Surgical Tumor-Derived Personalized Photothermal Vaccine

Feb 21, 2019 - Personalized cancer vaccines show great potential in cancer immunotherapy by inducing an effective and durable antitumor response...
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Surgical Tumor-Derived Personalized Photothermal Vaccine Formulation for Cancer Immunotherapy Xinyu Ye, Xin Liang, Qian Chen, Qianwei Miao, Xiuli Chen, Xudong Zhang, and Lin Mei ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b07371 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Surgical Tumor-Derived Personalized Photothermal Vaccine Formulation for Cancer Immunotherapy Xinyu Ye1, 2, 4 #, Xin Liang1, #, Qian Chen5, Qianwei Miao6, Xiuli Chen2, 4, Xudong Zhang1, 3, *, Lin Mei1, 4, *

1School

of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Guangzhou 510275, P.R. China

2School

of Life Sciences, Tsinghua University, Beijing 100084, P.R. China.

3School

of Medicine (Shenzhen), Sun Yat-sen University, Guangzhou 510080, China

4Graduate

School at Shenzhen, Tsinghua University, Shenzhen, China. Shenzhen 518055, P.R. China.

5Department

6School #

of Bioengineering, University of California, Los Angeles, California 90095, USA.

of Life Science, Lanzhou University, Lanzhou 730000, P.R. China

These authors contributed equally to this work.

*Correspondence to: Xudong Zhang, Email: [email protected]; Lin Mei, Email: [email protected]

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Abstract Personalized cancer vaccines show great potential in cancer immunotherapy by inducing an effective and durable anti-tumor response. However, the limitation of neoantigen identification, low immunogenicity and weak immune response hamper the development of personalized cancer vaccines. The surgically removed tumor contains tumor antigens specific to the patient, which provides promising source for personalized cancer vaccines. Here, we utilized the surgically removed tumor to prepare a personalized photothermal vaccine combined with the PD-1 checkpoint blockade antibody to prevent tumor relapse and metastasis. Black phosphorus quantum dot nanovesicles (BPQD-CCNVs) coated with surgically removed tumor cell membrane were prepared and loaded into a thermosensitive hydrogel containing GM-CSF and LPS.

The sustained

release of GM-CSF from the hypodermic injection of Gel-BPQD-CCNVs effectively recruited dendritic cells to capture tumor antigen. NIR irradiation and LPS stimulated the expansion and activation of DCs, which then travelled to the lymph nodes to present antigen to CD8+ T cells. Moreover, the combination with PD-1 antibody significantly enhanced tumor-specific CD8+ T cell elimination of the surgical residual and lung metastatic tumor. Hence, our work may provide a promising strategy for the clinical development of a personalized cancer vaccine.

Key words: black phosphorous, immunotherapy, photothermal, cancer cell membrane, personal vaccine

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Cancer immunotherapy refers to a mechanism to boost the host immune system to respond to the tumor and achieve a durable antitumor response.1,2 The checkpoint blockade antibody and chimeric antigen receptor T cell (CAR-T) have achieved good clinical responses in many types of cancers.3 Cancer vaccines including neoantigen can activate immune system to generate a robust anti-tumor immune response.4, 5 However, the huge cost of personal mutant neoantigens identification has hindered its clinical application.6 In addition, neoantigens differ between patients, making it difficult to design a common commercial vaccine.7 Thus, neoantigen identification-based personalized cancer vaccines still have many limitations to their clinical application.6 Surgery is the main option for the treatment of most solid tumors. However, surgery often suffers the risk of relapse because of incomplete resection of the tumor.8 Importantly, the removed tumor holds important information about the patient, including tumor-penetrating lymphocytes, mutations of genes that function as neoantigens, and the metabolism checkpoint signature.9 Thus, the removed tumor harbors neoantigens specific to the patient.6 Whole cell lysate-based cancer vaccines have been developed with a limited response in the past decade, which may be hindered by the formulation of the vaccine and the immune checkpoint.10-12 Whole-cell vaccine contains all antigens that can be recognized by the immune system, but it usually fails to cause significant therapeutic benefits because of the interference from the vast majority of normal cellular constituents, like housekeeping genes, carbohydrates and lipids. On the other hand, there are also many antigenic motifs on the cell surface, using cancer cell membrane to develop vaccine can reduce the contamination of intracellular materials and improve the specific response of immune system.13, 14

Nanoparticles have been extensively used as carriers of tumor antigens because they can be efficiently taken up by antigen presenting cells (APCs), including DCs and macrophages.15, 16 Thus, the nanoparticle is an immune adjuvant for cancer vaccines.16-18 Local heat can cause the release of immunomodulatory molecules to enhance DC functions.19, 20 The mild temperature increase upregulates the expression of MHC-I and MHCII molecules, as well as CD80 and CD86 (co-stimulatory molecules) of DCs, augments migration to draining ACS Paragon Plus Environment

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lymph nodes, and it further enhances the ability to cross-present antigens and stimulate T cells.19 In addition, local heat can increase blood flow, vascular permeability and interstitial pressure to facilitate T cell entry into tumors, which leads to an improved antitumor immune response and delayed tumor growth.19However, the activation of tumor-infiltrated CD8+ T cells are inhibited by the tumor cell ligand PD-L1 through interactions with PD-1.21 Thus, using PD-1 checkpoint blockade antibodies can revitalize the cancer immunotherapy and enhance the anti-tumor effect.22, 23

Black phosphorus quantum dots (BPQDs), a photothermal material with a high photothermal conversion efficiency and good biodegradable properties, are irradiated by NIR to allow the local release of heat.15, 16, 24, 25

Herein, we utilized surgically removed tumor to prepare a personalized photothermal vaccine and combined

it with the checkpoint blockade antibody PD-1 to prevent tumor relapse and metastasis (Figure 1a). Surgically removed tumor cell membrane-coated black phosphorus quantum dot nanovesicles (BPQD-CCNVs) were prepared and loaded into a thermosensitive hydrogel containing GM-CSF and lipopolysaccharide (LPS). Moreover, sustained release of GM-CSF and LPS from the hydrogel could recruit and activate DCs, respectively. Subsequently, the mature DCs activated the tumor-specific T cells to attack cancer cells through cross-presentation in peripheral lymph organs. Furthermore, PD-1 antibody reinvigorated the exhausted CD8+ T cells. Thus, the vaccine could induce a strong and durable immunological response to eliminate residual and metastatic cancer cells (Figure 1b). The present surgically removed tumor-derived photothermal vaccine formulation combined with immune checkpoint therapy holds promise for clinical applications in cancer therapy.

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Results Preparation and characterization of Gel-BPQD-CCNVs. The BPQDs were prepared using a modified liquid exfoliation method.25,

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The morphology of BPQDs

(Figure 1c) was characterized by transmission electron microscopy (TEM). High resolution TEM image of BPQDs showed lattice fringes with a spacing of 0.33 nm, which was ascribed to the plane of the BP crystal (Figure S1a). In addition, the surface structure of the BPQDs were examined by atomic force microscopy (AFM). The height profiles range is from 1.2 to 3.2 nm, corresponding to BPQDs with 2-5 layers (Figure S1d). To prepare the BPQD-CCNV formulation, cancer cell membranes (CCMs) were collected from the surgical tumors from 4T1 tumor-bearing mice. The BPQDs were mixed with the CCMs and subjected to a Dounce and ultrasonicated to form BPQD-CCNVs. To determine whether the BPQDs had been coated with the CCMs, we stained BPQDs and CCMs with FITC and Cy5.5, respectively. The mixture was then analyzed by confocal microscopy. As shown in Figure 1d, the overlap of the CCMs and BPQDs indicated that the BPQDs were coated with CCMs. Furthermore, TEM was used to further visualize the structure of the BPQDCCNVs. Indeed, BPQDs had been sequestered into the CCNV (Figure 1e). Moreover, the diameter and zeta potentials were measured by dynamic light scattering (DLS). The average diameter of BPQD-CCNVs was approximately 120 nm (Figure 1f). Meanwhile, BPQD-CCNVs and CCNVs had similar zeta potentials of approximately -23 mV, suggesting successful coating of the cancer cell membrane. Compared with the BPQDs, the zeta potentials of BPQD-CCNVs were significantly higher, showing an improved stability of the nanoparticles in suspension (Figure S1b, c). The lysate of BPQD-CCNVs showed a similar protein profile compared with the cancer cell lysate and purified cancer cell membrane, indicating that the BPQD-CCNVs harbored the tumor membrane-associated antigens (Figure 1g).

Pluronic F-127 is a widely used thermosensitive biomaterial that is approved by the FDA and has excellent biocompatibility, including mild gelation conditions and effective protection of the loaded protein.27, ACS Paragon Plus Environment

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However, the low mechanical strength and poor gel stability of pluronic F-127 (F-127) limit its applicatio2n.29 Therefore, hyaluronic acid (HA) was used to enhance the mechanical strength of the hydrogel for sustained drug release. To investigate the optimal ratio of HA and F-127, we tested the mechanical strength and payload release of various formulations and finally determined a HP gel (hyaluronic acid-pluronic F-127 hydrogel) concentration of 2 wt% HA and 25 wt% PF-127. The SEM images of the F-127 hydrogel and HP hydrogel are shown in Figure 2 b, c. BPQD-CCNVs, GM-CSF and LPS were then encapsulated into the HP hydrogel to form Gel-BPQD-CCNVs (Figure 2a). Next, we examined the in vitro release of BPQD-CCNVs, GM-CSF and LPS from the hydrogel. Of note, the release profiles of BPQD-CCNVs, GM-CSF and LPS showed similar release kinetic curves. More than 60% of the payloads were released from the hydrogel within 5 days, and there was a quick release period during the first 2 days (Figure 2f). Next, we evaluated the photothermal effect of Gel-BPQD-CCNVs in vitro through NIR irradiation. The temperature change was recorded by infrared thermal camera (Figure 2 d, e). Compared with PBS, the surface temperature of Gel-BPQD-CCNVs increased rapidly within 2 min. We then assessed the stability and degradability of the HP hydrogel in vivo by labelling it with FITC, and the fluorescence of the scaffold was monitored daily using a fluorescence imaging system. As shown in Figure 2g, h, the fluorescence density decreased on a daily basis and completely disappeared on day 6.

In vitro and in vivo immune response of BPQD-CCNVs. Dendritic cells are the most important antigen-presenting cells in mammalian systems and play crucial roles in initiation of the adaptive immune response. Therefore, bone marrow-derived dendritic cells (BMDCs) from Balb/c mice were used to test the interaction of the different formulations with antigen-presenting cells. To examine cellular uptake of the BPQD-CCNVs, we stained the CCMs and BPQDs with Cy5.5 and FITC, respectively. The BMDCs were then incubated with BPQD-CCNVs at 37°C for 24 hours (Figure 3a). Confocal microscopy showed that BMDCs could efficiently take up BPQD-CCNVs (Figure 3b). To confirm the effect ACS Paragon Plus Environment

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of the BPQD-CCNVs on BMDC maturation in vitro, the costimulatory markers CD11c, CD80, CD86, and MHC-II were analyzed by flow cytometry 24 h after administering the different treatments: blank (#1), GelBPQD-CCNVs (LPS-free, #2), Gel-BPQD-CCNVs + NIR (LPS-free, #3), Gel-LPS (#4) or Gel-BPQDCCNVs + NIR (#5). Compared with blank control (#1), the Gel-BPQD-CCNVs + NIR (LPS-free, #3), GelLPS (#4) or Gel-BPQD-CCNVs + NIR (#5)-treated group showed a higher proportion of mature DCs, indicating that heat could promote the maturation of DCs (Figure 3c and Figure S2, S3a). In contrast, the GelBPQD-CCNVs (LPS free, #2)-treated group showed no effect on the change in mature DCs. Moreover, FACS analysis of CD11c+, MHC-II+ cells in each group also showed similar results (Figure 3d and Figure S3b).

To test the photothermal effect in vivo, mice were anesthetized and irradiated with 808-nm (1 W cm-2) for 10 min daily after a subcutaneous injection with different formulations (Figure S4 a, b): (1) PBS, (2) hydrogel, (3) Gel-BPQDs, (4) Gel-BPQD-CCNVs. The temperature change in the injected area was recorded by infrared thermal camera. As shown in Figure 3e, g, the temperature of the injected area of the mice treated with GelBPQDs, Gel-BPQD-CCNVs quickly rose to ~45°C, indicating that being encapsulated in hydrogel or cancer cell membrane did not affect the photothermal effect on the BPQDs. In the in vivo experiment, mice were randomly separated into 4 groups and received different treatments: PBS (#1), Gel-BPQD-CCNV+ NIR (GMCSF free, #2), Gel-BPQDs-CCNVs (#3), Gel-BPQD-CCNVs + NIR (#4). In the NIR-treated groups, mice were anesthetized and irradiated with 808-nm (1 W cm-2) for 10 min daily for 3 days. To confirm the recruitment of DCs, the skin of the treated site in each group was harvested 24 h after irradiation and analyzed by immunofluorescence and flow cytometry. The density of CD11c+ dendritic cells in mice treated with HP hydrogel containing GM-CSF (#3) increased by 3~4-fold compared with the control group (#1). Moreover, the mature CD11c+ dendritic cells in the Gel-BPQD-CCNVs + NIR (#4)-treated group was even higher, which indicated that NIR and the release of GM-CSF from HP hydrogel could recruit DCs to the treated site (Figure 3h). Immunostaining of CD11c+ cells provided similar results to those of the FACS analysis (Figure 3f). To ACS Paragon Plus Environment

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analyze the activation extent of the immune system after treatment in vivo, draining lymph nodes, spleens and peripheral blood were collected and analyzed by flow cytometry. Compared with the Gel-BPQD-CCNVs (#3) group, a significantly higher level of CD11c+ MHC+-positive mature DCs was observed in lymph nodes of mice treated with Gel-BPQD-CCNVs + NIR (#4), indicating that local heat could promote the maturation of DCs in vivo (Figure 3i,j and Figure S5a, b). Cytotoxic T lymphocytes (CD3+ CD8+) play a crucial role in tumor killing, which is regulated by helper T cells (CD3+ CD4+). The percentages of both CD4+ T cell and CD8+ T cell showed an enormous increase in both group #3 and group #4, especially group #4 (Figure 3k and Figure S5c). Furthermore, the percentage of Ki67, the proliferation marker of T cells, was also dramatically increased in Gel-BPQD-CCNVs + NIR (#4)-treated mice (Figure 3l, Figure S5d, e and S6). The percentages of Ki67, CD4+ and CD8+ T cells in spleen and peripheral blood of Gel-BPQD-CCNVs + NIR (#4)-treated mice was similar to that in lymph nodes (Figure S7-S9). Taken together, these data suggested that injection of Gel-BPQD-CCNVs with NIR could trigger a robust immune response and that the cooperation of local heat and LPS adjuvant was necessary for inducing a robust immune response.

In vivo therapeutic efficacy of Gel-BPQD-CCNVs with NIR combined with PD-1 blockade for antitumor recurrence. Immunosuppression in the tumor microenvironment leads to CD8+ T cell exhaustion, which can lead to immune escape and greatly reduce the efficacy of cancer immunotherapy.30 The PD1 antibody can block the PD-1 checkpoint of the T cells, which could prevent T cell exhaustion.31 To examine whether the treatment of Gel-BPQD-CCNVs with NIR could inhibit tumor recurrence after surgery, a 4T1-luc mouse breast cancer tumor model was used in this experiments. Most of the tumor (~99%) was surgically removed on day 7, and the mice were randomly assigned into 6 different treatment groups (Figure 4a): PBS (#1), CCNVs (#2), aPD1 (#3), Gel-BPQD-CCNVs (#4), Gel-BPQD-CCNVs + NIR (#5), Gel-BPQD-CCNVs + NIR + a-PD1 (#6). The cancer cell membrane collected from surgical resected tumors was used to coat the BPQDs to form the ACS Paragon Plus Environment

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vaccine. Tumor growth was monitored by bioluminescence of the 4T1-luc cells, and tumor size was simultaneously measured. Notably, mice treated with Gel-BPQD-CCNVs + NIR (#5) showed significant inhibition of tumor growth compared with the PBS (#1) control group, indicating that the immune response induced by Gel-BPQD-CCNVs with NIR could be translated into an anti-tumor response. The mice treated with Gel-BPQD-CCNVs + NIR + a-PD1 (#6) exhibited the strongest anti-relapse-tumor effect according to the tumor bioluminescence, tumor volume and harvested tumor weight (Figure 4b-d and Figure S10). In addition, a limited antitumor effect was observed in those mice treated with a-PD1 (#3), or Gel-BPQD-CCNVs (#4). Mouse survival time was significantly increased following the treatment with Gel-BPQD-CCNVs + NIR + a-PD1 (#6). In contrast, all mice in the other groups died by day 42 after challenge (Figure 4e). Additionally, the mice in each group also showed normal body weight growth, indicating that no toxicity occurred during treatment with the different formulations (Figure S11).

To further investigate the mechanism responsible for the synergistic anti-tumor effect triggered by Gel-BPQDCCNVs + NIR in combination with a-PD1 therapy, tumor-infiltrating lymphocytes (TILs) of relapsed tumors harvested from the mice were analyzed by immunofluorescence and flow cytometry on day 23. Compared with the control group (#1), the mice that were treated with the different formulations showed a promotion of CD8+ and CD4+ T cell infiltration into the relapsed tumors. In addition, the percentages of CD8+ T cells in relapsed tumors of mice treated with a-PD1 + Gel-BPQD-CCNVs + NIR (#6) were significantly increased to ~14.9%, which was much higher than in the groups treated with CCNVs (mean 2.05%, #2) a-PD1 (mean 8.62%, #3), Gel-BPQD-CCNVs (mean 6.14%, #4) or Gel-BPQD-CCNVs + NIR (mean 9.72%, #5) (Figure 5a, b). Furthermore, the trends of CD4+ T cells were similar to those of CD8+ T cells (Figure S12). In addition, immunostaining of the CD8+ and CD4+ T cells in the tumor tissue showed similar results compared with those of the FACS analysis (Figure 5c, d). IFN-γ and TNF-α, which are mainly secreted by Th1 cells, play important ACS Paragon Plus Environment

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roles in immunotherapy against cancer. Therefore, we measured the concentration of IFN-γ and TNF-α in serum. Mice treated with a-PD1 + Gel-BPQD-CCNVs + NIR (#6) showed significantly higher serum levels of IFN-γ and TNF-α (Figure 5e, f). These results indicated that the anti-tumor immune response triggered by Gel-BPQD-CCNVs + NIR combined with PD1 blockade could successfully prevent tumor relapse.

In vivo therapeutic efficacy for metastatic tumors. To analyze the potential of the photothermal vaccine to treat metastatic tumors, we established a metastatic model that 4T1-luc cells were intravenously injected into mice to mimic the escape of cancer cell from the surgical site. The mice then received the same treatment as described above (Figure 6a). Considering the increased aggressiveness of the lung metastatic tumor model, the dose of a-PD1 used herein was doubled. The spreading and growth of tumors were monitored by bioluminescence of the 4T1-luc cells. Based on the bioluminescence imaging, we found that mice treated with PBS (#1) showed obvious cancer metastasis 7 days after intravenous injection of 4T1-luc cells. Mice treated with CCNVs (#2), a-PD1 (#3), Gel-BPQD-CCNVs (#4) or Gel-BPQD-CCNVs + NIR (#5) also showed significant bioluminescence signals at later stages. In contrast, mice treated with a-PD1 + Gel-BPQD-CCNVs + NIR (#6) showed almost no metastasis (Figure 6b). The lungs of mice in each group were harvested and stained with India ink, and the numbers of metastatic tumor nodules were counted (Figure S13). Only a few metastatic tumor nodules were found in the lungs of mice treated with Gel-BPQD-CCNVs + NIR + a-PD1 (#6) (Figure 6c). These results were also confirmed by hematoxylin and eosin (H&E) staining of the lungs (Figure 6f). The mouse survival rate was 40% for mice treated with Gel-BPQD-CCNVs + NIR + a-PD1 (#6) by day 60. In contrast, all the mice in the other groups ended the experiment by day 52 after challenge (Figure 6d). The mice in each group also showed normal body weight growth (Figure 6e).

In vivo therapy for recurrence of melanoma tumor ACS Paragon Plus Environment

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To further verify the effectiveness of this treatment in another type of cancer, we performed tests in a melanoma cancer tumor model (B16F10-luc carcinoma). C57BL/6 mice were subcutaneously injected with B16F10-luc cells, and the 99% tumor was removed by surgery 7 days later, then, the mice received the treatment as above (Figure 7a). In this model, as shown in the Figure 7, the mice of Gel-BPQD-CCNVs + NIR + a-PD1 treated group (#6) showed the most significant tumor recurrence inhibition (Figure 7b-d) and longest survival time (Figure 7f), which show a similar result to 4T1-luc model. Furthermore, mice in all groups showed a normal weight growth (Figure 7e). The relapsed tumors harvested from the mice were analyzed by FACS and immunofluorescence as above on day 19. The percentages of CD8+ T and CD4+ T cells in relapsed tumors of mice treated with a-PD1 + Gel-BPQD-CCNVs + NIR (#6) were significantly higher than those of other groups (Figure S15a, b).In addition, immunostaining of the CD8+ and CD4+ T cells in the tumor tissue showed similar results compared with those of the FACS analysis (Figure S15c, d). Mice treated with a-PD1 + Gel-BPQD-CCNVs + NIR (#6) showed significantly higher serum levels of IFN-γ and TNF-α (Figure S15e, f). All these results indicated that our strategy could reduce the tumor recurrence significantly in different types of tumor.

Conclusion In this study, we integrated 4T1-luc or B16F10-luc cancer cell membrane-coated BPQDs, GM-CSF, and LPS into a thermosensitive hydrogel that was subcutaneously injected for sustained delivery of the vaccine. The percentage of mature DCs improved both in vitro and in vivo after treatment, which showed the ability of the vaccine to activate DCs. The enhanced recruitment and migration of DCs upon local NIR irradiation were also observed in vaccinated mice. The upregulated expression of Ki67 in the main immune cells, like DCs, CD4+ T cells and CD8+ T cells, in draining lymph node and spleen revealed a significant mobilization of the immune system in the vaccinated mice. Combined with PD-1 blockade immunotherapy, the vaccine could markedly ACS Paragon Plus Environment

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prolong the survival rate of 4T1-luc or B16F10-luc tumor cell-challenged mice after surgery. These results together indicated that our strategy could induce a robust anti-tumor immune response against recurrence and metastasis.

Compared with traditional single-antigen vaccines that are limited by personalized epitope identification, the cancer cell membrane-based vaccine can provide a wealth of autologous tumor antigens. Compared with whole-cell vaccines, our vaccine does not include many housekeeping proteins or other nontumor-related antigens that could interfere with the immune response. Furthermore, the cancer cell membrane can also protect BPQDs from degradation in vitro. For traditional administration, vaccines are repeatedly subcutaneously or intravenously injected into mice to achieve the optimum efficiency, and they are captured by APCs passively via the circulation system. In contrast, we placed a biodegradable and biocompatible hydrogel under the skin as a sustained delivery platform to avoid repeated injection, and local recruitment and stimulation of DCs upon NIR and immunogenic adjuvant can also enhance the uptake rate of vaccines and reduce the auto immune toxicity caused by systemic exposure.

In summary, the anti-tumor immune response triggered by Gel-BPQD-CCNVs + NIR combined with PD1 antibody blockade can effectively reduce cancer recurrence and metastasis after surgical removal of the primary tumor. Tumors are caused by an accumulation of genetic abnormalities that differ between patients, and therefore the identification of individual tumor mutations is necessary for the development of a personalized vaccine; however, despite great advances in genomics and other technologies in science, it remains a great challenge. In in the future, we can potentially use the tumor resected from the patient to form a cancer-cell-membrane-based vaccine to provide individual cancer-specific neoepitopes that can be recognized by autologous T cells. Thus, our treatment strategy may have a meaningful impact on personalized treatment for cancer and help to promote the application of personalized anti-cancer vaccines in the clinic. ACS Paragon Plus Environment

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Methods Materials. Bulk black phosphorous (BP) was obtained from Smart-Elements (company) and stored in a dark Ar glovebox. The FITC-PEG-NH2 was purchased from ToYong Bio (Shanghai, China). Cy5.5 NIR fluorescent dye was purchased from Invitrogen (Carlsbad, USA). PBS (pH 7.4), trypsin-EDTA, RPMI 1640 Basic Media, fetal bovine serum and penicillin-streptomycin, were purchased from Life Technologies (AG, Switzerland). Granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased from Sino Biological Inc. (Beijing, China). Recombinant mouse interleukin-4 (IL-4) was purchased from Invitrogen (Carlsbad, USA). Lipopolysaccharide (LPS) and PF-127 were purchased from Sigma-Aldrich (Santa Barbara, USA). Antimouse PD-1 antibody (a-PD1) and antibodies for the flow cytometry assay were purchased from BioLegend (San Diego, USA). Antibodies used for immunostaining were purchased from Abcam (Cambridge, UK). All other chemicals used were analytical reagent grade without further purification.

Preparation of BP quantum dots. The BPQDs were prepared by a modified liquid exfoliation technique. In brief, 20 mg of the bulk BP powder was added to 40 mL of NMP in a 50-mL conical tube and sonicated in ice water with a probe sonicator for 4 h (Amplifier: 25%, 5 s on/5 s off), followed by sonication for 8 h in an ice bath. The resulting dispersion was centrifuged at 3000 rpm for 30 min to remove the bulk BP, and the supernatant with the BPQDs was carefully transferred to another tube. The supernatant was subsequently centrifuged at 20000 rpm for 2 h to remove the NMP. The obtained BPQDs were stored in 1 ml of NMP at 4°C.

Cancer cell membrane derivation. Cancer cell membranes were collected from either cultured 4T1-luc cells, B16F10-luc cells or surgically ACS Paragon Plus Environment

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resected tumor. The 4T1-luc cells and B16F10-luc were purchased from Cobioer (Nanjing, China) and maintained in RPMI 1640 and DMEM supplemented with 10% FBS, 1% penicillin-streptomycin and 1 µg mL-1 puromycin, respectively. Tumors were cut into piece and pressed gently with a homogenizer containing PBS (7.4) and 2% FBS to obtain a single-cell suspension. To harvest the membrane, cells were grown in 150mm cell culture dishes to full confluency, collected by scraping and washed three times with PBS at 500 g for 5 min. The cells were resuspended in HM lysis buffer containing 0.25 M sucrose, 1 mM EDTA, 20 mM HEPES-NaOH, pH 7.4 and 1.0 mM phenylmethanesulfonyl fluoride (PMSF, Beyotime), and then they were subjected to a glass Dounce homogenizer in an ice bath 200 times. The entire solution was centrifuged at 3000 × g for 5 min, and the supernatant was subsequently centrifuged at 15000 × g for 1 h. The resulting vesicles of cancer cell membrane were washed once with PBS and stored in HM lysis buffer with PMSF at -20°C.

Preparation and characterization of cancer cell membrane-coated BPQD nanovesicles (BPQD-CCNVs). To synthesize BPQD-CCNVs, 1 mL 200 µg/ml BPQDs which were dispersed in PBS mixed with the vesicles of cancer cell membrane and 200 µg of membrane protein was subjected to gentle pressure in a glass Dounce homogenizer and then sonicated in a 2-mL Eppendorf safe-lock tube for 5 min by KQ-300 GDV bath sonicator (Kunshan Ultrasonic Instruments, China) at a frequency of 40 kHz and power of 100 W. The obtained nanoparticles were stored at -20°C.

Preparation of Gel-BPQD-CCNVs. The HP hydrogel was prepared using a cold method. In brief, HA (2 wt%) and Pluronic F-127 (25 wt%) were simply mixed in cold distilled water with 20 ng/ml GM-CSF and 1 μg/ml LPS and then stored in a refrigerator (4°C) overnight until the polymer had completely dissolved. All hydrogels used in this research included GMCSF and LPS without any special instructions. The BPQD-CCNVs were then added in the HP hydrogel and blown to blend by pipette, and the mixture was stored at 4°C before use. ACS Paragon Plus Environment

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Morphology and characterization. The TEM images of BPQDs, CCNVs and BPQD-CCNVs were obtained by transmission electron microscopy (Tecnai G2 Spirit120kV). CCNVs and BPQD-CCNVs were first stained with uranyl acetate. The SEM images of hydrogels and Gel-BPQD-CCNVs were obtained using a scanning electron microscope (MDTC-EQ-M1801). The size and zeta potential of the BPQD-CCNVs were measured by dynamic light scattering (Nano-ZS, model MPT2; Malvern Instruments, Worcestershire, UK).

In vitro release study. BPQD-CCNVs were first labeled with Cy5.5 for the in vitro release study. Next, 500 μL of Gel-BPQDCCNVs solution was added to an 8-µm pore-size cell culture insert and then incubated in a 37°C incubator until the hydrogel formed. PBS (1 ml) and the inserts were then added to each well, followed by gentle shaking at a constant temperature of 37°C. The medium (50 μL) was then removed, and the same volume of PBS was added back at each at each time point. The GM-CSF level was measured using an ELISA kit according to the manufacturer’s protocol. BPQD-CCNVs were evaluated by fluorescence at an excitation of 675 nm and emission of 695 nm.

Mice. Female BALB/c mice and C57BL/6 (6-8 weeks old) used in the experiments were obtained from the Guangdong Medical Laboratory Animal Center (Guangdong, China). All animal experiments followed the animal protocols approved by the Animal Care and Use Committee of Sun Yat-Sen University.

In vitro uptake and activity. Dendritic cells were collected from the bone marrow of 6~8-week-old female BALB/c mice. The harvested ACS Paragon Plus Environment

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cells were cultured in RPMI 1640 supplemented with 10% FBS, 1% penicillin-streptomycin, 10 ng/ml IL-4 and 20 ng/ml GM-CSF in 24-well plates at a concentration of 1x106. Half of the medium was replaced every two days, and the nonadherent cells were harvested as dendritic cells on day 6. To generate fluorescent BPQDCCNVs, BPQDs were stirred with FITC-PEG-NH2 for 5 h, and Cy5.5 was added to the CCNVs with shaking at 4°C overnight. To each well was added an 8-µm pore-size cell culture insert containing 500 μL of the GelBPQD-CCNVs suspension, followed by incubation at 37°C until the hydrogel formed. The cells were incubated with the insert at a constant temperature of 37°C with shaking for 48 h and then stained with DAPI and analyzed by confocal microscope (Olympus, FV1000, Olympus Optical, Japan).

To study the activation of DCs, the cells were divided into 5 groups to receive different treatments: blank (#1), Gel-BPQD-CCNVs (LPS free, #2), Gel-BPQD-CCNVs + NIR (LPS-free, #3), Gel-LPS (#4) and GelBPQD-CCNVs + NIR (#5). The DCs were incubated with the formulation for 48 h before irradiation. After irradiation with an 808-nm NIR laser to keep the temperature of medium around 42°C for 10 min, the cells were incubated at a constant temperature of 37°C with shaking for 24 h and then collected, washed three times with PBS and stained with anti-CD11c-APC, anti-CD80-FITC, anti-CD86-PE, and anti-MHC II-Percp/Cy5.5 antibodies according to the manufacturer’s protocols. The stained cells were sorted using a Becton Dickinson FACSCanto-II flow cytometer and analyzed with FlowJo software.

In vivo activation. The mice were randomly separated into 4 groups (n=4) and subjected to different treatments: PBS (200 μL, #1), Gel-BPQD-CCNVs + NIR (200 μL, 50 μg/ml BPQDs, 1 μg/ml LPS #2), Gel-BPQD-CCNVs (200 μL, 50 μg/ml BPQDs, 20 ng/ml GM-CSF and 1 μg/ml LPS ,#3), Gel-BPQD-CCNVs + NIR (200 μL, 50 μg/ml BPQDs, 20 ng/ml GM-CSF and 1 μg/ml LPS, #4). All the formulations were injected subcutaneously in the left back of the mice. For NIR treatment, the mice were anaesthetized and irradiated with 808-nm NIR (1 W ACS Paragon Plus Environment

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cm-2) across the entire region of the formulation for 10 min daily for 3 days. The highest temperature was controlled at approximately 42°C in real time through an infrared thermal camera.

Twenty-four hours after the prior irradiation, the skin of the irradiated area was harvested. To assess dendritic cell recruitment, the skin was cut into piece and pressed gently with a homogenizer containing PBS (7.4) and 2% FBS to obtain a single-cell suspension. These cells were stained with anti-CD11c-APC, sorted using a Becton Dickinson FACSCanto-II flow cytometer and analyzed with FlowJo software.

The popliteal lymph nodes and spleens were collected from each mouse, and single-cell suspensions were obtained using the method described mentioned. Lymphocytes in peripheral blood were obtained according to the manufacturer’s protocols. To assess dendritic cell maturation, cells were stained with anti-CD11c-APC, anti-CD80-FITC, anti-CD86-PE, and anti-MHC II -Percp/Cy5.5 antibodies. To examine T cell proliferation, cells were stained with anti-CD3-FITC, anti-CD4-PE, anti-CD8-APC and anti-Ki67-PE/Cy7 antibodies. All samples were sorted using a Becton Dickinson FACSCanto-II flow cytometer and analyzed with FlowJo software.

In vivo tumor model. To study the antitumor therapeutic effect, female BALB/c mice or C57BL/6 mice (6-8 weeks old) were challenged with 1x106 4T1-luc cells or B16F10-luc cells on the right side of the back on day 0. After 7 days, 99% of the resulting tumor (~200 mm3 in volume) was resected with 1% remaining to mimic the presence of the residual microtumor in the surgical bed in the clinic. Mice were anesthetized with isoflurane and maintained using a nose cone. All surgical instruments were sterilized with high-pressure stream. The wound was closed with a surgical suture. For the metastasis model, mice were injected intravenously with 2x105 4T1luc via the tail vein after surgery. The mice were separated randomly into 5 groups (n=7 or 10) and received ACS Paragon Plus Environment

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one of the following treatments: (1) PBS (200 μL), (2) CCNVs, (3) a-PD1, (4) Gel-BPQD-CCNVs (200 μL, 50 μg/ml BPQDs), (5) Gel-BPQD-CCNVs + NIR (200 μL, 50 μg/ml BPQDs), (6) PD1 (5 mg aPD-1 kg-1) + Gel-BPQD-CCNVs + NIR (200 μL, 50 μg/ml BPQDs). For NIR treatment, the mice were anaesthetized and irradiated with 808-nm NIR (1 W cm-2) across the entire region of the formulation for 10 min daily for 3 days. The highest temperature was controlled at approximately 42°C in real time through an infrared thermal camera. Anti-PD-1 antibody (5 mg kg-1) was injected on days 9, 12, 15 and 18 via the tail vein. Bioluminescence Images of tumors were obtained by fluorescence imaging system (Xenogen IVIS Spectrum, USA). Tumor size was measured by digital caliper and the volume (mm3) was calculated as follows: width2 × length × 0.5. Tumors were measured every 3 days, and the mice were euthanized when the tumor volume exceeded 2000 mm3. For the metastatic lung tumor, mice were sacrificed and injected intratracheally with India ink. The lung tumors were fixed in Fekete’s solution (1 ml formalin, 0.5 ml glacial acetic acid, 10 ml 70% ethanol). After 4 h, tumor lesions appeared white, while normal lung tissue remained stained. Tumor metastatic sites were counted carefully. The lung tissue sections of mice with lung tumors were stained with H&E.

Cytokine detection. Serum samples of each group were collected and diluted for analysis after the different treatments. IFN-γ (Invitrogen) and TNF-α (Invitrogen) levels were measured by ELISA kits according to the manufacturer’s protocols.

Analysis of different T cell groups in the recurrent tumor. To study the mechanism, the immune cells in the recurrent tumor were analyzed. Tumors were collected from each mouse, and single-cell suspensions were obtained according to the method described above and then stained with anti-CD3-FITC, anti-CD4-PE, anti-CD8-APC antibodies. Cells were sorted by Becton Dickinson FACS Canto-II flow cytometer and analyzed with FlowJo software. In addition, the tumors were snap frozen ACS Paragon Plus Environment

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in OCT (optimal cutting temperature) medium, cut into 10-μm-thick sections, and mounted on slides. Sections were fixed in 4% polyoxymethylene for 15 min, then washed with PBS three times and dried at room temperature. After blocking with BSA (3%) for 30 min, the sections were incubated with anti-CD4 and antiCD8 primary antibodies at 4°C overnight. On the following day, the sections were washed with PBS 3 times, then incubated with secondary antibodies at room temperature for 2 h in the dark, stained with DAPI and analyzed by confocal microscope.

Statistical analysis. All results are presented as the mean ± s.d. or the mean ± s.e.m. Comparisons between the study groups (multiple comparisons) were performed using one-way ANOVA and the Tukey posttest. Survival curves were compared using a log-rank test. All experiments, unless otherwise stated, were performed in biological replicates. All statistical analyses were performed using GraphPad Prism 5 software (* P