Hybrid Ginseng‐derived Extracellular Vesicles‐Like Particles with Autologous Tumor Cell Membrane for Personalized Vaccination to Inhibit Tumor Recurrence and Metastasis

Abstract Personalized cancer vaccines based on resected tumors from patients is promising to address tumor heterogeneity to inhibit tumor recurrence or metastasis. However, it remains challenge to elicit immune activation due to the weak immunogenicity of autologous tumor antigens. Here, a hybrid membrane cancer vaccine is successfully constructed by membrane fusion to enhance adaptive immune response and amplify personalized immunotherapy, which formed a codelivery system for autologous tumor antigens and immune adjuvants. Briefly, the functional hybrid vesicles (HM‐NPs) are formed by hybridizing ginseng‐derived extracellular vesicles‐like particles (G‐EVLPs) with the membrane originated from the resected autologous tumors. The introduction of G‐EVLPs can enhance the phagocytosis of autologous tumor antigens by dendritic cells (DCs) and facilitate DCs maturation through TLR4, ultimately activating tumor‐specific cytotoxic T lymphocytes (CTLs). HM‐NPs can indeed strengthen specific immune responses to suppress tumors recurrence and metastasis including subcutaneous tumors and orthotopic tumors. Furthermore, a long‐term immune protection can be obtained after vaccinating with HM‐NPs, and prolonging the survival of animals. Overall, this personalized hybrid autologous tumor vaccine based on G‐EVLPs provides the possibility of mitigating tumor recurrence and metastasis after surgery while maintaining good biocompatibility.


Figure S3 .
Figure S3.Particle size distribution of TM-NPs, G-EVLPs and HM-NPs determined by DLS.a. Particle size distribution of TM-NPs (derived from 4T1 cells).b.Particle size distribution of G-EVLPs.c. Particle size distribution of HM-NPs (derived from 4T1 cells).d.Particle size distribution of TM-NPs (derived from CT26 cells).e. Particle size distribution of G-EVLPs.f.Particle size distribution of HM-NPs (derived from CT26 cells).g.Particle size distribution of TM-NPs (derived from MB49 cells).h.Particle size distribution of G-EVLPs.i. Particle size distribution of HM-NPs (derived from MB49 cells).

Figure S4 .
Figure S4.SDS-PAGE protein analysis of Marker (M), G-EVLPs (G), TM-NPs (T) and HM-NPs (H).a. Images of SDS-PAGE protein analysis.b.Images of SDS-PAGE protein analysis marked for the same protein (The red box indicates the same G-EVLPs protein band, while the yellow box indicates the same tumor cell membrane protein band).

Figure S5 .
Figure S5.Flow cytometry analysis of cellular uptake of DiD-labeled tumor membrane after incubation with different NPs for 24 hours.a. Principles of flow cytometry data processing.b.Flow cytometry uptake data for different groups.

Figure S7 .
Figure S7.Western blot analysis of TLR2 and TLR4 proteins of BMDCs receiving different process.The gels were loaded with equal amounts of the proteins (10 μg).

Figure S8 .
Figure S8.Flow cytometry analysis of CD45 + CD3 + CD8 + T cells of the specific immune activation experiments in vitro.

Figure S9 .
Figure S9.Specific immune activation determined by LDH kit.LDH concentration in the supernatant after co-incubation of the splenic T lymphocytes with B16F10 tumor cells (a), CT26 tumor cells (b), MB49 tumor cells (c) and 4T1 tumor cells (d).Data are representative or pooled and are expressed as Mean ± SE.Asterisks indicate statistically significant differences as analyzed by One-Way ANOVA (N.S. p>0.05).

Figure S10 .
Figure S10.Photographs of the inguinal lymph nodes after vaccination after 24 h.

Figure S11 .
Figure S11.Photographs of the spleens and the spleens weight after vaccination after 24 h.Data are representative or pooled and are expressed as Mean ± SE.Asterisks indicate statistically significant differences as analyzed by One-Way ANOVA (N.S. p>0.05).

Figure S13 .
Figure S13.Distribution of different NPs in lymph nodes and spleens.a. Fluorescence images of LNs after vaccination (DiR-labeled tumor membrane).b.Fluorescence images of LNs after vaccination (DiD-labeled G-EVLPs).c. Fluorescence intensity analysis of inguinal lymph nodes after vaccination (DiR-labeled tumor membrane).d.Fluorescence intensity analysis of inguinal lymph nodes after vaccination (DiD-labeled G-EVLPs).e. Fluorescence images of spleens after vaccination (DiR-labeled tumor membrane).f.Fluorescence images of spleens after vaccination (DiD-labeled G-EVLPs).g.Fluorescence intensity analysis of spleens after vaccination (DiR-labeled tumor membrane).h.Fluorescence intensity analysis of spleens after

Figure S14 .
Figure S14.Flow cytometry analysis of DiD high cells of CD11c + DCs in inguinal lymph nodes after vaccination (DiR-labeled tumor membrane).a. Principles of flow cytometry data processing and flow cytometry uptake data for different groups.b.Relative cellular uptake of DiR-labeled tumor membrane of CD11c+ DC in inguinal lymph nodes, as assessed by flow cytometry.

Figure
Figure S15.HM-NPs (B16F10) promote splenic T cells activation after vaccination.a-d.IFN-γ concentration in the supernatant after co-incubation of the splenic T lymphocytes with B16F10 tumor cells (a), CT26 tumor cells (b), MB49 tumor cells (c) and 4T1 tumor cells (d).e-f.LDH concentration in the supernatant after co-incubation of the splenic T lymphocytes with B16F10 tumor cells (e), CT26 tumor cells (f), MB49 tumor cells (g) and 4T1 tumor cells (h).Data are representative or pooled and are expressed as Mean ± SE.Asterisks indicate statistically significant differences as analyzed by One-Way ANOVA ( * * * p < 0.001, N.S. p>0.05).

Figure S16 .
Figure S16.Repertoire analysis of splenic IgM B lymphocytes after vacation.a.The average mutation rate per base pair in the BCR V region.b.The comparison of the clone number and clone size distribution of CDR3 region reveals differences in the converging rate among different groups under the same RNA-UMI sequencing amount.c.At the levels of CDR3 clone number, RNA clone number, and unique RNA number, we calculate the extent to which the immune system diversity changed from control groups to HM-NPs.d.Visualizing the clone size distribution after clustering based on the CDR3 region similarity by Igraph package.e. Visualizing the clustering results that define clone size based on the number of UMIs linking to the same RNA, indicating the occurrence of polyclonal reactions.Data are representative or

Figure S18 .
Figure S18.HM-NPs (B16F10) vaccination enhances anti-tumor immune response in the murine B16F10 tumor model.a. Proinflammatory IFN-γ concentration in the serum of the mice receiving each treatment determined by ELISA assay.b.Proinflammatory TNF-α concentration in the serum of the mice receiving each treatment determined by ELISA assay.c.Proinflammatory IL-6 concentration in the serum of the mice receiving each treatment determined by ELISA assay.d.Proinflammatory IL-1β concentration in the serum of the mice receiving each treatment determined by ELISA assay.e. Proinflammatory IFN-γ concentration in the tumor homogenate receiving each treatment determined by ELISA assay.

f.
Proinflammatory TNF-α concentration in the tumor homogenate receiving each treatment determined by ELISA assay.g.Proinflammatory IL-6 concentration in the tumor homogenate receiving each treatment determined by ELISA assay.h.Proinflammatory IL-1β concentration in the tumor homogenate receiving each treatment determined by ELISA assay.Data are representative or pooled and are expressed as Mean ± SE.Asterisks indicate statistically significant differences as analyzed by One-Way ANOVA ( * * p < 0.01, N.S. p>0.05).

Figure S23 .
Figure S23.HM-NPs vaccination inhibits specific tumor recurrence.a. Tumor weight of each group in the murine CT26 tumor model at day 41.b.Tumor weight of each group in the murine 4T1 tumor model at day 41.c.Photographs of the murine CT26 tumor model at day 41.d.Photographs of the murine 4T1 tumor model at day 41.Data are representative or

Figure S24 .
Figure S24.HM-NPs vaccination provides long-term anti-tumor protection.a. Tumor growth curves of each group in the murine tumor model.b.Tumor weight of each group in the murine 4T1 tumor model at day 99.c.Body weight curves of the Control group in the murine 4T1 tumor model.d.Body weight curves of the HM-NPs group in the murine 4T1 tumor model.e. Photographs of the murine 4T1 tumor model at day 99.Data are representative or pooled and are expressed as Mean ± SE.Asterisks indicate statistically significant differences as analyzed by One-Way ANOVA ( * * * p < 0.001, * * p < 0.01)

Figure S31 .
Figure S31.HM-NPs vaccination establishes protection against orthotopic mouse MB49 bladder tumor.a. Photographs of the murine MB49 tumor at day 36.b.Photographs are the H&E staining, TUNEL staining, and immunohistochemical images of the tumors post treatments of the tumors receiving different treatments

Figure S33 .
Figure S33.Localization of M-NPs and HM-NPs determined by CLSM.a.The Pearson Correlation coefficient of M-NPs.b.The Pearson Correlation coefficient of HM-NPs.c.The Pearson Correlation coefficient of M-NPs and HM-NPs.Data are representative or pooled and are expressed as Mean ± SE.Asterisks indicate statistically significant differences as analyzed by One-Way ANOVA ( * * * p < 0.001).

Figure S34 .
Figure S34.Analysis of the proportion of DiD and DiR double-positive particles using flow cytometry to determine the fusion efficiency of HM-NPs.Data are representative or pooled and are expressed as Mean ± SE.Asterisks indicate statistically significant differences as analyzed by One-Way ANOVA ( * * * p < 0.001).

Figure S35 .
Figure S35.Flow cytometry analysis of CD45 + CD11c + CD83 + cells in BMDCs.a. Figures of flow cytometry analysis.b.Statistical analysis of Figure a.Data are representative or pooled and are expressed as Mean ± SE.Asterisks indicate statistically significant differences as analyzed by One-Way ANOVA ( * * * p < 0.001).

Figure S36 .
Figure S36.Photographs are the white filed images of the H&E staining of the tumors receiving different treatments.

Figure S37 .
Figure S37.Flow cytometry analysis of CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + cells in LNs after vaccination.a. Flow cytometry analysis of CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + cells in LNs after vaccination.b.Statistical analysis of CD45 + CD3 + CD8 + cells of Figure a. c.Statistical analysis of CD45 + CD3 + CD4 + cells of Figure a.Data are representative or pooled and are expressed as Mean ± SE.Asterisks indicate statistically significant differences as analyzed by One-Way