyse the changes of various cytokines including IL-6, TNF-a and IL-12p70. Similarly, although PLGA-ICG-R837 injection alone or PTT with PLGAICG was able to increase the secretion of pro-inammatory cytokines, their secretions induced by PLGA-ICG-R837-based PTT were obviously higher and lasted longer, favourable for triggering anti-tumour immune response (Fig. 2ik and Supplementary Fig. 6). The non signicant increase of IL-4 secretion, an important indicator for humoral immunity, in those treated mice (Supplementary Fig. 7), indicates that humoral immunity may play a less important role in this system. Considering the risk of high cytokine levels to induce possible harmful effects to normal organs, serum biochemistry assay and complete blood panel test were conducted for 4T1-tumour-bearing mice at 1, 7 and 14 days after PLGA-ICG-R837-based photothermal therapy. All measured parameters fell within normal ranges, indicating that such elevated cytokine levels post PTT with PLGA-ICG-R837 should be well tolerable by those mice (Supplementary Table 1). These results suggest that PLGA ICG R837-based photothermal therapy is able to induce immunological stimulation effects in vivo. We thus hypothesize that the in vivo adjuvant activities of those R837-containing nanoparticles in combination with tumour-associate antigens released after tumour ablation therapy could act together as a safe tumour vaccine potentially useful for cancer immunotherapy.
PTT plus CTLA4 blockade to inhibit growth of distant tumours. The majority of cancer deaths are caused by metastases, which if occurred can hardly be effectively treated by conventional therapies such as surgery, chemotherapy and radiotherapy47,48. Therefore, we wondered if photothermal immunotherapy with our PLGAICG-R837 could provide any opportunity in treating metastatic cancer. Cytotoxic T lymphocyte-associate antigen-4 (CTLA-4) is a critical negative regulator of immune responses49,50, and its blockade by antibodies (for example, anti-CTLA-4) to inhibit the activities of immune-suppressive Tregs has been approved by FDA as a cancer immunotherapy approach currently used in the clinic5154. Therefore, in our animal experiments, CTLA-4 blockade therapy was introduced, aiming at enhancing the anti-cancer therapeutic efcacy of tumour vaccines that are in situ generated after PLGA-ICG-R837-based photothermal ablation of primary tumours.
The design of our animal experiment is shown in Fig. 3a. Tumour cells including breast cancer (4T1) and colorectal cancer (CT26) were inoculated on the left ank of each mouse. A week later, a second tumour was inoculated on the right ank of the same mouse as an articial mimic of metastasis. In the following
day, the rst tumours were eliminated by PLGA-ICG-R837-based photothermal therapy or surgery. Afterwards, mice were intravenously (i.v.) injected with anti-CTLA4 (clone 9H10) at doses of 10 mg per mouse three times on day 1, 4 and 7 (ref. 50).
The growth of secondary tumours in different groups was measured by a caliper every other day (Fig. 3b,c). For mice with their primary tumours removed by surgery, the secondary tumours in both tumour model showed rather rapid growth, whose speed could only be slightly delayed if the mice were either s.c. injected with PLGA-ICG-R837, or i.v. injected with anti-CTLA4. The combination treatment by both PLGA-ICGR837 and anti-CTLA4, however, could signicantly slow down the growth of secondary tumours (especially in the rst 20 days) on mice with their primary tumours dissected by surgery, indicating that such non-specic combined immunotherapy could be effective in cancer treatment. On the other hand, for mice with their primary tumours ablated by PLGA-ICG-R837-based PTT, the growth of their secondary tumours was also partly delayed. We found that for mice with their primary tumours eliminated by PLGA-ICG-R837-based PTT in combination with CTLA-4 blockade, their secondary tumours showed almost completely inhibited growth for the 4T1 model, and disappeared for the C26T model, achieving efcacies much better than that obtained in the surgery groups receiving the combined PLGAICG-R837 plus anti-CTLA4 treatment. As another control, the immunological response induced by PLGA-ICG-based PTT of primary tumours (no R837) together with CTLA-4 blockade therapy could only inhibit the growth of secondary tumours in the early days, demonstrating the important role of the immune-adjuvant R837 in those nanoparticles to trigger strong immune responses.
In addition to the above subcutaneous tumour models, we further tested the efcacy of our method in the treatment of a more aggressive whole-body spreading tumour model. In this experiment, while the treatment plan was not changed, the second wave of tumour cells was induced by i.v. injection of 4T1 cells expressing rey luciferase(fLuc-4T1) into mice before their primary tumours are eliminated either by surgery or PLGA-ICGR837-based PTT (Fig. 3a). In the following days, mice were i.v. injected with anti-CTLA-4 antibody as aforementioned (dose 20 mg per mouse for each time). Considering the much
higher aggressiveness of the lung metastasis tumour model, the dose of anti-CTLA-4 antibody used here was doubled. In vivo bioluminescence imaging was conducted to track the spreading and growth of fLuc-4T1 cancer cells in different groups of mice. From bioluminescence imaging, it was found that mice with their primary tumours removed by surgery showed obvious cancer metastasis 10 days after i.v. injection with fLuc-4T1 cells. For the other groups of mice with surgical removal of primary tumours and treated with PLGA-ICG-R837 alone, anti-CTLA4 alone or even the combination of PLGA-ICG-R837 and anti-CTLA4, signicant bioluminescence signals, indication of tumour metastases, also showed up, although at later stages. In contrast, while mice treated with PLGA-ICG-R837-based PTT to ablate their primary tumours showed delayed metastases, the group with PLGA-ICG-R837-based PTT together with CTLA-4 blockade therapy showed nearly no metastasis (Fig. 3d). Photographs of India-ink stained whole lungs harvested at different days also conrmed that different from all control groups in which many metastatic tumour nodules were found in the mouse lung, no noticeable sign of lung metastasis was noted in the last group with PLGA-ICG-R837-based PTT combined with anti-CTLA-4 therapy (Supplementary Fig. 8).
To further evaluate the therapeutic outcomes of combined PLGA-ICG-R837-based PTT and anti-CTLA-4 therapy, mice after various treatments were closely monitored. We found 7
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13193
a
808 nm laser
8 d 0 d
1 d
1st tumour inoculation
2nd tumour inoculation (s.c. or i.v.)
Remove the first tumour by PTT or surgery
Anti-CTLA4 injection
Analysis and measurement of the 2nd tumour
b c
Subcutaneous 4T1 model
Subcutaneous CT26 model
Surgery
700
Surgery
600
600
500
***
***
Tumour volume (mm3)
Tumour volume (mm3)
500
***
**
**
**
*
400
400
300
***
** *
**
300
200
200
100
100
0
0
12 14 16 18 20 22 24 26
Day 17 Day 21 Day 25 Day 29 Day 36 Day 50
10 12 14 16 18 20 22 24 26 10 Day
Day
d
Surgery (1)
Surgery + anti-CTLA4 (2)
PLGA-ICG-R837 + laser (4)
Surgery + PLGA-ICG-R837 + anti-CTLA4 (5)
PLGA-ICG-R837 + laser + anti-CTLA4 (6)
Day 10
Surgery + PLGA-ICG-R837 (3)
e f
Metastatic 4T1 model
Orthotopic 4T1 model
100
Morbidity-free survival (%)
Surgery
Surgery + anti-CTLA4
PLGA-ICG-R837 + laser
Surgery + PLGA-ICG-R837 + anti-CTLA4
100
Morbidity-free survival (%)
80
80
60
60
40
40
20
20
PLGA-ICG-R837 + laser + anti-CTLA4
0
0
20
30 40 50 60 70 30 40 50 60 70 80 90
20
Time (day)
Time (day)
Figure 3 | Anti-tumour effect of PLGA-ICG-R837-based PTT plus anti-CTLA-4 therapy. (a) Schematic illustration of PLGA-ICG-R837-based PTTand anti-CTLA-4 combination therapy to inhibit tumour growth at distant sites. (b,c) Tumour growth curves of different groups of mice (six mice per group) with s.c. inoculation of secondary 4T1 (b) or CT26 (c) tumours after various treatments to eliminate their primary tumours. (d) In vivo bioluminescence images to track the spreading and growth of i.v. injected fLuc-4T1 cancer cells in different groups of mice after the cancer cells after various treatments to eliminate their primary tumours. (e) Morbidity-free survival of different groups of mice with metastatic 4T1 tumours in d after various treatments indicated to eliminate their primary tumours (10 mice per group). (f) Morbidity-free survival of different groups of mice-bearing orthotopic 4T1 tumours with spontaneous metastases after various treatments indicated to eliminate their primary breast tumours (10 mice per group). PLGA-ICG-R837-based photothermal ablation of the rst primary tumours in combination with anti-CTLA4 treatment would be able to induce strong anti-tumour immunological effects to inhibit the growth of tumour cells spread into other organs. P values in b and c were calculated by Tukeys post-hoc test (***Po0.001, **Po0.01 or *Po0.05) by comparing other groups with the last group (PLGA-ICG-R837 laser anti-CLTA-4). Data are presented
as the means.e.m.
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seven out of 10 mice receiving i.v. injection of 4T1 cells could survive for 70 days after PLGA-ICG-R837-based PTT plus anti-CTLA4 therapy (Fig. 3e), in marked contrast to mice in the other ve control groups which all died within 2540 days. The survived seven mice in this last group behaved normally and were killed at day 70 for careful necropsy, which uncovered no noticeable metastatic tumours in these seven mice.
In addition to the metastatic tumour model induced articially, an orthotopic murine breast cancer model with spontaneous metastasis was created and used to evaluate the therapeutic efcacy of our treatment strategy. As shown in Supplementary Fig. 9a, fLuc-4T1 tumour cells were inoculated into the breast pad of each mouse. Two weeks later, when spontaneous metastases of tumour cells should have occurred, the primary tumour on each mouse was eliminated by PLGA-ICG-R837-based photothermal therapy or surgery. Afterwards, anti-CTLA4 with the same dose was i.v. injected (dose 20 mg per mouse for each time). Then,
the in vivo bioluminescence imaging was conducted to track the metastases of fLuc-4T1 tumour cells after different treatments. It was also found that mice after PTT based on PLGA-ICG-R837 together with CTLA-4 blockade therapy showed effective inhibition of cancer metastasis, in marked contrast to other control groups in which detectable metastases were observed, sooner or later after treatments (Supplementary Fig. 9b). Those mice after various treatments were closely monitored. In comparison with different control groups, in which the majority or large proportions of mice died from spontaneous metastases within 80 days, combining PLGA-ICG-R837-based PTT with anti-CTLA-4 therapy resulted in 90% of survival rate in our observation period of 80 days (Fig. 3f). Therefore, it is obviously that immunological responses triggered after PLGA-ICG-R837-based photothermal ablation of primary tumours in combination with anti-CTLA-4 therapy can effectively inhibit cancer metastasis and prolong the survival of mice with spreading tumour cells.
Besides CTLA-4, programmed death 1 (PD-1), as another important T-cell inhibitory receptor, and PD-L1, one of its ligands, play important roles in helping cancer cells to evade the immune attack. Blockade of PD-L1, sometimes in combination with CTLA-4 blockade, to enhance anti-tumour activity has been approved for cancer immunotherapy in the clinic50,55. In our experiments, mice post spreading of 4T1 tumour cells(i.v. injection) with surgical removal of their primary tumours and treated with the combination of CTLA-4 and PD-L1 co-blockade, or even together with the treatment of PLGA-ICGR837 but no laser irradiation, showed obvious cancer metastases as indicated by bioluminescence imaging (Supplementary Fig. 10). Therefore, our current strategy by combining nano-adjuvant-based PTT with anti-CTLA-4 appears to be more effective than the clinically adopted PD-L1 CTLA-4
co-blockade therapy. However, when we combined anti-PD-L1 and anti-CTLA-4 to treat mice with tumours after NIR-induced photothermal ablation with PLGA-ICG-R837, more than a half of mice died while no mice died after PLGA-ICG-R837-based PTT combined with single checkpoint blockade. Considering the critical roles of PD-L1 and CTLA-4 in the immune homoeostasis, the simultaneous blockade of these two checkpoints may lead to the immune-related adverse events or cytokine release syndrome56, which after PLGA-ICG-R837-based PTT may be beyond the limit that mice could tolerant.
The mechanism study. To understand the mechanism of synergistic anti-tumour effect triggered by PLGA-ICG-R837-based PTT in combination with anti-CTLA-4 therapy, immune cells in secondary 4T1 tumours were studied on day 10. While
cytotoxic T lymphocytes (CTL) (CD3 CD4-CD8 ) could
directly kill targeted cancer cells, helper T cells (CD3 CD4
CD8-) play important roles in the regulation of adaptive immunities. In our experiments, for mice with primary tumours removed by surgery, either PLGA-ICG-R837 injection alone or anti-CTLA-4 treatment alone failed to promote CD8 CTL
inltration into the secondary tumours. In contrast, the percentage of CD8 CTL in the secondary tumours of mice after
the PLGA-ICG-R837-based PTT plus anti-CTLA-4 treatment signicantly increased to B19.7%, which appeared to be higher than that in groups treated with PLGA-ICG-R837-based PTT (B8.96%) or surgery plus anti-CTLA4 treatment (B12.96%)
(Fig. 4a,c). On the other hand, compared with the surgery only group, the percentages of helper T cells in the secondary tumours of the other ve groups all showed dramatic increase (Fig. 4a,c). Moreover, the percentage of total T cells in the secondary tumour also showed remarkable increase after PLGA-ICG-R837-based PTT (Supplementary Fig. 11).
With Foxp3 as the marker, CD4 helper T cells could be
classied into effective T cells that are helpful to promote immune responses (CD3 CD4 Foxp3-), as well as
regulatory T cells (Tregs) (CD3 CD4 Foxp3 ) which could
hamper effective anti-tumour immune responses. Immune cells in secondary tumours were collected for further analysis after co-staining with CD4 and Foxp3. Among the greatly enriched CD4 helper T cells in the secondary tumours of mice
post PLGA-ICG-R837-based PTT ablation of primary tumours, most of these increased helper T cells were immune-suppressive Tregs. Therefore, although vaccine-like immune responses have been generated after PLGA-ICG-R837-based PTT, the anti-tumour efcacy in this group remained to be less effective owing to the presence of high numbers of Tregs. It was found that CTLA-4 blockade therapy could greatly reduce the percentages of Tregs (CD3 CD4 Foxp3 ) in secondary tumours (Fig. 4b).
Therefore, both CD8 CTL/Treg ratio and CD4 Teff/Treg
ratios were greatly enhanced in secondary tumours of mice after PLGA-ICG-R837-based PTT plus anti-CTLA-4 treatment (Fig. 4c,d). Moreover, comparing the last two groups in Fig. 4 (groups 5 and 6), PLGA-ICG-R837-based PTT plus anti-CTLA4 induced the highest percentage of CD8 CTLs (also the CD8
CTL/Treg ratio), which are primarily responsible for cell immunity in cancer immunotherapy.
Long-term immune-memory effects. An important feature of immune systems is their ability to remember pathogens for several decades, critical for disease prevention. Therefore, it is necessary to evaluate immune memory induced generated by PLGA-ICG-R837-based PTT. In our experiment, the secondary 4T1 tumours were inoculated 40 days after PLGA-ICG-R837-based PTT or surgery to remove their rst 4T1 tumours. Mice were i.v. injected with anti-CTLA-4 antibody at different days (20 mg per mouse each time) for two rounds of treatment, with the rst round given right after their rst tumours were eliminated (days 1 and 5), and the second round given right after their secondary tumours were re-inoculated (days 41, 44 and 47) (Fig. 5a). It was found that the re-inoculated tumours in mice after their rst tumours were ablated by PLGA-ICG-R837-based PTT plus CTLA4 blockade therapy showed inhibited growth (Fig. 5b), and anti-CTLA4 treatment in the rst round (after PTT ablation of rst tumours) appeared to be not necessary to induce the immune-memory effect. In marked contrast, surgical removal of rst tumours plus two rounds of anti-CTLA4 treatment (pre and post), or even anti-CTLA4 (pre and post) in combined with s.c. injected PLGA-ICG-R837, exerted no appreciable inhibitory effect to the rechallenged
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13193
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10.88%
1.36% 0.00%
0.00%
0.00% 1.14%
35.64%
40.54%
43.56%
43.5%
33.8% 63.6%
25.5%
17.1%
4.67%
5.41%
Surgery (1)
Surgery + anti-CTLA4 (2) Surgery + anti-CTLA4 (2)
Surgery (1)
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52.63%
67.16%
0.00%
5.26%
8.96%
42.11%
23.88%
Surgery + PLGA-ICG-R837 (3)
Surgery + PLGA-ICG-R837 + anti-CTLA4 (5)
PLGA-ICG-R837 +laser (4)
PLGA-ICG-R837 + laser + anti-CTLA4 (6)
Surgery + PLGA-ICG-R837 (3)
Surgery + PLGA-ICG-R837 + anti-CTLA4 (5)
PLGA-ICG-R837 + laser (4)
104
103
102
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38.89%
11.1%
12.96%
19.70%
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CD4
Foxp3
PLGA-ICG-R837 + laser + anti-CTLA4 (6)
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CD8
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CD8+ CTL
CD8+ CTL / Treg
CD4+ Teff / Treg
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CD4 + Foxp3 + (Treg)
Figure 4 | The mechanism study. (a) Representative ow cytometry plots showing different groups of T cells in secondary tumours. Tumour cell suspensions were analysed by ow cytometry for T-cell inltration (gated on CD3 Tcells). (b) Representative ow cytometry plots showing percentages
(gated on CD4 cells) of CD4 FoxP3 T cells in secondary tumours after various treatments indicated. (c) Proportions of tumour-inltrating
CD8 killer T cells, CD4 FoxP3- effector T cells and CD4 FoxP3 regulatory T cells according to data in a and b. (d) CD8 CTL: Treg ratios and
CD4 effector T cells: Treg ratios in the secondary tumours upon various treatments to remove the rst tumours. Both ratios were signicantly enhanced
after combination treatment with PLGA-ICG-R837-based PTTand anti-CTLA4 therapy. Three mice were measured in each group in ad. Data are presented as the means.e.m. Error bars are based on triplicated experiments.
tumours. Although s.c. injected PLGA-ICG-R837 in combination with anti-CLTA4 therapy could signicantly delay the growth of existing tumour cells in the body likely via non-specic immune responses (Fig. 3b,c), such a treatment without generating tumour-associate agents has no immune memory effect to protect mice from tumour rechallenge (Fig. 5b).
We next carried out a series of analyses to understand the robust anti-tumour immune memory generated after PLGA-ICGR837-based photothermal tumour ablation. Based on the effector function, proliferative capacity and migration potential, memory T cells are classied into central memory T cells (TCM) and
effector memory T cells (TEM) (ref. 57). While TCM mainly locates in the secondary lymphoid tissues and only provides protections after antigen-stimulated clonal expansion, differentiation and trafcking, TEM residing in both lymphoid and non-lymphoid
tissues can elicit immediate protections by producing cytokines like IFN-g (refs 5860). Therefore, we measured the proportions of both TCM and TEM cells at day 40 after the removal of the primary tumours with different treatments (right before rechallenging mice with secondary tumours). It was found that the percentage of TEM cells (CD3 CD8 CD62L-CD44 ) was
much higher in the group of mice with their primary tumour removed by PLGA-ICG-R837-based PTT (Fig. 5c), whereas the percentage of TCM cells (CD3 CD8 CD62L CD44 ) that
are less important for immune memory decreased after PLGAICG-R837-based PTT together with anti-CTLA treatment in the rst round (Supplementary Fig. 12). Furthermore, 1 week after secondary tumours were introduced, cytokines in sera of mice with different treatments were analysed by ELISA. It is known that Th1 cytokines including TNF-a and IFN-g (ref. 61), the
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a
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Figure 5 | Long-term immune-memory effects. (a) Schematic illustration of PLGA-ICG-R837-based PTT and anti-CTLA-4 combination therapy to inhibit cancer relapse. (b) tumour growth curves of rechallenged tumours inoculated 40 days post eliminated of their rst tumours (eight mice per group). (c) Proportions of effector memory T cells (TEM) in the spleen analysed by ow cytometry (gated on CD3 CD8 T cells) at day 40 right before
rechallenging mice with secondary tumours (groups 4 and 5 would be identical at this point). (d,e) Cytokine levels in sera from mice isolated 7 days after mice were rechallenged with secondary tumours (after the second round of anti-CTLA4 treatment). Three mice were measured in each group in ce. Data are presented as the means.e.m.
typical markers of cellular immunity, play vital roles in immunotherapy against cancer. The serum levels of TNF-a and
IFN-g were signicantly increased in the mice treated with PLGA-ICG-R837-based PTT, particularly for those with PTT plus the second round of anti-CTLA-4 treatment (post), indicating the successful establishment of anti-tumour immune responses triggered by the rechallenging of cancer cells 40 days later in this group (Fig. 5d,e).
Reformulated nanoparticles for systemic administration. Finally, we would like to further explore the possibility of realizing PTT-triggered cancer immunotherapy by intravenous (i.v.) systemic administration of nanoparticles (Fig. 6a). Polyethylene glycol (PEG) grafted PLGA co-polymer (mPEG-PLGA), instead of PLGA, was used to encapsulate both ICG and R837 (Supplementary Fig. 13). As revealed by in vivo and ex vivo uorescence imaging, such PLGA-PEG-ICG-R837 nanoparticles showed rather high accumulation in CT26 tumours upon i.v. injection (Fig. 6b), owing to their prolonged blood circulation behaviour (Fig. 6c) favourable for tumour passive uptake via the enhanced permeability and retention effect.
Utilizing the high tumour accumulation of PLGA-PEG-ICGR837, photothermal ablation of the rst tumour was carried out. After i.v. injection with PLGA-PEG-ICG-R837 (6 mg kg 1 R837, 8 mg kg 1 ICG) for 24 h, CT26-tumour-bearing mice were irradiated by the 808 nm laser for 10 min (0.8 W cm 2), which led to the rise of tumour surface temperature to B52 C (Fig. 6d,e). To evaluate the immunotherapeutic efcacy of PLGA-PEG-ICG-R837-based PTT combined with CTLA-4 blockade, animal experiments were carried out as illustrated in Fig. 6a. After the rst tumour of each mouse was removed by PTT (with i.v. injection of PLGA-PEG-ICG-R837) or simply by surgery, the growth of the secondary tumour was carefully
monitored (Fig. 6f). As expected, PTT based on PLGA-PEG-ICGR837 together with CTLA-4 blockade could obviously delay the growth of secondary tumours, offering an obviously prior therapeutic effect compared with other control groups (Fig. 6f).
To further understand the immune effects triggered by PTT with i.v. injected PLGA-PEG-ICG-R837, mice after different treatments were killed and their DCs were collected from the nearest lymph nodes for assessment by ow cytometry (Supplementary Fig. 14). Serum of mice after different treatments was collected at 24, 72 and 168 h post treatment to analyse the changes of various cytokines (Supplementary Fig. 15). It was found that PTT with i.v. injected PLGA-PEG-ICG-R837 was able to induce a high level of DC maturation as well as enhanced secretion of multiple pro-inammatory key cytokines. Even with systemic administration of our nanoparticles, we did not observe any notable cytokine-storm-like side effect. All mice behaved normally after treatment with i.v. injected PLGA-PEG-ICG-R837 without signicant body weight uctuation or accidental death. Although further studies are still required to carefully evaluate the safety and efcacy of this treatment approach with i.v. injected PLGA-PEGICG-R837 nanoparticles, achieving PTT-triggered cancer immunotherapy with systemic injection of all therapeutic agents may have a great value for clinical uses, especially for certain tumours that can hardly be reached via local injection.
DiscussionCompared with currently existing immunotherapeutic strategies, our method by photothermal tumour ablation with immune-adjuvant nanoparticles together with checkpoint-blockade therapy may overcome several critical issues in cancer immunotherapy. Compared with conventional cancer vaccines with specic proteins or peptides as antigens, whose efcacies may vary signicantly between different groups of patients because of the
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a
808 nm laser
8 d 1 d 0 d
1st tumour inoculation
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i.v. injection of PLGA-PEGICG-R837 and PTT
Anti-CTLA4 injection
Analysis and measurement of the 2nd tumour
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Figure 6 | PTT-triggered immunotherapy via systemic injection of nanoparticles. (a) Schematic illustration showing the design of animal experiments. (b) In vivo uorescence images of CT26-tumour-bearing mice taken at different time points post i.v. injection of PLGA-PEG-ICG-R837. The right column shows an ex vivo uorescence image of major organs and tumour dissected from the mouse 24 h post injection. Tu, Li, Sp, Ki, H and Lu stand for tumour, liver, spleen, kidney, heart and lung, respectively. (c) Blood circulation curve of PLGA-PEG-ICG-R837 in mice by measuring the uorescence of ICG in blood at different time points post i.v. injection (three mice per group). (d) IR thermal images of CT26-tumour-bearing mice injected with PLGA-PEG-ICG-R837 or PBS under the 808 nm laser (0.8 Wcm 2) irradiation. (e) The tumour temperature changes based on IR thermal imaging date in d. (f) The growth curves of secondary tumours in different groups of CT26-tumour-bearing mice after various treatments to eliminate their primary tumours (six mice per group).
Data are presented as the means.e.m.
varied antigen expression levels in their tumours, such in situ generated tumour vaccines utilizing tumour residues as tumour-associated antigens after ablation therapy may induce anti-tumour immune responses against a broad spectrum of solid tumours. For traditional whole cancer cell vaccine strategies, cell lysates made from dissected tumour tissues are usually re-injected into patients to generate immune responses. In contrast, our strategy to generate vaccine-like functions in situ does not need sophisticated procedures, has no ethic concerns, and is able to efciently elicit strong protections owing to the synergistic effects between photothermally generated tumour residues and nanoadjuvants pre-injected into the primary tumour, or delivered into tumours after systemic administration. Compared with adoptive T-cell therapy or DC-based therapy, which usually require sophisticated techniques and a lot of hands on experiences, our approach is obvious simpler and cheaper, favourable for future clinical practices. Distinguished from previously reported inorganic materials-based photothermal strategies62,63, all the components within our nanoparticles have already been
approved by US FDA for clinical use. Lastly, although the penetration depth of light in tissues could be limited even with NIR lasers, endoscope-based clinical devices with imbedded laser optical bres may be used for laser treatment of tumours located deeply inside the body. Therefore the clinical translation of our nanoparticles and proposed technique may indeed be realistic.
In summary, we propose that photothermal ablation of tumours with multifunctional nanoparticles encapsulating both NIR heaters and immune-adjuvant TLR agonists could induce vaccine-like immune responses that may be combined with CTLA4 checkpoint blockade to realize highly effective cancer immunotherapy. Great anti-tumour efcacies have been observed using this strategy to treat two types of subcutaneous tumour models, an articial whole-body metastasis tumour model, as well as an orthotopic tumour model with spontaneous metastasis. Furthermore, a strong immune-memory effect is observed 40 days later after photothermal tumour ablation with those nanoparticles, which together with anti-CLTA4 therapy would be able to effectively protect mice from tumour rechallenge. Using
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reformulated nanoparticles, we further demonstrate that such a treatment strategy may also be realized with systemic injection of all agents. Therefore, our study presents a new cancer treatment strategy, which may be able to eliminate primary tumours, attack and kill spreading metastatic tumours, and nally offer immune-memory protection to prevent tumour relapse. Considering that all components in our nanoparticles are FDA-approved ones, our technique may indeed have a good chance for future clinical translation.
Methods
Materials. PLGA, ICG, R837(TLR7 ligand) and polyvinyl alcohol (PVA) were obtained from Sigma-Aldrich. Polyethylene glycol (PEG) grafted PLGA co-polymer (mPEG-PLGA), 50:50 (w:w), (Mw B5,000:10,000 Da) was purchased from PolySciTech. Dimethyl sulfoxide (DMSO) and dichloromethane (CH2Cl2) were obtained from Sinopharm Chemical Reagent Co. Anti-CTLA-4 used in vivo was obtained from Bioxcell. Antibodies against cell surface markers for ow cytometry (uorescent-activated cell sorting) assay were purchased from eBioscience.
Synthesis of PLGA-ICG-R837 nanoparticles. PLGA-ICG-R837 nanoparticles were formed using o/w single-emulsion method64. Briey, R837 (TLR7 ligand) was dissolved in DMSO at 2.5 mg ml 1. Photothermal agent ICG was dissolved at10 mg ml 1 in DMSO. A total of 38 ml R837 and 6.25 ml ICG were added to 1 ml
PLGA (5 mg ml 1) dissolved in dichloromethane. Then, the mixture was homogenized with 0.4 ml 5% w/v PVA solution for 10 min using Selecta Sonopuls.
The o/w emulsion was then added to 2.1 ml of a 5% w/v solution of PVA to evaporate the organic solvent for 4 h at room temperature. PLGA-ICG-R837 nanoparticles were obtained after centrifugation at 3,500g for 20 min.
Synthesis of PLGA-PEG-ICG-R837 nanoparticles. PLGA-PEG-ICG-R837 nanoparticles were formulated based on a previously described protocol with slight modications65. Briey, R837 was dissolved in DMSO at 2.5 mg ml 1 and ICG was dissolved in DMSO at 10 mg ml 1. 60 ml R837 and 25 ml ICG solutions in DMSO were added to 1 ml mPEG-PLGA (10 mg ml 1) dissolved in acetonitrile.
Then, the mixture was dropwisely added into 5 ml water. After 1 h stirring and 12 h standing, PLGA-PEG-ICG-R837 nanoparticles were obtained after centrifugation at 22,000g for 5 min.
Nanoparticle characterization. The morphology and structure of PLGA-ICGR837 were characterized by transmission electron microscopy using a FEI Tecnai F20 transmission electron microscope. The ultravioletvisibleNIR absorbance spectra were recorded by a PerkinElmer Lambda 750 ultravioletvisibleNIR spectrophotometer. The dynamic diameters of nanoparticles were determined by a Zetasizer Nano-ZS (Malvern Instruments, UK). The encapsulation efciency of R837 was determined by a HPLC (Agilent 1260) with a ultravioletvisible detector at 325 nm. Acetonitrile was used as the mobile phase. The ICG encapsulated in nanoparticles was measured by ultravioletvisibleNIR.
Cellular experiments. 4T1 murine breast cancer and CT26 colorectal cancer cell lines were originally obtained from American Type Culture Collection (ATCC) and cultured under recommended conditions. Dendritic cells were isolated from the bone marrow of B8-week-old BALB/c mice purchased from Nanjing Peng Sheng
Biological Technology Co. Ltd. according to an established method66. For in vitro DC stimulation experiments, DCs were treated with free R837, PLGA-ICG or PLGA-ICG-R837 for 12 h. Alternatively, residues of 4T1 cells after photothermal ablation with either PLGA-ICG or PLGA-ICG-R837 were also added into DC culture using a transwell system. Lipopolysaccharide (LPS, Sigma) at 1 mi ml 1 was used as the positive control. After various treatments, DCs were stained with anti-CD11c FITC, anti-CD86 PE and anti-CD80 APC, and then sorted by ow cytometry (BD FACSCalibur).
In vivo experiments. Female BALB/c mice (68 weeks) were purchased from Nanjing Peng Sheng Biological Technology Co Ltd and used under protocols approved by Soochow University Laboratory Animal Center. Mice were divided into groups randomly. For the rst tumour inoculation, 4T1 cells or CT26 cells (1 106) suspended in PBS were subcutaneously injected into the left ank of
each female BALB/c mouse. For the second tumour inoculation, which was conducted 7 days later, 4T1 cells (2 105) or CT26 cells (4 105) suspended in
PBS were subcutaneously injected into the right ank of each female BALB/c mouse. The tumor volume was calculated according to the following formula: width2 length 0.5.
To establish lung metastases, fLuc-4T1 cells (1 105), a gift from PerkinElmer
Inc., were administered intravenously via tail vein infusion into each BALB/c mouse. Mice were injected with the relevant substrate before bioluminescence
imaging, which was carried out using an in vivo imaging instruments (IVIS) spectrum system with 60 s exposure time. Besides, lungs were analysed ex vivo at different days after i.v. injection of 4T1 tumour cells. Mice were killed right after being injected with India ink through the trachea. Tumour metastasis sites subsequently appeared as white nodules on the surface of black lungs and were counted under a microscope.
To establish 4T1 orthotopic murine breast cancer model with spontaneous metastasis, fLuc-4T1 cells (5 105) suspended in PBS were inoculated into the
breast pad of each mouse. Two weeks later, the primary tumour on each mouse was removed by PLGA-ICG-R837-based photothermal therapy or surgery. In the following days, mice were imaged by an IVIS spectrum system to monitor the spontaneous metastasis. For anti-metastasis treatment, mice were i.v. injected with 20 ml anti-CTLA-4 in 0.2 ml PBS on day 1, 4 and 7, after their primary visible tumours were removed by photothermal therapy or surgery.
To study PTT-triggered cancer immunotherapy upon systemic administration, mice bearing CT26 tumours were i.v. injected with PLGA(-PEG)-ICG-R837 nanoparticles (6 mg kg 1 R837, 8 mg kg 1 ICG). 24 h later, their rst tumours were removed by surgery or PTT (808 nm laser, 0.8 W cm 2, 10 min ). For surgery or PTT with nanoparticles i.v. injected with 10 mg anti-CTLA4 in 0.2 mL PBS on days 1, 4 and 7. Afterwards, the growth of the secondary tumour was carefully monitored.
Cytokine detection. Serum samples were isolated from mice after various treatments and diluted for analysis. Tumour necrosis factor (TNF-a, Dakewe biotech), interferon gamma (IFN-g, Dakewe biotech), IL-12 (Dakewe biotech) and IL-6 (Dakewe biotech) were analysed with ELISA kits according to vendors protocols.
Ex vivo analysis of different groups of T cells. To study the immune cells in secondary tumours, tumours were harvested from mice in different groups and stained with anti-CD3-FITC (Biolegend, Clone: 17A2, Catalog: 100204), anti-CD8a-APC (Biolegend, Clone: 53-6.7, Catalog: 100712), anti-CD4-PerCP (Biolegend, Clone: GK1.5, Catalog: 100432) antibodies according to the manufacturers protocols. Briey, tumour tissues were cut into small pieces and put into a glass homogenizer containing PBS (pH7.4) with 2% heat-inactivated fetal bovine serum67. Then, the single-cell suspension was prepared by gentle pressure with the homogenizer without addition of digestive enzyme. Finally, cells were stained with uorescence-labelled antibodies after the removal of red blood cells (RBC) using the RBC lysis buffer. Cytotoxic T lymphocytes (CTL) and helperT cells were CD3 CD4-CD8 and CD3 CD4 CD8-, respectively. To analyse
CD4 helper T cells, cells in the secondary tumour were further stained with
anti-CD3-FITC (eBioscience, Clone: 145-2C11, Catalog: 11-0031), anti-CD4-PerCP (Biolegend, Clone: GK1.5, Catalog: 100432), and anti-Foxp3-PE (eBioscience, Clone: NRRF-30, Catalog: 12-4771) antibodies according to the standard protocols. CD4 helper T cells were classied into effective T cells
(CD3 CD4 Foxp3-) and regulatory T cells (Tregs) (CD3 CD4 Foxp3 ).
For analysis of memory T cells, lymph nodes harvested from mice after various treatment were stained with anti-CD3-FITC (eBioscience, Clone: 145-2C11, Catalog: 11-0031), anti-CD8-PerCP-Cy5.5 (eBioscience, Clone: 53-6.7, Catalog: 45-0081), anti-CD62L-APC (eBioscience, Clone: MEL-14, Catalog: 17-0621) and anti-CD44-PE (eBioscience, Clone: IM7, Catalog: 12-0441) antibodies according to the manufacturer) antibodies. The single-cell suspension from lymph nodes was prepared using the same protocol to that of tumour tissues67. Central memoryT cells (TCM) and effector memory T cells (TEM) were CD3 CD8 CD62L
CD44 and CD3 CD8 CD62L-CD44 , respectively. All these antibodies
used in our experiments were diluted B200 times.
Data availability. All relevant data are available from the authors.
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Acknowledgements
This article was partially supported by the National Basic Research Programs of China (973 Program) (2012CB932601), the National Natural Science Foundation of China (51525203, 51132006, 31300824), a Juangsu Natural Science Fund for Distinguished Young Scholars (BK20130005), the Collaborative Innovation Center of Suzhou Nano Science and Technology, and a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.
12 NATURE COMMUNICATIONS | 7:13193 | DOI: 10.1038/ncomms13193 | http://www.nature.com/naturecommunications
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Author contributions
Q.C. and L.X. contributed equally to this work. Q.C. and Z.L. conceived the project. Q.C.,L.X., C.L. and C.W. performed the experiments and analysed the results. R.P. provided useful suggestions to this work. Q.C., L.X.and Z.L. wrote the manuscript.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications
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How to cite this article: Chen, Q. et al. Photothermal therapy with immune-adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapy. Nat. Commun. 7, 13193 doi: 10.1038/ncomms13193 (2016).
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Copyright Nature Publishing Group Oct 2016
Abstract
A therapeutic strategy that can eliminate primary tumours, inhibit metastases, and prevent tumour relapses is developed herein by combining adjuvant nanoparticle-based photothermal therapy with checkpoint-blockade immunotherapy. Indocyanine green (ICG), a photothermal agent, and imiquimod (R837), a Toll-like-receptor-7 agonist, are co-encapsulated by poly(lactic-co-glycolic) acid (PLGA). The formed PLGA-ICG-R837 nanoparticles composed purely by three clinically approved components can be used for near-infrared laser-triggered photothermal ablation of primary tumours, generating tumour-associated antigens, which in the presence of R837-containing nanoparticles as the adjuvant can show vaccine-like functions. In combination with the checkpoint-blockade using anti-cytotoxic T-lymphocyte antigen-4 (CTLA4), the generated immunological responses will be able to attack remaining tumour cells in mice, useful in metastasis inhibition, and may potentially be applicable for various types of tumour models. Furthermore, such strategy offers a strong immunological memory effect, which can provide protection against tumour rechallenging post elimination of their initial tumours.
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