Research ArticleAPPLIED SCIENCES AND ENGINEERING

An implantable blood clot–based immune niche for enhanced cancer vaccination

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Science Advances  25 Sep 2020:
Vol. 6, no. 39, eabb4639
DOI: 10.1126/sciadv.abb4639

Abstract

Cancer immunotherapy using cancer vaccines has shown great potential in the prevention and treatment of cancer. Here, we report an implantable autologous blood clot scaffold for enhanced cancer vaccination. It comprises a gel-like fibrin network formed by coagulation of blood to trap a large number of red blood cells. Upon implantation, the cross-linked RBCs in the blood clot can attract and recruit a great number of immune cells, leading to the formation of an “immune niche.” Encapsulated with tumor-associated antigen and adjuvant, the blood clot vaccine (BCV) can induce a robust anticancer immune response. The BCV combined with immune checkpoint blockade effectively inhibits tumor growth in B16F10 and 4T1 tumor models. The proposed implantable blood clot cancer vaccine can be readily made by mixing the blood from patients with cancer with immunomodulating agents ex vivo, followed by reimplantation into the same patient for personalized cancer immunotherapy in future clinical translation.

INTRODUCTION

Cancer immunotherapy represents a change in paradigm for cancer treatment by using the host immune system to control cancer progression (14). Such therapy, including cancer vaccines, immune checkpoint blockade (ICB), and T cell adoptive therapy, is becoming one of the mainstream approaches for cancer treatment (5). Among them, cancer vaccines aim to generate long-lasting antitumor immunity by providing multiple tumor antigens for immune recognition and stimulating specific immunity against tumors. However, the research on tumor vaccine is still at an early stage. Except for sipuleucel-T, a dendritic cell (DC) vaccine for prostate cancer, most cancer vaccines have shown little clinical efficacy. Possible reasons include (i) absence of a high immunogenic tumor antigen pool, (ii) immunosuppressive nature of the tumor microenvironment [e.g., a high level of programmed death-ligand 1 (PD-L1) expression and the lack of a durable CD4 and CD8 T cell expansion], (iii) low efficacy to recruit and activate antigen-presenting cells (APCs), and (iv) an effective delivery technology for the vaccine components. Previous studies have reported that cancer vaccines can cooperate with ICB therapy to exert synergistic therapeutic effects by enhancing the number of infiltrating tumor-specific T cells and by reducing the immune checkpoint molecules (4, 6, 7). However, further efforts are still demanded to address the current challenges in the field of cancer vaccines.

Advances in biomaterials engineering have been offering new opportunities to improve cancer vaccine design (8, 9). Various sophisticated biomaterials have been exploited, from micro-/nanodelivery systems to depot platforms for vaccine delivery (1014). These systems augment current cancer vaccine strategies by sustained delivery of multiple immunomodulating agents, targeting the specific site and prolonging the in vivo bioactivity, thereby producing more robust and lasting immune effects compared with naked vaccination (15). Among them, gel systems have been widely studied as platforms for diverse therapeutic cargo delivery, including immunomodulatory factors and cancer vaccines (16). Although promising, the clinically used gel drug delivery systems are still limited. Formulation of many gel delivery systems often requires multiple synthetic steps. In addition to the high cost of developing gel-based drug delivery systems, the biocompatibility of synthetic biomaterials may also hinder clinical translation because of the lengthy regulatory process; the physiological interaction and metabolism of such systems and/or their degradation products still need further investigation before clinical practice (1719).

Coagulation or clotting is a natural process that transforms blood from a liquid to a gel-like material. Fibrin is generated in coagulation and intertwined into a hydrated fibrous network, which traps blood cells to form a blood clot scaffold (20). Red blood cells (RBCs) account for the most abundant cell type in the blood clot scaffold (21). RBCs have a prominent role in modulating the innate immune system (22, 23). For example, RBCs may promote immune activation when inflammation or oxidative stress occurs. Oxidized or senescent RBCs cause lipopolysaccharide-induced DCs to exhibit a fully mature phenotype, which is contrary to the function of healthy RBCs (24). Resident macrophages tend to work as a scavenger to remove old or damaged RBCs, while hemoglobin and heme released from damaged RBCs induces the polarization of macrophage to the proinflammatory (M1) state with the secretion of proinflammatory cytokines (25). Besides, the proliferation and survival of T cells could be enhanced by oxidized/aged RBCs as reported (2628).

Here, inspired by the coagulation process, we have developed an implantable natural blood clot scaffold for cancer vaccine delivery by using the autologous blood (Fig. 1A). Because of the intrinsic immune-stimulating effect, we found blood clot itself could attract and recruit various types of immune cells, including macrophage, DCs, B cells, T cells, and natural killer (NK) cells, forming an immune niche with a rich variety and abundant immune cells. We hypothesized that the implantable blood clot scaffold could be used to load tumor antigens and adjuvants, thereby stimulating the recruited immune cells to become tumor antigen–specific immune cells and form a potent, personalized cancer vaccine. In this initial vaccine design, we chose Toll-like receptor 9 (TLR9) agonists as the adjuvant because of its demonstrated efficacy in a number of cancer studies (29). Here, we demonstrated that a cancer vaccine constructed by encapsulating TLR9 agonist CpG oligodeoxynucleotide (CpG-ODN), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor-associated antigen (TAA) evoked a long-term prophylactic immunity and strong antitumor effects in mouse B16F10 and 4T1 tumor models. The potent immune-memory effect induced by postimmunization of combination strategy could effectively protect treated mice from cancer relapse. For therapeutic purposes, the combination of ICB with the blood clot vaccine notably augmented the ICB responses and achieved a synergistic anticancer effect. Because of its efficacy, excellent biocompatibility, and ease of manufacturing, the proposed cancer vaccine warrants further investigation and translational consideration.

Fig. 1 Preparation and characterization of the blood clot vaccine.

(A) Schematic of implantable blood clot vaccine. (B) Photographs of the blood clot. (C) SEM image of the blood clot. Scale bar, 2 μm. (D) Fluorescence image of FITC-OVA distributed in the blood clot (green and yellow) and rhodamine-labeled RBC (orange). Scale bar, 10 μm. (E) Rheology behavior of the blood clot and blood clot vaccine. (F) Loading efficacy of OVA and CpG into the blood clot. (G) In vitro degradation of the blood clot in PBS. Photo credit: Qin Fan, Soochow University. (H) Cumulative release behavior of OVA and CpG from the blood clot. (I) Volume change of blood clot after subcutaneous implantation. (J) Mass of blood clot taken from mice on different days after implantation. (K) Appearance of blood clot after extraction ex vivo. (L) SEM images of the implanted blood clots over 7 days. Scale bars, 10 μm. Values represent means ± S.E.M. (n ≥ 3). mAb, monoclonal antibody.

RESULTS

The construction and characterization of blood clot vaccine

The therapeutic blood clot vaccine was obtained by mixing the mouse blood with immunomodulating agents followed by a vacuum treatment (Fig. 1B). The components in the clot appeared evenly distributed as observed by scanning electron microscopy (SEM) (Fig. 1C). The immunomodulators could be effectively entrapped into the blood clot (Fig. 1D). The gel-like scaffold could be quickly formed through the coagulation process under 37°C (Fig. 1E and fig. S1A) via a complex cascade of sequential reactions to convert liquid blood to gel and trapping many RBCs along the way. The loading efficiency for OVA and CpG is about 95 and 90%, respectively (Fig. 1F). The ovalbumin (OVA) and CpG-ODN–loaded blood clot retained superior mechanical properties similar to the blank ones (Fig. 1E). A vacuum treatment at 37°C for 2 hours promoted the aging/damage of RBCs, which was confirmed by the decrease in CD47 marker on the cell membrane (fig. S1, B and C).

To study the degradability of the blood clot in vitro, samples were incubated with phosphate-buffered saline (PBS) at 37°C and observed for degradation over time (Fig. 1G). Correspondingly, the OVA and CpG were released in a similar profile from the blood clot (Fig. 1H). The sustained release over 7 days was consistent with the degradation behavior of the blood clot (Fig. 1G). The in vivo degradable pattern was investigated by monitoring the changes of the in situ blood clot over time after implantation (fig. S1D). The volume of the blood clot decreased relatively slowly in the first 3 days and then disappeared entirely in about 15 days, suggesting their excellent biodegradability and biocompatibility (Fig. 1I and fig. S1E). We further took out the implanted blood clots from mice to compare the morphology on different days after implantation. The reduction in size of blood clots correlated with the decrease in mass over time (Fig. 1, J and K). Blood clots retrieved from different time points were further investigated by SEM, which showed that the blood clots were gradually engulfed by phagocytes, and the underlying structure, especially vaccine components, were utterly obscured over time (Fig. 1L).

Immune stimulation and recruitment in vitro and in vivo

The capacity of blood clot vaccines in immune stimulation was next carried out in vitro. Bone marrow–derived DCs (BMDCs) were cultured with the blood clot vaccine for about 20 hours. The blood clot vaccine had a similar effect as the free vaccine in promoting maturation of DCs, with about 45% of BMDCs staining positive for CD11c, CD80, and CD86 (fig. S2, A and B). Other stimulatory molecules, including major histocompatibility complex II (MHCII) and CD40, expressed on BMDCs were significantly up-regulated as well (fig. S2, C to F). Activated DCs can secrete inflammatory cytokines to regulate the immune response. The levels of various cytokines such as tumor necrosis factor–α (TNF-α), interleukin-12 (IL-12), and IL-6 were elevated after coculturing with blood clot vaccine (fig. S2, G to I).

Macrophages, another type of APCs, play a critical role in initiating immune defense responses by the recruitment of other immune cells. After incubation for 24 hours, the blood clot itself stimulated the high expression of CD80 marker on the surface of peritoneal macrophages, indicating the generation of a proinflammatory M1-like phenotype (fig. S3, A and B). The level of proinflammatory expression was further increased, probably because of the release of immune agonists from the blood clot. Stimulatory marker MHCII expressed on the macrophages was also increased (fig. S3, C and D). Meanwhile, the blood clot vaccine triggered the production of proinflammatory cytokines, with markedly increased concentrations of TNF-α and IL-6 in the supernatants compared with the control groups (fig. S3, E and F). Furthermore, the phagocytic efficiency of macrophage was enhanced as determined by flow cytometry and confocal imaging of antigen OVA phagocytosis. The intensity of fluorescein isothiocyanate (FITC)–labeled OVA was found to be higher in cells from the blood clot vaccine group (fig. S4, A and B), and more OVA was internalized into the macrophages (fig. S4C). Together, the above data demonstrated that the immune modulators released from blood clot could effectively stimulate the activation of APCs.

Blood clot induced a proinflammatory immune niche by recruiting and activating various types of immune cells. In our in vivo experiment, the implanted blood clot was collected from the mice at different time points for hematoxylin and eosin (H&E) staining and immunofluorescence staining (Fig. 2, A to E). Fibrin clot was used as a control (30, 31). The blood clot itself could lead to a rapid influx of leukocytes from the edge to the inside over time, whereas the leukocytes were mostly confined to the surface of fibrin clot (Fig. 2A and fig. S6, A and B). The finding that the cell recruitment ability of blood clots was substantially stronger than that of fibrin clots indicates that the damaged RBCs in the clots play a critical role in the recruitment of immune cells. Higher numbers of CD45+ leukocytes were recruited toward the scaffold at days 3 and 7 compared with that on day 0 (Fig. 2, B to E). Flow cytometry analysis showed that about 60% of cells in the clot were CD45+ cells at days 3 and 7 after implantation, including macrophages (F4/80+), T cells (CD3+), B cells (CD19+), NK cells (NK1.1+), and DCs (CD11c+) (Fig. 2, F to L). The abundance of blood clot–infiltrating immune cells should facilitate antigen recognition and presentation as well as antigen processing to stimulate naïve T cells. Besides, the implantation of blood clot significantly increased the number of WBCs (e.g., lymphocytes, monocytes, and granulocytes) in peripheral blood without affecting other blood parameters, indicating the systemic innate immune responses could be induced by the blood clot local implantation (Fig. 2M and fig. S6, D to J).

Fig. 2 Blood clot recruited a high number of immune cells.

(A) H&E images of cell infiltration in blood clots over 7 days. Scale bars, 200 μm. (B) Representative immunofluorescence images of CD45+ cells in cryosection of blood clots over 7 days after implantation. Scale bars, 50 μm. (C) Proportion of CD45+ cells in all infiltrated cells recruited by blood clots on days 0, 3, and 7. (D) Intensity of CD45+ and DAPI signals from the edge of the cryosection of blood clot to the core. (E) Quantitative analysis of infiltrated CD45+ cells shown in (B). (F to K) Proportion of immune cells recruited in blood clot analyzed by flow cytometry. (F) Leukocytes (CD45+), (G) T cells (CD3+), (H) macrophages (F4/80+), (I) B cells (CD19+), (J) NK cells (NK1.1+), and (K) DCs (CD11c+). (L) Total proportion of different immune cells in leukocytes according to the flow cytometry results (n = 3 to 4). (M) Clinical chemistry of mice before and after blood clot implantation. Values represent means ± S.E.M. Statistical significance was calculated by Student’s t test and one-way ANOVA using the Tukey posttest. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. a.u., arbitrary units.

We further analyzed the magnitude of immune responses triggered by immunomodulating agent–loaded blood clots in vivo. The blood clot vaccines were subcutaneously implanted into the flanks of C57BL/6 mice and harvested for immunofluorescence imaging and flow cytometry analysis over 7 days. The encapsulation of immunoregulators in the blood clot vaccine significantly further increased the number of recruited immune cells. The fluorescent signal intensity of CD45 in the vaccine scaffold was enhanced with time, as shown in the confocal imaging (Fig. 3A and fig. S7A). Nearly 80% of cells inside the blood clot vaccine were CD45+ cells at day 3 (Fig. 3B). Furthermore, the total numbers of lymphocyte subpopulations cells, including macrophages, DCs, and T cells in the blood clot, were all elevated significantly. Infiltrated F4/80+ cells increased from 15% to nearly 40% (Fig. 3, C and D, and fig. S7, B and D), along with the up-regulation of CD80 (Fig. 3, E and F) and MHCII (Fig. 3, G and H), indicating a distinct proinflammatory M1 phenotype. The number of CD11c+ cells was increased almost fivefold in comparison with the blank blood clot (Fig. 3, I and J, and fig. S7, C and E). According to the flow cytometry results, the expression of various stimulatory markers, including CD80, CD86 (Fig. 3K), MHCII (Fig. 3L), and CD40 (Fig. 3M), on infiltrated DCs was up-regulated compared with the blank blood clot, indicating the maturation and activation of DCs, which is a crucial step to present antigen and initiate the subsequent tumor-specific immune response. Tissue-resident CD103+ DCs are specialized in priming and cross-expressing antigen to CD8+ T cells, leading to tumor-specific CD8+ T cell differentiation (32). Blood clot vaccine recruited more CD103+ DCs into the scaffolds (Fig. 3, N and O), which were potent at cross-presenting tumor-related antigens loaded in blood clots. The total proportion of CD3+ cells in all cells increased to about 15% from the 10% observed in blank blood clots (Fig. 3P). As the amount of CD8+ T cells was almost 10% higher than the blank blood clot (Fig. 3, Q and R) and the proliferation marker Ki67 of CD8+ T cells was increased under the stimulation of immunoregulators and infiltrated APCs (Fig. 3, S and T), these indicate an activation of T cells within the blood clot vaccine. When the clots and surrounding tissues were removed and analyzed for local cytokine profiles, we observed a much higher concentration of proinflammatory cytokines and chemokines in the immunomodulator-loaded clots than in the blank clots (Fig. 3U). Collectively, all the above data demonstrated that the blood clot vaccine generated an “immunological niche” in which a large number of immune cells, including APCs and T cells, stimulate the infiltrated cells into antigen-specific immune cells. Consequently, this immunologic niche could produce effector immune cells to induce a robust anticancer immune response.

Fig. 3 Blood clot vaccine stimulated and activated the infiltrated immune cells.

(A) Representative immunofluorescence images of CD45+, F4/80+, and CD11c+ cells in cryosection of blood clot vaccine after implantation. Scale bars, 50 μm. (B) The proportion of CD45+ cells at day 3 after immunization extracted from implanted blood clot vaccine. (C) Representative flow cytometric analysis of F4/80+ cells in infiltrated cells and (D) corresponding quantitative analysis. (E) Mean fluorescence intensity (MFI) of CD80 in F4/80+ cells, and (F) MFI of CD206 in F4/80+ cells. (G) Flow cytometric analysis of MHCII expression on F4/80+ cells and (H) corresponding quantitative analysis. (I) Flow cytometric analysis for CD11c+ cells in infiltrated cells and (J) corresponding quantitative measurement. (K) Fraction of CD80+CD86+ DCs in the blood clot vaccine. (L and M) Expression of (L) MHCII and (M) CD40 in infiltrated DCs. (N) Proportion of CD103+ in DCs and (O) representative flow cytometric analysis. (P) Proportion of CD3+ cells in infiltrated cells. (Q) Flow cytometric analysis for CD8+ in CD3+ T cells and (R) corresponding quantitative measurement. (S) Representative flow cytometric analysis for Ki67 in CD8+ T cells and (T) quantitative measurement of Ki67 in CD8+ T cells. (U) Concentration of proinflammatory cytokines and chemokine factors in the blood clot at day 3 after implementation. Values represent means ± S.E.M. Statistical significance was calculated by Student’s t test and one-way ANOVA using the Tukey posttest. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. ns, not significant; SSC, side scatter; MDC, macrophage-derived chemokine; MIP-3α, macrophage inflammatory protein-3 alpha; BLC, B lymphocyte chemoattractant; RANTES, regulated upon activation, normal T cell expressed and presumably secreted; EOTAXIN, eosinophil chemotactic chemokines; TARC, thymus and activation regulated chemokine.

Long-term prophylactic immunity of blood clot–based vaccination

We next studied the antitumor effects of the blood clot–based cancer vaccine containing antigen (OVA), chemokine (GM-CSF), and adjuvant (CpG) in a prophylactic model using B16-OVA tumor-bearing mice (fig. S8A). Healthy C57BL/6 mice were implanted subcutaneously with blood clot vaccine 7 days before tumor inoculation, while other mice injected or implanted with the equivalent amount of PBS, blank blood clot, or free immunomodulates served as controls. On the basis of the detailed analysis of the cocultured cells or recruited cells, the blood clot–based cancer vaccine not only activated various immune cells by up-regulating the stimulatory molecules (e.g., CD80, CD86, MHCII, and CD40) but also induced the negative feedback regulation as evidenced by the increment of the checkpoint ligand programmed death 1 (PD-1) on CD8+ T cells (fig. S7, F and G), and PD-L1 on CD11c+ cells (fig. S5, A and B, and fig. S7, H and I) and F4/80+ (figs. S5, C and D, and S7, J and K). In addition, an increase in CD103+ DCs is associated with the promotion of antitumoral effects upon blockade of ligand PD-L1 (33). Therefore, we further included an immune checkpoint inhibitor after the implantation of the blood clot vaccine on day 5 (fig. S8A). As shown in fig. S8B, there was no difference between the PBS group and blank blood clots. Free soluble immunomodulator–treated mice showed a mild delay of tumor growth (fig. S8B). In contrast, mice receiving the blood clot vaccine showed a significant delay in tumor growth. This immunization strategy promoted a noticeable antitumor response compared with soluble immunomodulators. Moreover, when we combined the immunization strategy with the checkpoint blockade antibody, the amplified immune responses produced a stronger antitumor efficacy than monotherapy, as observed in the tumor growth (fig. S8C). In addition, B16-OVA tumors were eradicated up to 100% within 40 days (fig. S8D). Meanwhile, the body weight of mice did not show significant change to suggest minimal side effects with our combination strategy (fig. S8E).

To further evaluate the immune memory effect generated by blood clot vaccine–based combination therapy, we assessed the memory CD8+ T cells in the peripheral circulation of tumor-free survival mice or naïve mice on day 120. Antigen-specific memory T cells against cancer could be found in both central memory T cell (Tcm; CD3+CD8+CD62L+CD44+) and effector memory T cell (Tem; CD3+CD8+CD62LCD44+) subsets (34). We found that the proportion of peripheral circulatory Tcm in the treated mice group was about three times higher than that of the naïve group (fig. S8, F and G). Tcm lymphocytes show a great ability to proliferate and have been proven to exhibit potent immunity against cancer (35). There was no significant difference in the percentage of Tem in the peripheral blood among groups in this study (fig. S9A).

When the tumor-free mice were rechallenged with B16-OVA tumor cells, a significant delay of the tumor growth was observed compared with the naïve mice (fig. S8H). Established tumors were rejected completely in 75% of mice in the treated group within 60 days (fig. S8I). In contrast, naïve mice all died within 20 days due to tumor progression. In addition, the lungs of the control group showed many tumor lesions from H&E immunostaining, while the lungs of the treated group were normal without any detectable micro tumors (figs. S8J and S9C), indicating that he blood clot vaccine treatment could inhibit tumor metastasis as well.

Next, the memory CD8+ T cells in the peripheral circulation and spleen tissues were analyzed at day 60 (fig. S8, K to Q). In peripheral circulatory lymphocytes, the proportion of Tcm and Tem in CD8+ T cells was increased significantly in the treated group compared with the naïve mice (fig. S8, K to M). In addition, splenocytes collected from survived mice and naïve mice were also analyzed. Similarly, both Tem and Tcm were elevated significantly (fig. S8, N to P). All these data suggested that the educated memory T cells were effectively generated by blood clot vaccine treatment and could respond quickly to the encountered tumor antigen to prevent cancer recurrence. Furthermore, after restimulating the splenocytes with B16-OVA cell lysis in vitro, a high level of interferon-γ (IFN-γ) was produced (fig. S8Q). Together, these results provided critical evidence that blood clot vaccine–based vaccination could effectively prevent tumor recurrence by generating potent memory immunity.

Blood clot–based vaccination for the treatment of established tumor

To establish how widely applicable the proposed vaccination technology is, we further evaluated its therapeutic efficacy in other tumor models. As OVA is an artificial antigen, it is not suitable for clinical translation. Therefore, we used tumor lysate in the therapeutic model without OVA to investigate the potency of our blood clot–based vaccine. For the treatment of B16F10 tumor, vaccination was conducted on day 8 after 1 × 106 B16F10 cells were injected into the flank of C57BL/6 mice. Anti–PD-1 was intravenously administered every other day starting on day 9 for a total of six injections (Fig. 4A). Empty blood clots and anti–PD-1 treatment elicited similar effects with PBS. Free immunomodulators and blood clot vaccines stimulated antitumor immune responses at various degrees. Blood clot vaccine was superior to soluble immunomodulators judging from therapeutic efficacy (Fig. 4, B and C). Mice treated with the blood clot vaccine prolonged the survival time significantly. Encouragingly, enhanced antitumor immunity was achieved when anti–PD-1 therapy was included in the therapeutic model (Fig. 4, B to D). None of the mice in the combination group died within 34 days of treatment. The body weights did not show notable changes (Fig. 4E). Besides, we also used the 4T1 tumor model to study the broad application of our proposed combination therapy. Encouragingly, blood clot vaccination together with anti–PD-1 induced an antitumor response as shown in the tumor growth kinetics and the survival curves of 4T1-bearing mice (fig. S10, A to D). Our data suggested that the proposed strategy could suppress tumors in multiple tumor models. The interaction analysis using the Bliss independent model showed that the observed effects (OE) value was greater than the perceived effects (PE) value in the B16 tumor model and the 4T1 tumor model. Considering the significant difference in survival rate, there was a conclusive synergistic effect on tumor inhibition with blood clot vaccine and anti–PD-1 (table S1).

Fig. 4 Blood clot vaccine induced an effective antitumor immune response against established B16F10 tumor.

(A) Schematic illustration of blood clot vaccination in conjunction with anti–PD-1 therapy. (B) Individual tumor growth kinetics and (C) the average tumor growth curve of each group after different treatments as indicated. (D) The survival curves of treated groups and (E) weight of mice (n = 5 to 7). (F) Mass of tumor after different treatments as indicated. (G) Representative flow cytometric analysis of the tumor-infiltrated T cells. (H) Quantitation of the percent of CD8+ T cells in the tumor, (I) the proportion of the tumor-infiltrated T cells CD8+ in CD3+ T cells and (J) intratumoral ratio of CD8+ T cells to Treg (CD3+CD4+Foxp3+). (K) Proportion of IFN-γ within CD8+ T cells. (L) Representative flow cytometric analysis and (M) quantitation of the percent of Ki67 within CD8+ T cells. (N) Quantitative analysis of intratumoral NK1.1+ cells. Values represent means ± S.E.M. Statistical significance was calculated by one-way ANOVA using the Tukey posttest. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.

To further study the immune mechanism underlying the robust antitumor effects, we collected and analyzed the tumors (Fig. 4F). Flow cytometry analysis on day 15 showed that more CD8+ cells infiltrated the tumor treated with the combination therapy compared with other control groups in terms of the absolute number (Fig. 4, G and H) or the percentage in CD3+ cells (Fig. 4I). The proportion of cytotoxic T lymphocytes (CTLs) (CD3+CD4CD8+), which could directly exert an effect of antitumor immunity, was markedly increased to ∼40% in tumors on mice after treating with combination treatment. The intratumoral ratio of CD8+ T cells to regulatory T cells (Treg) (CD3+CD4+Foxp3+) was also increased more than 1.5-fold compared with the other three control groups (Fig. 4J). In addition to the higher number of infiltrated CTLs, elevated levels of IFN-γ+ and Ki67+ in CD8+ T cells (Fig. 4, K to M) were also associated with the outcome of the therapy. Besides, the amount of NK cells was increased in both the blood clot vaccine and the combination group (Fig. 4N), indicating that in addition to adaptive immune responses, blood clot–based vaccination could also promote innate immune responses, such as activation of NK cells.

Blood clot–based vaccination for the treatment of postsurgical tumor recurrence

To further assess the potency of the blood clot–based vaccination, we used a more clinically relevant model of tumor recurrence after surgery as we reported before (Fig. 5A) (36). In our experiment, 1 × 106 B16F10 cells were injected subcutaneously into the back of C57BL/6 mice. At day 10 after inoculation of tumor cells, a secondary tumor was injected into the other side of the mice to establish the ectopic tumor recurrence model to mimic cancer metastasis in clinical scenarios. Meanwhile, surgery was conducted to remove all the primary tumor tissue (fig. S11, A and B). Tumor lysis from the removed tumor was obtained. Blood was collected from the same tumor-bearing mice and mixed with tumor lysis, CpG, and GM-CSF to generate an autologous blood clot vaccine for reimplantation. The blood clot–based vaccine was implanted into the resection cavity directly during the surgery. Five doses of anti–PD-1 were injected into mice via the tail vein on days 3, 5, 7, 9, and 11 following the surgery (Fig. 5A). In this model, mice receiving blood clot–based vaccination delayed the growth of the tumor (Fig. 5, B and C). When combined with anti–PD-1 blockade, the synergistic strategy showed greater ability to suppress tumor growth and higher survival rates in comparison with other control groups (Fig. 5, D and E and fig. S11C), indicating a more potent ability in resisting tumor recurrence. There was no significant change in the weight of the mice among different treatments (Fig. 5F). In addition, we further evaluated the cytokines in the serum of mice after the final injection of anti–PD-1 (Fig. 5, G to J). The secretion of TNF-α, IFN-γ, immunoglobulin G (IgG), and IgM was markedly enhanced in the mice with combination therapy and higher than the control groups, suggesting that both humoral immunity and cell-mediated immunity contributed to the success of this cancer recurrence inhibition.

Fig. 5 Blood clot vaccine effectively inhibited the postsurgical recurrence.

(A) Experimental design of the tumor recurrence model. (B) Individual tumor growth kinetics in each group and (C) representative bioluminescence images of the B16F10-luc tumor after different treatments as indicated. Photo credit: Qin Fan, Soochow University. (D) Average tumor growth curve, (E) survival curve, and (F) body weight of mice after different treatments as indicated (n = 5 to 7). (G) TNF-α, (H) IFN-γ, (I) IgG, and (J) IgM concentrations in the serum of mice in each group on day 7 after treatments. Values represent means ± S.E.M. Statistical significance was calculated by one-way ANOVA using the Tukey posttest. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.

DISCUSSION

Different from previously reported biomaterials for vaccine delivery (37, 38), this report describes a new approach of leveraging on blood clots as a biological delivery system instead. Blood clots have the potential to be more advantageous over polymer/synthesized hydrogels for translation with respect to carrier development, formulation complexity, regulatory hurdles, and cost of commercialization (16). Blood clot with inherent biocompatibility can be effectively metabolized in vivo without producing any unknown or toxic by-products and side effects caused by some of the synthesized materials. The safety profile of such systems is favorable and guaranteed. As the cross-linked RBCs in the blood clot showed reduced CD47 levels and activation of the complement system, they are targets for phagocytosis. As a result, blood clot was capable of recruiting a great number of immune cells to form an “immune niche” in situ. Previous studies have shown that aged or damaged RBCs can activate the classical M1 polarization of macrophage toward proinflammatory immunostimulation. Although in this work we mainly studied the role of cross-linked RBCs in the recruitment of immune cells into blood clots, other cells including platelets, monocytes, neutrophils, and lymphocytes (less than 1% in the clots) may also play a role in immune regulation, which should be investigated in future work.

Inspired by the endogenous immune response of blood clots, we hypothesized that blood clot could be formulated as an implantable scaffold for enhanced cancer vaccination by loading with tumor antigens and adjuvants. The infiltrated naïve immune cells can be stimulated to become tumor-specific immune cells when encountering the tumor antigen inside the blood clot. With the help of immunoadjuvants, the blood clot vaccine generated potent tumor-specific immune responses compared with the blank blood clot. Adaptive immune responses can be activated by increasing the proportion and activity of CD8+ T cells. Besides, the infiltrating APCs were effectively activated, as evidenced by the boosted proportion of the matured DCs and stimulated macrophage with M1 state. The production of proinflammatory cytokines and chemotactic factors was significantly higher in the local site of blood clot and blood clot vaccine compared with the control groups. However, we also found that the blood clot vaccine also stimulate the expression of immunosuppressive markers, such as the PD-1 in CD8+ T cells and PD-L1 in F4/80+ and CD11c+ cells, along with the stimulatory markers. This could be due to the intrinsic homeostasis regulation mechanism that the body would up-regulate the negative regulatory pathway to avoid the excessive immune response. The expression of immunosuppressive markers indicates the importance of the combination with ICB to achieve effective anticancer responses.

A combination with checkpoint blockade therapy effectively released the brake of negative immune regulation produced by stimulation of blood clot–based vaccination (39). This synergistic therapy promoted powerful anticancer immune responses in prophylactic tumor models. Significantly, the generation of memory T cells can provide long-term immune memory effects against relapse and recurrence. Compared with free immunomodulators, the blood clot served as a controlled-release niche to recruit immune cells in a sustained manner. In addition, this vaccine design facilitates combination therapy for synergistic effects, such as the ICB therapy demonstrated here.

Regarding future translation, adjuvants and antigens loaded in the blood clot gel can be changed and optimized for personalized cancer immunotherapy to treat different cancer patients. Blood content in patients can be different among individuals (e.g., age or emergency myelopoiesis). For example, CD71+ erythroid cells in neonatal mice and human cord blood have distinctive immunosuppressive properties (40), which are not suitable for the preparation of blood clot cancer vaccine. One solution is to use blood from healthy donors to minimize patient-to-patient variability. Last, there is ample room for improvement on this vaccine design with respect to dosage, drug combination, and treatment frequency.

In summary, we report a blood clot–based immune niche for enhanced cancer vaccination. Capable of encapsulating immunomodulators and trapping deactivated RBCs in the blood clot, this vaccine design forms an immune niche in situ to recruit immune cells and stimulate them to initiate tumor-specific adaptive immunity. Combination with ICB therapy further potentiates the immune response, leading to efficacy in both prophylactic and therapeutic settings against B16F10 and 4T1 tumor progression, including inhibition of metastasis and recurrence in a clinically relevant model of surgery and reinoculation of tumor cells. Amenable to the use of autologous blood, this vaccine design renders personalized immunotherapy feasible with loading of specific tumor neoantigens. Potential safety and ease of formulation add to the attraction of this cancer vaccine. Overall, the potency, versatility, and translational potential of this new cancer vaccine technology warrant further investigation and development.

MATERIALS AND METHODS

Materials

CpG oligonucleotide 1826, 5′-TCC ATG ACG TTC CTG ACG TT-3′ (InvivoGen, catalog no. tlrl-1826-5), was purchased from Suzhou Alpha Biological Experimental Equipment Co. Ltd. Murine GM-CSF (PeproTech, catalog no. 315-03-50) was purchased from Dakewe Biotech Co. Ltd. FITC-OVA (Solarbio, catalog no. SF21) was purchased from Jiangsu Bomeida Life Science Co. Ltd., and Cy5.5-CpG was synthesized and purified by Sangon Biotech (Shanghai) Co. Ltd. Other reagents were listed in table S2.

Cell lines and mice

Murine melanoma B16-OVA cells were gifted by H. Liu at Soochow University. Murine melanoma B16F10 or luciferase-labeled B16F10 cells (B16F10-luc) were gifted by W. Zhu at Soochow University. B16-OVA cells, B16F10 cells, and B16F10-luc cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco), penicillin (100 U/ml; Invitrogen), and streptomycin (100 U/ml; Invitrogen). Murine 4T1 breast cancer cell line was originally purchased from the American Type Culture Collection. The 4T1 cells were maintained in RPMI 1640 medium containing 10% FBS (Gibco), penicillin (100 U/ml; Invitrogen), and streptomycin (100 U/ml; Invitrogen). BMDCs were isolated from bone marrow cavities of 9-week-old C57BL/6 mice according to an established method. Peritoneal macrophages were extracted from the perineal cavity of healthy C57BL/6 mice and then cultured in DMEM with 10% FBS before use. RAW264.7 cells were cultured in DMEM with 10% FBS. C57BL/6 and BALB/c mice were obtained from Nanjing Peng Sheng Biological Technology Co. Ltd. We performed all mice studies in accordance with the animal protocol approved by our university laboratory animal center.

Blood clot and blood clot vaccine fabrication

The blood clot and the blood clot vaccine were formed through natural agglutination and mild vacuum drying technology. In detail, for the preparation of blood clot, 200 μl of fresh blood taken from the fundus venous plexus of mice was injected into a sterilized round vial, allowed to stand at room temperature for 10 min, and then dried at 37°C for 2 hours under optimized vacuum. For the fabrication of the blood clot vaccine, 100 μg of CpG OND-1826, 1 μg of GM-CSF, and 100 μg of OVA or 500 μg of tumor lysate (protein amount) were pre-added into a sterilized round vial. Then, about 200 μl of fresh blood taken from the fundus venous plexus of mice was mixed with immunomodulating agents immediately. The mixed blood complex was dried at the optimized vacuum condition for 2 hours. The entire preparation process is performed in a sterile environment. For the construction of fibrin clot, the fibrin solution (50 mg/ml) was mixed with thrombin (500 IU/ml) in a volume ratio of 2:1.

Implantable scaffold characterization

Formed blood clot was photographed under the light-emitting diode light. The internal morphology of the blood clot was characterized by using a scanning electron microscope (Carl Zeiss, Supra 55). The expression of CD47 on the RBC membrane was measured by flow cytometry. Blood clot was prepared into the single-cell suspension and then labeled with FITC-CD45 and phycoerythrin (PE)–CD47. Free blood cells were used as a control group. Mean fluorescence intensity (MFI) of PE-CD47 on the FITC-CD45 cell population was compared between the two groups. For drug loading, 5 μl of FITC-OVA (5 mg/ml) or 5 μl of Cy5.5-CpG (3.3 mg/ml) was added to a round vial and then mixed with 200 μl of fresh blood followed by vacuum drying. To visually observe the drug loading status, blood clots with or without FITC-OVA were embedded in optimal cutting temperature compound (OCT gel) and cut into slices. Membranes of blood cells were stained with rhodamine (dilution 1:100). The samples were determined by fluorescence confocal microscopy. To test the loading capacity, the fluorescence intensity in the supernatant was determined by a microplate reader after quantifying the volume of the extra precipitated liquid (Thermo Fisher Scientific, Variskan). The loading capacity of FITC-OVA or Cy5.5-CpG in blood clot was calculated according to the following equation: loading percentage of FITC-OVA or Cy5.5-CpG (%) = 1 – the weight of unloaded agents/weight of added agents × 100%. The rheological properties of the blood clot and blood clot vaccine were determined by Haake Rheo Stress 6000 rotational rheometer.

In vitro degradation and release behavior of blood clot

To study the in vitro degradation, 2 ml of sterilized PBS (pH 7.0 to 7.2) was added to the blood clot, and then photos were taken on days 0, 1, 3, 5, and 7. To study the release behavior, immunomodulating agent–loaded samples (50 μg of FITC-OVA and 33 μg of Cy5.5-CpG) were incubated with 2 ml of sterilized PBS (pH 7.0 to 7.2) at 37°C while agitating constantly. At different time points, 200 μl of liquid was collected for quantitative fluorescence analysis, and then the same volume of prewarmed PBS was resupplied. Collected samples were centrifuged at high speed under low temperature and then tested with a microplate reader.

In vivo degradation of blood clot

To study the in vivo degradation, blood clots were implanted into the flank of BALB/c mice and photos were taken until the blood clots disappeared. The volume of the blood clot was measured with a digital caliper and calculated following the formula: short diameter2 × long diameter × 0.5. To further visualize the degradation, blood clots removed from the mice on days 0, 1, 3, and 7 were weighed with an electronic scale and then fixed with 4% paraformaldehyde after taking photos. SEM was used to characterize the morphology of the blood clot after extraction ex vivo.

In vitro cellular immune stimulation experiment

For DC and macrophage stimulation experiments, 1 × 106 myeloid-derived DCs (BMDCs) were cocultured with PBS, blank blood clot, soluble-free vaccine (5 μg of CpG ODN, 0.05 μg of GM-CSF, and 5 μg of OVA), or blood clot vaccine with the same immunomodulating agents for 20 hours. After different treatments, cell culture supernatants were collected immediately and saved at −80°C before measurement. To test the stimulation on BMDCs, cells were stained with FITC-CD11c, APC-CD80, and PE-CD86, FITC-CD11c and PerCP-MHCII, FITC-CD11c and APC-CD40, and FITC-CD11c and PE-PD-L1 and then were analyzed by flow cytometry (BD FACSCalibur). The concentration of cytokines TNF-α, IL-6, and IL-12 secreted in the culture medium was tested by enzyme-linked immunosorbent assay (ELISA) following the operation manuals.

For macrophage stimulation experiments, 5 × 105 peritoneal macrophages were cocultured with PBS, blank blood clot, soluble-free vaccine, or blood clot vaccine for 24 hours. After different treatments, cell culture supernatants were withdrawn immediately and saved at −80°C before measurement. Macrophages stained with FITC-F4/80, PE-CD206 and APC-CD80, FITC-F4/80 and PerCP-MHCII, or PE-PD-L1 was sorted by flow cytometry (BD FACSCalibur). The concentration of TNF-α and IL-6 secreted in the culture medium was tested by ELISA following the manufacturer’s instructions.

To study the phagocytic ability of macrophages after immunostimulation, 1 × 105 RAW264.7 cells were cocultured with PBS, blank blood clot, soluble-free vaccine (5 μg of CpG ODN, 0.05 μg of GM-CSF, and 25 μg of FITC-OVA), or blood clot vaccine with the same of immunomodulating agents for 24 hours. The amounts of FITC-OVA phagocytized by macrophage were evaluated by flow cytometry and confocal microscopy, respectively. For flow cytometry analysis, RAW264.7 cells were stained with FITC-F4/80, and then MFI of FITC-OVA on the FITC-F4/80+ was measured. For confocal analysis, RAW264.7 cells were recultured with 100 nM LysoTracker after washing with sterilized PBS twice. Then, the cell nucleus was labeled with 4′,6-diamidino-2-phenylindole (DAPI) for about 15 min after the cells were fixed with 4% paraformaldehyde. The phagocytosis of stimulated macrophages was observed by confocal microscopy (Zeiss LSM 800).

In vivo cell recruitment

C57BL/6 mice received subcutaneous implantation of blank blood clots, fibrin clot, or blood clot vaccines after being anesthetized with 2.5% isoflurane in a sterile environment. In addition, the unimplanted blood clots were used as a control group to demonstrate the background signal of blank blood clots. To evaluate the cell recruitment capability of blank blood clot, explanted blood clots that were implanted into mice at days 0, 1, 3, and 7 were embedded in OCT gel for immunofluorescence staining or embedded in paraffin for H&E histological analysis. Specific immune cells infiltrating the blood clot were analyzed by flow cytometry. For immunofluorescence staining, collected samples were dissected into 8 μm at −20°C by microtome (Leica CM1860). Sections attached on the adhesive slide were fixed with cold 4% paraformaldehyde. After rehydrating with PBS, sections were blocked with bovine serum albumin (3%) and then stained with primary antibodies for about 12 hours in 4°C condition.

For the detection of leukocytes, the blood clot sections were stained with CD45 primary antibody and Cy3 anti-rat IgG secondary antibody according to the official instruction, and antibodies were diluted 200-fold. APCs were stained with FITC-CD11c and FITC-F4/80 (dilution 1:100), respectively. Cell nuclei were stained with DAPI. Confocal microscopy was used to analyze the sealed slides (Zeiss LSM 800). H&E slides were prepared by Wuhan Servicebio Technology Co. Ltd. and observed using a fluorescence optical microscope (Leica DM4000M). For flow cytometry study, immune markers like PE-CD45, FITC-CD3, FITC-CD11c, APC-F4/80, APC-CD19, and APC-NK1.1 were used to label infiltrated immune cells after lysing RBCs.

To study the immune stimulation of blood clot vaccines, C57BL/6 mice received subcutaneous implantation of blank blood clots or blood clot vaccines (10 μg of CpG ODN, 0.1 μg of GM-CSF, and 10 μg of OVA) after being anesthetized with 2.5% isoflurane in a sterile environment. Anticoagulated blood was taken from mice, and then, blood routine tests were performed on day 0 or day 3. Besides, on day 3, the implanted samples were excised and prepared into single-cell suspensions. The RBCs were completely lysed and then stained for analysis. Recruited leukocytes were stained with FITC-CD45. To analyze macrophage, cells were stained with FITC-F4/80, PE-CD206 and APC-CD80, FITC-F4/80 and PE-MHCII, or PE–PD-L1. To delineate DC cells, cells were stained with FITC-CD11c, APC-CD80 and PE-CD86, FITC-CD11c and PE-MHCII, or APC-CD40 or PE-PD-L1. To determine the T cell recruitment and the cell differentiation in blood clot vaccine, recruited immune cells were stained with FITC-CD3, APC-CD8, and peridinin chlorophyll protein/Cyanine5.5 (PerCP/Cy5.5) or PE–PD-1.

The concentration of different cytokines and chemotic factors secreted in the implanted sites was tested after implanting about 3 days. Blood clot scaffolds and adjacent tissues were removed and extracted with 1 ml of tissue protein extraction reagent. Then, the concentration of cytokines in the extracting solutions was analyzed with Mouse Proinflammatory Chemokine Panel and Mouse Th Cytokine Panel. The concentration of cytokines was normalized over the weight of excised tissue.

Prophylactic experiment

C57BL/6 mice were weighed and randomly divided into six groups: PBS group, blood clot (BC) group, free vaccine group, blood clot vaccine group (BC-vacc), anti–PD-1 group (αPD-1), and blood clot vaccine and anti-PD-1 combination group (combination). On day −7, the prepared blank blood clot and blood clot vaccine (100 μg of CpG OND, 1 μg of GM-CSF, and 100 μg of OVA) were implanted into the left flank of mice. Mice in the PBS group were injected with about 200 μl of sterilized PBS (pH 7.0 to 7.2). For the free vaccine group, the same doses of immunomodulating agents and tumor-associated antigen were diluted with 200 μl of PBS and then injected directly into the mice subcutaneously. On days 5, 7, 9, and 11, mice were injected with anti–PD-1 (20 μg per mice), respectively. B16-OVA melanoma cells (3.5 × 105) were injected subcutaneously on the right flank of mice. The tumor sizes and mice weights were measured every 2 days. The tumor volumes were calculated following the formula: short diameter2 × long diameter × 0.5. The animal was euthanized when the tumor exceeded 1.5 cm3 or when the tumor exhibited broken signals such as rupture and bleeding.

To assess the inoculation effect on tumor recurrence, surviving mice were reinjected with 1.5 × 106 of B16-OVA cells at day 120 after the first tumor challenge, and age-matched untreated mice were used as naïve control. The memory CD8+ T cells in the peripheral blood cells (PBs) of survival mice, and naïve mice were tested before and after rechallenging with B16-OVA tumor cells. The tumor volumes and body weights of mice were recorded every 2 days. At day 60 of tumor rechallenge, spleens were digested into single splenocyte, the composition of Tem and Tcm in mice splenocytes was studied, and the levels of IFN-γ secretion were measured by stimulating the spleen cells with or without antigen restimulation. The lungs of mice were prepared for H&E staining to observe the tumor metastasis. For the analysis of memory CD8+ T cells, the PBs and spleen cells that lysed RBCs were stained with FITC-CD3, PerCP-CD8, APC-CD62L, and PE-CD44 antibodies.

Therapeutic experiment

The therapeutic efficacy of the blood clot vaccine was studied by using B16 and 4T1 tumor–bearing mice. To establish the B16F10 tumor model, 1 × 106 B16F10 cells were injected into the right flank of C57BL/6 J mice. Mice were treated 8 days after tumor inoculation, when the tumor reached 100 mm3 in volume. To mimic a personalized vaccine, 100 μg of CpG OND, 1 μg of GM-CSF, and 500 μg of B16F10 tumor lysate (protein amount) were loaded into the blood clot. Mice were divided into six groups and treated as mentioned above. Mice in the combination group and in the free anti–PD-1 group were injected with anti–PD-1 antibody (50 μg) at days 9, 11, 13, 15, 17, and 19, respectively. To assess the therapeutic efficacy, tumor growth and body weight of mice were monitored. For the 4T1 mice model, a similar therapeutic experiment was carried out on day 3 after 1 × 106 4 T1 tumor cells were injected subcutaneously. 4T1 tumor lysate (500 μg; protein amount) was used as the tumor-associated antigens. For the combination group, vaccinated mice were injected anti–PD-1 antibody (50 μg) at days 5, 7, 9, and 11, respectively. Other control groups were set as follows: PBS group, blood clot group, free vaccine group, and free anti–PD-1 group. Mice in the free vaccine group and the free anti–PD-1 group were administrated locally or systemically at the same dose with blood clot vaccine. To assess the therapeutic efficacy of vaccination, tumor growth and body weight of mice were monitored. The volume of the tumor was measured with a digital caliper every 2 days.

The interaction between blood clot vaccine and anti–PD-1 was quantitatively analyzed using the Bliss independent model. Blood clot vaccine inhibited PB percent of tumor growth, and anti–PD-1–inhibited PA percent of tumor growth was calculated by the equation PB=ControlBControl, PA=ControlAControl, respectively. The theoretical combination inhibition PE (predicted effects) was predicted: PE = PB + PAPBPA. The observed combined inhibition OE (observed effects) was compared with PE. If OE < PE, then the combination would be thought antagonistic; if OE > PE, then the combination would be synergistic; and if OE = PE, then the combination would be additive.

To study the amplifying immune responses of combination strategy, B16-OVA–bearing mice were randomly divided into four groups: PBS, anti–PD-1, blood colt vaccine, and combination group, respectively. After day 15 posttreatment, mice were euthanized, and tumors were harvested from mice for the immunological evaluation. Tumor tissues were processed by a protocol reported before. The prepared single-cell suspension was stained with fluorescent dye–labeled antibodies. For assessing CTL infiltration, cells were stained with FITC-CD3, APC-CD4, and PE-CD8. For Treg evaluation, cells were stained with FITC-CD3, APC-CD4, and PE-Foxp3. For NK cell evaluation, cells were stained with FITC-CD45 and PE-NK1.1. In addition, IFN-γ and Ki67 in CTLs were stained with FITC-CD3, APC-CD8, and PE–IFN-γ or FITC-CD3, PE-CD8, and PerCP/Cy5.5-Ki67, respectively.

Tumor recurrence model

To study the inhibit behavior of blood clot vaccine on tumor recurrences, 1 × 106 of luciferase-tagged B16F10 (B16F10-luc) were transplanted into the right flank of mice 10 days prior. Tumors in all groups were completely resected. Different formulations, including PBS, blood clot, free vaccine, blood clot vaccine, and combination, were implanted or injected into the resection site directly. The prepared free vaccine and blood clot vaccine contained 100 μg of CpG OND, 1 μg of GM-CSF, and 500 μg of tumor lysates (protein amount). Mice were rechallenged with 1 × 106 B16F10-luc on the other flank of mice. Five doses of anti–PD-1 were injected into mice in the anti–PD-1 group and combination group via the tail vein on days 3, 5, 7, 9, and 11 after surgery. The animals were treated with isoflurane anesthesia during the whole treatment process. Surgical instruments and the environment were sterile for removing the first tumor. The wound was sutured by the Autoclip wound clip system with 9-mm nails. The serums of mice were collected from mice at day 7 after different treatments and analyzed to evaluate the level of immune-related cytokines. The concentrations of TNF-α, IFN-γ, IgG, and IgM secreted in the medium were tested by ELISA following the manufacturer’s instructions. Tumor growth and body weight of mice were monitored with a digital caliper and electronic scales every 2 days, respectively.

In vivo fluorescence imaging

The growth of the B16F10-luc tumor in C57BL/6 mice was monitored by the in vivo IVIS Spectrum Imaging System (PerkinElmer Ltd.). After intraperitoneal injection of fluorescent substrates for about 10 min, mice were exposed for 2 min to obtain bioluminescence imaging. IVIS Living Image 4.2 software was used to perform bioluminescence signals quantified as average radiance (photons s−1 cm−2 sr−1).

Statistical analysis

All data in the present study are means ± S.E.M. The significance of differences between two groups was calculated by a two-tailed unpaired Student’s t test. Besides, analysis of variance (ANOVA) comparison and Tukey post hoc tests were performed between more than two groups (multiple comparisons). For animal survival, the statistical differences were determined by log-rank test. Values of P = 0.05 or less were considered significant. The standard symbols were presented as *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001.

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/39/eabb4639/DC1

https://creativecommons.org/licenses/by-nc/4.0/

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REFERENCES AND NOTES

Acknowledgments: We thank the use of instrumentation facility at FUNSOM and Soochow University. Funding: This work was supported by grants from startup supports of Soochow University and the Program for Jiangsu Specially-Appointed Professors to C.W. This work was also supported by the National Natural Science Foundation of China (no. 31900988), the Natural Science Foundation of Jiangsu Province (no. SBK2019040088), and Jiangsu Province Six Talent Peaks Project (no. SWYY-110). This work was partly supported by Collaborative Innovation Center of Suzhou Nano Science & Technology, the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the 111 Project. Author contributions: C.W. and Q.F. designed the project. Q.F. and Q.M. performed the experiments and collected the data. Q.F. and Q.M. analyzed and interpreted the data. All authors contributed to the writing of the manuscript, discussed the results and implications, and edited the manuscript at all stages. Competing interests: C.W. and Q.F. are inventors on a pending patent related to the technology described here, filed by Soochow University (no. 202010085502.4, filed 8 February 2020). The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.
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