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

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.

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.

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.

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.

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 T_reg (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.

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.