rWTC-MBTA: autologous vaccine prevents metastases via antitumor immune responses

Abstract

Background: Autologous tumor cell-based vaccines (ATVs) aim to prevent and treat tumor metastasis by activating patient-specific tumor antigens to induce immune memory. However, their clinical efficacy is limited. Mannan-BAM (MB), a pathogen-associated molecular pattern (PAMP), can coordinate an innate immune response that recognizes and eliminates mannan-BAM-labeled tumor cells. TLR agonists and anti-CD40 antibodies (TA) can enhance the immune response by activating antigen-presenting cells (APCs) to present tumor antigens to the adaptive immune system. In this study, we investigated the efficacy and mechanism of action of rWTC-MBTA, an autologous whole tumor cell vaccine consisting of irradiated tumor cells (rWTC) pulsed with mannan-BAM, TLR agonists, and anti-CD40 antibody (MBTA), in preventing tumor metastasis in multiple animal models.

Methods: The efficacy of the rWTC-MBTA vaccine was evaluated in mice using breast (4T1) and melanoma (B16-F10) tumor models via subcutaneous and intravenous injection of tumor cells to induce metastasis. The vaccine's effect was also assessed in a postoperative breast tumor model (4T1) and tested in autologous and allogeneic syngeneic breast tumor models (4T1 and EMT6). Mechanistic investigations included immunohistochemistry, immunophenotyping analysis, ELISA, tumor-specific cytotoxicity testing, and T-cell depletion experiments. Biochemistry testing and histopathology of major tissues in vaccinated mice were also evaluated for potential systemic toxicity of the vaccine.

Results: The rWTC-MBTA vaccine effectively prevented metastasis and inhibited tumor growth in breast tumor and melanoma metastatic animal models. It also prevented tumor metastasis and prolonged survival in the postoperative breast tumor animal model. Cross-vaccination experiments revealed that the rWTC-MBTA vaccine prevented autologous tumor growth, but not allogeneic tumor growth. Mechanistic data demonstrated that the vaccine increased the percentage of antigen-presenting cells, induced effector and central memory cells, and enhanced CD4⁺ and CD8⁺ T-cell responses. T-cells obtained from mice that were vaccinated displayed tumor-specific cytotoxicity, as shown by enhanced tumor cell killing in co-culture experiments, accompanied by increased levels of Granzyme B, TNF-α, IFN-γ, and CD107a in T-cells. T-cell depletion experiments showed that the vaccine's antitumor efficacy depended on T-cells, especially CD4⁺ T-cells. Biochemistry testing and histopathology of major tissues in vaccinated mice revealed negligible systemic toxicity of the vaccine.

Conclusion: The rWTC-MBTA vaccine demonstrated efficacy in multiple animal models through T-cell mediated cytotoxicity and has potential as a therapeutic option for preventing and treating tumor metastasis with minimal systemic toxicity.

Keywords: Mannan-BAM; Metastasis; T-cell cytotoxicity; TLR agonists; rWTC-MBTA vaccine.

Fig. 1

Schematic illustration of rWTC-MBTA vaccine for cancer immunotherapy (by Biorender). a, Fabrication process of the rWTC-MBTA vaccine. b, Simplified mechanism of MBTA-mediated cancer immunotherapy to prevent post-operative tumor recurrence and metastasis

Fig. 2

The rWTC-MBTA vaccine prevents tumor metastasis in breast and melanoma metastasis animal models. a, Treatment schedule for immunotherapy. b&e, Representative photographs of lung tissue from animal models of 4T1 or B16 metastasis. The black arrows indicate tumor metastasis foci (n = 5/group). c&f, Representative H&E staining of lung tissues collected on day 45 from (c) 4T1 or (f) B16 metastatic animal models (2× scale bar = 600 μm; 20 × scale bar = 60 μm) (n = 5/group). d&g, Quantification of metastatic foci in the lung mice using (d) 4T1 or (g) B16 metastasis model (n = 5/group). Ordinary one-way ANOVA with multiple comparisons was used to calculate statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001,**** P < 0.0001.All data were represented as mean ± SD. P-values shown are for each treated group comparison

Fig. 3

rWTC-MBTA vaccine inhibits postoperative tumor metastasis in 4T1 breast cancer animal model. a, Treatment schedule for immunotherapy in breast animal model with surgical tumor removal. b, Represantative H&E staining of lung tissue collected each at various time points. Scale bar = 100 μm (n = 3/group). c, IHC for CD4 and CD8 staining of lung tissue collected at different time points (n = 3/group). Scale bar = 100 μm. d, Overall survival curve of mice after control and vaccine treatments (n = 7/group). Survival rate was analyzed using the Mantel-Cox (log-rank) test. e, Treatment schedule for rechallenge with subcutaneous tumor cells in mice that survived the initial challenge. f, Mean and individual tumor growth curves for mice receiving different treatments (n = 4 for control, n = 2 for re-challenged survival mice). The mean tumor volumes were analyzed using Kruskal-Walllis test with multiple comparisons. All data were represented as mean ± SD. P-values are shown for control versus rWTC-MBTA vaccine

Fig. 4

The rWTC-MBTA vaccine against autologous tumor metastasis and growth but not allogeneic tumor metastasis and growth. a, Treatment schedule for tumor growth and metastasis inhibition by the rWTC-MBTA vaccine. b, Mean and individual tumor growth curves for mice receiving different treatments (n = 5/group). The mean tumor volumes were analyzed using one-way ANOVA. c, Representative H&E staining of lung tissue collected at day 45 from the primary mammary animal model (2× scale bar = 600 μm; 20 × scale bar = 60 μm) (n = 3). d, Quantification of lung metastasis nodes in each group (n = 5/group). Ordinary one-way ANOVA with multiple comparisons was used to assess statistical significance. All data were represented as mean ± SD. P-values were shown for each treated group compared to rWTC-MBTA vaccine or r4T1. e, Cross-vaccination experiment design. f, Mean and individual tumor growth curves of the tumors receiving different treatments (n = 3/group). The data were presented as mean ± SD. g, Representative H&E staining of lung tissue collected on day 45 (2× scale bar = 600 μm; 20 × scale bar = 60 μm) (n = 3/group). h, Quantification of lung metastasis nodes in each group (n = 3/group). Ordinary one-way ANOVA with multiple comparisons was used to assess statistical significance. All data are represented as mean ± SD. P-values shown are for each treated group comparison. *P < 0.05, **P < 0.01, ***P < 0.001,**** P < 0.0001

Fig. 5

rWTC-MBTA vaccine enhances antigen presentation by increasing the proportion of antigen-presenting cells. After one or multi-dose treatments, lymph nodes (LNs) were collected to check the percentage of APCs by flow cytometry. a, Schematic diagram of the experimental design. b, Percentage of dendritic cells. c, Percentage of monocytes. d, Percentage of B cells. e, Percentage of mature DCs (gated as CD80⁺CD86⁺) after one or multi-dose of treatments. f, Percentage of MHCII⁺ monocytes in total monocytes after one or multi-dose treatments. g, CD137L⁺ B cells in total B cells after one or multi-dose treatments (gated on CD45 + cells). h, Serum concentrations of IFN-γ and i, IL-6 after one or multi-dose treatments. Ordinary one-way ANOVA with multiple comparisons was used to assess statistical significance. All data are represented as mean ± SD. P-values shown for each treated group compared to the rWTC-MBTA vaccine group (n = 5/group). *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001

Fig. 6

The rWTC-MBTA vaccine stimulates anti-metastasis adaptive immunity. Spleens were collected and isolated at the endpoint of the 4T1 subcutaneous metastasis animal model (a). CD4⁺ and CD8⁺ T cells were examined by flow cytometry in splenocytes from different treatment groups. b, Percentage of T-cells (CD4 ⁺ or CD8⁺) in splenocytes from different groups. c, Percentage of central memory (CD44⁺CD62L⁺) T cells in splenocytes of different treatment groups. d, Percentage of effector memory (CD44⁺CD62L⁻) CD4 and CD8 T cells in splenocytes. e-i, All the analyses are based on the co-culture 4T1 tumor cells and indicated splenocytes from different treated animals. e, 4T1 tumor cell number count after co-culture. f & g, Concentrations of cytokines IFN-𝛾 (f) and TNF-𝛼 (g) from co-culture supernatants as measured by ELISA. h&i, Percentage of Gran B⁺, IFN-𝛾⁺, TNF-𝛼⁺, or CD107⁺ CD4⁺ (h) or CD8⁺ T cells (i) was determined by flow cytometry. The analysis from e to i was conducted after co-culturing splenocytes (from different treated mice) with 4T1 tumor cells. Ordinary one-way ANOVA with multiple was used to assess statistical significance. All data are represented as mean ± SD. P-values are shown for each treated group compared to the rWTC-MBTA vaccine group (n≥3/group). *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001

Fig. 7

rWTC-MBTA vaccine against early tumor metastasis depends on T cells. 4 weeks post-vaccination were treated with three doses of anti-CD8α or anti-CD4 antibody (250 µg/mouse) as shown in the schematic diagram (a). b&c Representative photographs and H&E staining of lung tissue from different treatment groups. d, Representative IHC staining of lung tissues for CD4 and CD8 markers from different treatment groups (scale bar = 100 μm). e, Representative photographs 48 h after co-culture of purified T cells from splenocytes and 4T1 tumor cells (scale bar = 400 μm). f, Tumor cell counting by flow cytometry 48 h after co-culture of T-cell and 4T1 tumor cells. Ordinary one-way ANOVA with multiple comparisons was used to assess statistical significance. All data are represented as mean ± SD. P-values are shown for each treated group compared to the rWTC-MBTA vaccine group. g, CD4⁺ T cells and CD8⁺ T cells from splenocytes from different treatments were detected via flow cytometry. Mann-Whitney test was used to assess statistical significance. All data were represented as mean ± SD (n≥3/group). *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001

Fig. 8

r4T1-MBTA vaccine toxicity evaluation. Serum levels of a, ALT; b AST; c cholesterol; d, triglycerides; e, amylase; f, glucose; g, BUN and h, uric acid after 1 or 12 dose injections with different treatments. All parameter values are in the normal range and there was no significant difference in either treatment (MBTA, IR only, and rWTC-MBTA vaccine) group compared with the control group using one-way ANOVA with multiple comparisons. i, Representative H&E staining of major organs after multiple dose injections. Scale bar = 60 μm. All data were represented as mean ± SD (n = 5/group)